Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.08.04.551608v1?rss=1 Authors: Gomez-Ferrer, M., Amaro-Prellezo, E., Albiach-Delgado, A., Ten-Domenech, I., Kuligowski, J., Sepulveda, P. Abstract: Premature infants (PIs) are at risk of suffering necrotizing enterocolitis (NEC), and infants consuming human milk (HM) show a lower incidence than infants receiving formula. The composition of HM has been studied in depth, but the lipid content of HM-derived small extracellular vesicles (HM sEVs) remains unexplored. We isolated HM sEVs from HM samples and analyzed their oxylipin content using liquid chromatography coupled to mass spectrometry, which revealed the presence of anti-inflammatory oxylipins. We then examined the efficacy of a mixture of these oxylipins in combating inflammation and fibrosis, in vitro and and in a murine model of inflammatory bowel disease (IBD). HM-related sEVs contained higher concentrations of oxylipins derived from docosahexaenoic acid, an omega-3 fatty acid. Three anti-inflammatory oxylipins, 14-HDHA, 17-HDHA, and 19,20-DiHDPA ({omega}3 OXLP), demonstrated similar efficacy to HM sEVs in preventing cell injury, inducing re-epithelialization, mitigating fibrosis, and modulating immune responses. Both {omega}3 OXLP and HM sEVs effectively reduced inflammation in IBD-model mice, preventing colon shortening, infiltration of inflammatory cells and tissue fibrosis. Incorporating this unique cocktail of oxylipins into fortified milk formulas might reduce the risk of NEC in PIs and also provide immunological and neurodevelopmental support. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.08.03.551824v1?rss=1 Authors: Qin, K., Wu, X. Abstract: The nuclear mitochondrial DNA (NUMT) is found in cancer cells, but the mitochondrial DNAs entering the nuclei in normal cells have not been captured. Here, we utilized super-resolution optical imaging to capture the phenomenon by the probe PicoGreen and found mitochondrial DNAs and mitochondria accumulated in the nucleoli by four probes and overexpressing the MRPL58-DsRed. Our results provide an new explanation for mtDNA carryover and lay the foundation for the involvement of nuclear export of nucleoli in de novo mitochondrial biogenesis in another of our unpublished articles. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.08.02.551383v1?rss=1 Authors: Cohen, M. L., Brumwell, A. N., Ho, T. C., Montas, G., Golden, J. A., Jones, K. D., Wolters, P. J., Wei, Y., Chapman, H. A., Le Saux, C. J. Abstract: Reciprocal interactions between alveolar fibroblasts and epithelial cells are crucial for lung homeostasis, injury repair, and fibrogenesis, but underlying mechanisms remain unclear. To investigate this, we administered the fibroblast-selective TGF{beta}1 signaling inhibitor, epigallocatechin gallate (EGCG), to Interstitial Lung Disease (ILD) patients undergoing diagnostic lung biopsy and conducted single-cell RNA sequencing on spare tissue. Unexposed biopsy samples showed higher fibroblast TGF{beta}1 signaling compared to non-disease donor or end-stage ILD tissues. In vivo, EGCG significantly downregulated TGF{beta}1 signaling and several pro-inflammatory and stress pathways in biopsy samples. Notably, EGCG reduced fibroblast secreted Frizzle-like Receptor Protein 2 (sFRP2), an unrecognized TGF{beta}1 fibroblast target gene induced near type II alveolar epithelial cells (AEC2s). In human AEC2-fibroblast coculture organoids, sFRP2 was essential for AEC2 trans-differentiation to basal cells. Precision cut lung slices (PCLS) from normal donors demonstrated that TGF{beta}1 promoted KRT17 expression and AEC2 morphological change, while sFRP2 was necessary for KRT5 expression in AEC2-derived basaloid cells. Wnt-receptor Frizzled 5 (Fzd5) expression and downstream calcineurin-related signaling in AEC2s were required for sFRP2-induced KRT5 expression. These findings highlight stage-specific TGF{beta}1 signaling in ILD, the therapeutic potential of EGCG in reducing IPF-related transcriptional changes, and identify the TGF{beta}1-non-canonical Wnt pathway crosstalk via sFRP2 as a novel mechanism for dysfunctional epithelial signaling in Idiopathic Pulmonary Fibrosis/ILD. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.08.04.552008v1?rss=1 Authors: Talavera, R. A., Prichard, B. E., Sommer, R. A., Leitao, R. M., Sarabia, C. J., Hazir, S., Paulo, J. A., Gygi, S., Kellogg, D. Abstract: Cell growth is required for cell cycle progression. The amount of growth required for cell cycle progression is reduced in poor nutrients, which leads to a reduction in cell size. In budding yeast, nutrients influence cell size by modulating the duration and extent of bud growth, which occurs predominantly in mitosis. However, the mechanisms are unknown. Here, we used mass spectrometry to identify proteins that mediate the effects of nutrients on bud growth. This led to the discovery that nutrients regulate numerous components of the Mitotic Exit Network (MEN), which controls exit from mitosis. A key component of the MEN undergoes gradual multi-site phosphorylation during bud growth that is dependent upon growth and correlated with the extent of growth. Furthermore, activation of the MEN is sufficient to over-ride a growth requirement for mitotic exit. The data suggest a model in which the MEN integrates signals regarding cell growth and nutrient availability to ensure that mitotic exit occurs only when sufficient growth has occurred. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.08.02.551232v1?rss=1 Authors: Nguyen, T. P., Otani, T., Tsutsumi, M., Fujiwara, S., Nemoto, T., Fujimori, T., Furuse, M. Abstract: Epithelia must be able to resist mechanical force to preserve tissue integrity. While intercellular junctions are known to be important for the mechanical resistance of epithelia, the roles of tight junctions (TJs) remain to be established. We previously demonstrated that epithelial cells devoid of the TJ membrane proteins claudins and JAM-A completely lack TJs and exhibit focal breakages of their apical junctions. Here, we demonstrate that apical junctions undergo spontaneous fracture when claudin/JAM-A-deficient cells are exposed to mechanical stress. The junction fracture was accompanied by actin disorganization, and actin polymerization was required for apical junction integrity in the claudin/JAM-A-deficient cells. Further deletion of CAR resulted in the disruption of ZO-1 molecule ordering at cell junctions, accompanied by severe defects in apical junction integrity. These results demonstrate that TJ membrane proteins regulate the mechanical resistance of the apical junctional complex in epithelial cells. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.08.04.551943v1?rss=1 Authors: Bagley, D. C., Russell, T., Ortiz-Zapater, E., Fox, K., Redd, P. F., Joseph, M., Rice, C. D., Reilly, C. A., Parsons, M., Rosenblatt, J. Abstract: Asthma is a common disease characterized by airway constriction, excess mucus, and inflammation. Although asthma is an inflammatory disease, subclassed by different endotypes, triggers, and immune responses, the defining diagnostic symptom is mechanical bronchoconstriction from uncontrolled smooth muscle contraction. We previously discovered a conserved process that drives epithelial cell death in response to mechanical cell crowding called epithelial cell extrusion (1,2). Because modest crowding triggers extrusion to maintain constant homeostatic epithelial cell densities, we reasoned that the pathological crowding from bronchoconstriction might potentially destroy the airway epithelial barrier, causing the typical inflammatory period that follows an asthma attack. Here, using immune-primed mice, we show that the crowding of bronchoconstriction causes excess epithelial cell extrusion and damage, resulting in inflammation in distal airways, and mucus secretion in proximal airways. Surprisingly, relaxing airways following bronchoconstriction with the current rescue treatment, albuterol, did not prevent epithelial extrusion and destruction, inflammation, or mucus secretion. However, inhibiting canonical live cell extrusion signaling during bronchoconstriction with stretch-activated/TRP channel or sphingosine 1-phosphate (S1P) inhibitors blocked all downstream symptoms. Our findings propose a new etiology for asthma where the extreme mechanical crowding from a bronchoconstrictive attack causes inflammation by wounding airway epithelium. Whereas most therapies focus on modulating downstream inflammatory symptoms, our studies suggest that blocking epithelial extrusion could prevent the feed-forward asthma inflammatory cycle. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.08.04.551992v1?rss=1 Authors: Quiroz, E. J., Kim, S., Gautam, L. K., Borok, Z., Kintner, C., Ryan, A. L. Abstract: A core pathophysiologic feature underlying many respiratory diseases is multiciliated cell dysfunction, leading to inadequate mucociliary clearance. Due to the prevalence and highly variable etiology of mucociliary dysfunction in respiratory diseases, it is critical to understand the mechanisms controlling multiciliogenesis that may be targeted to restore functional mucociliary clearance. Multicilin, in a complex with E2F4, is necessary and sufficient to drive multiciliogenesis in airway epithelia, however this does not apply to all cell types, nor does it occur evenly across all cells in the same cell population. In this study we further investigated how co-factors regulate the ability of Multicilin to drive multiciliogenesis. Combining data in mouse embryonic fibroblasts and human bronchial epithelial cells, we identify RBL2 as a repressor of the transcriptional activity of Multicilin. Knockdown of RBL2 in submerged cultures or phosphorylation of RBL2 in response to apical air exposure, in the presence of Multicilin, allows multiciliogenesis to progress. These data demonstrate a dynamic interaction between RBL2 and Multicilin that regulates the capacity of cells to differentiate and multiciliate. Identification of this mechanism has important implications for facilitating MCC differentiation in diseases with impaired mucociliary clearance. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.08.02.551716v1?rss=1 Authors: Ilyinsky, N. S., Bukhalovich, S. M., Bagaeva, D. F., Nesterov, S. V., Alekseev, A. A., Tsybrov, F. M., Bogorodskiy, A. O., Moiseeva, O. V., Vlasova, A. D., Kovalev, K. V., Mikhailov, A. E., Rogachev, A. V., Bamberg, E., Ivanovich, V., Borshchevskiy, V. I. Abstract: Lysosome protective, metabolic, signaling functions are highly dependent on their pH. A lack of tools of high spatial and temporal resolution for pH control is a bottleneck of lysosome related cell research. Light-driven inward proton pump NsXeR, targeted to the lysosomes of mammalian cells, produces lysosome alkalization simply by light. Complementary use of outward proton pumping Arch3 rhodopsins in lysosomes offers an approach to vary pH in a range from around 5 to 6.5 in both directions (alkalization and acidification). Lyso-NsXeR optogenetics efficiency was demonstrated, in particular, by its ability to inhibit lysosome proteolytic enzymes. Unprecedented time resolution of the optogenetic approach allowed direct in situ monitoring of vATPase activity. Thus, optogenetic monitoring and regulation of the lysosome function, through pH control over a wide range, could serve as an approach to studying fundamental cell processes, and rational drug design. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.08.04.551988v1?rss=1 Authors: Zhang, Z., Huang, Y., Tao, W., Wei, Y., Xu, L., Gong, W., Zhang, Y., Han, Y., Kuang, C., Liu, X. Abstract: Stimulated emission depletion microscopy (STED) is a powerful tool for studying nanoscale cell structure and activity, but the difficulties it encounters in multicolor imaging limit its application in biological research. To overcome the disadvantages of limited number of channels and high cost of multicolor STED imaging based on spectral identity, we introduced lifetime into live-cell multicolor STED imaging by separating selected dyes of the same spectrum by phasor analysis. Experimental results show that our method enables live-cell STED imaging with at least 4 colors, enabling observation of cellular activity beyond the diffraction limit. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.08.03.551916v1?rss=1 Authors: Takenaka, Y., Kakinuma, Y., Inoue, I. Abstract: We have previously identified the novel splicing variant of mouse Ppar{gamma} (Ppar{gamma}1sv) and proposed the synergistic regulation of the early stage of adipocyte differentiation by Ppar{gamma}1sv and Ppar{gamma}2. Here, we report the finding of PPAR{gamma}-binding sites within the Ppar{gamma} gene locus and its importance in adipogenesis and propose the positive feedback regulation of Ppar{gamma}1sv and Ppar{gamma}2 expression during the adipocyte differentiation of 3T3-L1 cells. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.08.03.551815v1?rss=1 Authors: Salvador-Garcia, D., Jin, L., Hensley, A., Golcuk, M., Gallaud, E., Chaaban, S., Port, F., Vagnoni, A., Planelles-Herrero, V. J., McClintock, M. A., Derivery, E., Carter, A. P., Giet, R., Gur, M., Yildiz, A., Bullock, S. L. Abstract: The cytoplasmic dynein-1 (dynein) motor organizes cells by shaping microtubule networks and moving a large variety of cargoes along them. However, dynein's diverse roles complicate in vivo studies of its functions significantly. To address this issue, we have used gene editing to generate a series of missense mutations in Drosophila Dynein heavy chain (Dhc). We find that mutations associated with human neurological disease cause a range of defects in larval and adult flies, including impaired cargo trafficking in neurons. We also describe a novel mutation in the microtubule-binding domain (MTBD) of Dhc that, remarkably, causes metaphase arrest of mitotic spindles in the embryo but does not impair other dynein-dependent processes. We demonstrate that the mitotic arrest is independent of dynein's well-established roles in silencing the spindle assembly checkpoint. In vitro reconstitution and optical trapping assays reveal that the mutation only impairs the performance of dynein under load. In silico all-atom molecular dynamics simulations show that this effect correlates with increased flexibility of the MTBD, as well as an altered orientation of the stalk domain, with respect to the microtubule. Collectively, our data point to a novel role of dynein in anaphase progression that depends on the motor operating in a specific load regime. More broadly, our work illustrates how cytoskeletal transport processes can be dissected in vivo by manipulating mechanical properties of motors. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.08.03.551853v1?rss=1 Authors: Chen, J., Yue, F., Kim, K. H., Zhu, P., Qiu, J., Tao, W. A., Kuang, S. Abstract: Mitochondria are not only essential for energy production in eukaryocytes but also a key regulator of intracellular signaling. Here, we report an unappreciated role of mitochondria in regulating cytosolic protein translation in skeletal muscle cells (myofibers). We show that the expression of mitochondrial protein FAM210A (Family With Sequence Similarity 210 Member A) is positively associated with muscle mass in mice and humans. Muscle-specific Myl1Cre-driven Fam210a knockout (Fam210aMKO) in mice reduces mitochondrial density and function, leading to progressive muscle atrophy and premature death. Metabolomic and biochemical analyses reveal that Fam210aMKO reverses the oxidative TCA cycle towards the reductive direction, resulting in acetyl-CoA accumulation and hyperacetylation of cytosolic proteins. Specifically, hyperacetylation of several ribosomal proteins leads to disassembly of ribosomes and translational defects. Transplantation of Fam210aMKO mitochondria into wildtype myoblasts is sufficient to elevate protein acetylation in recipient cells. These findings reveal a novel crosstalk between the mitochondrion and ribosome mediated by FAM210A. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.08.03.551216v1?rss=1 Authors: Zaharija, B., Bradshaw, N. J. Abstract: An emerging approach to studying major mental illness is through proteostasis, with the identification of several proteins that form insoluble aggregates in the brains of patients. One of these is Disrupted in Schizophrenia 1 (DISC1), a neurodevelopmentally-important scaffold protein, and the product of a classic schizophrenia risk gene. DISC1 was seen to aggregate in post mortem tissue from patients with schizophrenia, bipolar disorder and major depressive disorder, as well as in a variety of model systems, although the mechanism by which it does so is still unclear. Aggregation of two other proteins implicated in mental illness, TRIOBP-1 and NPAS3, was shown to be dependent on very specific structural regions of the protein. We therefore looked to the recently determined domain structure of DISC1, and investigated which structural elements were key for its aggregation. While none of the known DISC1 regions (named D, I, S and C respectively) formed aggregates individually when expressed in neuroblastoma cells, the combination of the D and I regions, plus the linker region between them, formed visible aggregates. Further refinement revealed that a region of approximately 30 amino acids between these two regions is critical to aggregation, with deletion of this region from full length DISC1 sufficient to abolish its aggregation propensity. This finding from mammalian cell culture contrasts with the recent determination that the extreme C-terminal of DISC1 can aggregate in vitro, although we did see some indication that combinations of C-terminal DISC1 regions can also aggregate in our system. It therefore appears likely that DISC1 aggregation, implicated in mental illness, can occur through at least two distinct mechanisms. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.08.03.550647v1?rss=1 Authors: Ibtisam, I., Kisselev, A. F. Abstract: Rapid recovery of proteasome activity may contribute to intrinsic and acquired resistance to FDA-approved proteasome inhibitors. Previous studies have demonstrated that the expression of proteasome genes in cells treated with sub-lethal concentrations of proteasome inhibitors is upregulated by the transcription factor Nrf1 (NFE2L1), which is activated by a novel DDI2 protease. Here we demonstrate that the recovery of proteasome activity is DDI2-independent and occurs before the upregulation of proteasome gene expression. The recovery requires protein translation, but the efficiency of translation of proteasomal mRNA does not increase upon proteasome inhibition. Based on this data, we propose that the increased efficiency of proteasome assembly is responsible for the recovery of proteasome activity. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.08.04.552058v1?rss=1 Authors: Lesiak, L., Dadina, N., Zheng, S., Schelvis, M., Schepartz, A. Abstract: Lysosomes have long been known for their acidic lumen and efficient degradation of cellular byproducts. In recent years it has become clear that their function is far more sophisticated, involving multiple cell signaling pathways and interactions with other organelles. Unfortunately, their acidic interior, fast dynamics, and small size makes lysosomes difficult to image with fluorescence microscopy. Here we report a far-red small molecule, HMSiR680-Me, that fluoresces only under acidic conditions, causing selective labeling of acidic organelles in live cells. HMSiR680-Me can be used alongside other far-red dyes in multicolor imaging experiments and is superior to existing lysosome probes in terms of photostability and maintaining cell health and lysosome motility. We demonstrate that HMSiR680-Me is compatible with overnight time lapse experiments, as well as time lapse super- resolution microscopy with a fast frame rate for at least 1000 frames. HMSiR680-Me can also be used alongside silicon rhodamine dyes in a multiplexed super-resolution microscopy experiment to visualize interactions between the inner mitochondrial membrane and lysosomes with only a single excitation laser and simultaneous depletion. We envision this dye permitting more detailed study of the role of lysosomes in dynamic cellular processes and disease. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.08.04.552031v1?rss=1 Authors: Lang, C. F., Anneken, A., Munro, E. Abstract: The PAR polarity network is a paradigmatic example of how systems of mutually antagonism interactions among peripheral membrane binding proteins allow them to form and maintain complementary polar domains in response to a transient polarizing cue. The oligomeric scaffolding protein PAR-3 has emerged as a keystone member of the PAR network in many different contexts. In early C. elegans embryos, PAR-3 is required for all other PAR asymmetries, and it can form stable unipolar asymmetries when its known inhibitors are absent and all other members of the PAR network are cytoplasmic or spatially uniform on the membrane. But how PAR-3 forms stable unipolar asymmetries absent mutual antagonism is unknown. Here we combine single particle analysis with quantitative modeling and experimental manipulations to determine how the dynamics of PAR-3 membrane binding, oligomerization and dissociation allow PAR-3 to maintain stable asymmetries in the one cell C. elegans embryo. We find that two forms of positive feedback contribute to sustaining PAR-3 asymmetries: First, a sharp size-dependent decrease in oligomer dissociation rates makes the effective dissociation rate of PAR-3 decrease sharply with its membrane density. Second, membrane-bound PAR-3 promotes additional binding of PAR-3 to the membrane through a mechanism that requires the presence of anterior polarity proteins CDC-42, PAR-6 and PKC-3. Through a combination of modeling and quantitative measurements, we show that these two feedback loops are sufficient to dynamically stabilize asymmetries of the magnitude observed in polarized C. elegans zygotes. These results establish a dynamic basis for stabilizing monopolar PAR-3 asymmetries; they underscore a crucial role for the oligomerization and add to the growing body of evidence that point to a central role for oligomerization of peripheral membrane proteins in the establishment and maintenance of cell polarity. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.08.03.551778v1?rss=1 Authors: Ohno, Y., Nakamura, T., Iwasaki, T., Katsuyama, A., Ichikawa, S., Kihara, A. Abstract: Protein-bound ceramides, specialized ceramides covalently bound to corneocyte surface proteins, are essential for skin permeability barrier function. However, their exact structure and target amino acid residues are unknown. Here, we found that epoxy-enone (EE) ceramides, precursors of protein-bound ceramides, as well as their synthetic analog, formed stable conjugates only with Cys among nucleophilic amino acids. NMR spectroscopy revealed that the beta-carbon of the enone was attached by the thiol group of Cys via a Michael addition reaction. We confirmed the presence of Cys-bound EE ceramides in mouse epidermis by mass spectrometry analysis of protease-digested epidermis samples. EE-ceramides were reversibly released from protein-bound ceramides via sulfoxide elimination. We found that protein-bound ceramides with reversible release properties accounted for approximately 60% of total protein-bound ceramides, indicating that Cys-bound EE ceramides are the predominant protein-bound ceramides. Our findings provide clues to the molecular mechanism of skin barrier formation by protein-bound ceramides. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.08.03.551868v1?rss=1 Authors: Lin, Z., Zhang, X., Nandi, P., Lin, Y., Wang, L., Chu, Y., Paape, T., Yang, Y., Xiao, X., Liu, Q. Abstract: X-ray tomography and x-ray fluorescence imaging are two non-invasive imaging techniques to study cellular structures and chemical element distributions, respectively. However, correlative X-ray tomography and fluorescence imaging for the same cell has yet to be routinely realized due to challenges in sample preparation and X-ray radiation damage. Here we report an integrated experimental and computational workflow for achieving correlative multi-modality X-ray imaging of a single cell. The method consists of the preparation of radiation-resistant single-cell samples using live-cell imaging-assisted chemical fixation and freeze-drying procedures, targeting and labeling cells for correlative x-ray tomography and x-ray fluorescence measurement, and computational reconstruction of the correlative and multi-modality images. With X-ray tomography, cellular structures including the overall structure and intracellular organelles are visualized, while X-ray fluorescence imaging reveals the distribution of multiple chemical elements within the same cell. Our correlative method demonstrates the feasibility and broad applicability of using X-rays to understand cellular structures and the roles of multiple chemical elements and related proteins in signaling and other biological processes. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.08.02.551028v1?rss=1 Authors: Vadakke-Madathil, S., Wang, B., Oniskey, M., Dekio, F., Brody, R., Gelber, S., Sperling, R., Chaudhry, H. W. Abstract: The human placenta is a reservoir of a multitude of cell types with immense regenerative potential. Caudal-type homeobox-2 (CDX2) is a conserved factor that regulates trophectoderm formation and placentation during early embryonic development and hence can play a vital role in understanding developmentally conserved regenerative mechanisms. Cdx2 lineage tracing in our previous study identified multipotent Cdx2 lineage cells in the mouse placenta capable of restoring cardiac function after intravenous delivery in male mice with experimental cardiac injury (myocardial infarction). Here we demonstrate that CDX2-expressing cells are prevalent in the human chorionic placenta and are uniquely committed to cardiovascular differentiation. We examined the term placentas from 106 healthy donors and showed that isolated CDX2 cells can spontaneously differentiate into cardiomyocytes, functional vascular cells, and retain homing ability in vitro. Functional annotation from transcriptomics analysis supports enhanced cardiogenesis, vasculogenesis, immune modulation, and chemotaxis gene signatures in CDX2 cells. CDX2 cells can be clonally propagated in culture with retention of cardiovascular differentiation. Bringing us a step closer to translation, our study identifies an easily accessible and ethically feasible cell source to facilitate therapeutic strategies for cardiovascular disease. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.08.03.551843v1?rss=1 Authors: Sun, S., Tranchina, D., Gresham, D. Abstract: Cells arrest growth and enter a quiescent state upon nutrient deprivation. However, the molecular processes by which cells respond to different starvation signals to regulate exit from the cell division cycle and initiation of quiescence remains poorly understood. To study the role of protein expression and signaling in quiescence we combined temporal profiling of the proteome and phosphoproteome using stable isotope labeling with amino acids in cell culture (SILAC) in Saccharomyces cerevisiae (budding yeast). We find that carbon and phosphorus starvation signals activate quiescence through largely distinct remodeling of the proteome and phosphoproteome. However, increased expression of mitochondrial proteins is associated with quiescence establishment in response to both starvation signals. Deletion of the putative quiescence regulator RIM15, which encodes a serine-threonine kinase, results in reduced survival of cells starved for phosphorus and nitrogen, but not carbon. However, we identified common protein phosphorylation roles for RIM15 in quiescence that are enriched for RNA metabolism and translation. We also find evidence for RIM15-mediated phosphorylation of some targets, including IGO1, prior to starvation consistent with a functional role for RIM15 in proliferative cells. Finally, our results reveal widespread catabolism of amino acids in response to nitrogen starvation, indicating widespread amino acid recycling via salvage pathways in conditions lacking environmental nitrogen. Our study defines an expanded quiescent proteome and phosphoproteome in yeast, and highlights the multiple coordinated molecular processes at the level of protein expression and phosphorylation that are required for quiescence. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.08.04.551922v1?rss=1 Authors: Silao, F. G. S., Valeriano, V. D., Uddström, E., Falconer, E., Ljungdahl, P. O. Abstract: Candida albicans has the remarkable capacity to neutralize acidic growth environments by releasing ammonia derived from the catabolism of amino acids. The molecular components and mechanisms controlling this capacity remain poorly understood. Here, we present an integrative model with the cytosolic NAD+-dependent glutamate dehydrogenase (Gdh2) as the principal component. We show that the alkalization defect of a strain lacking the SPS-sensor regulated transcription factor STP2 is due to the inability to fully derepress GDH2 and the two proline catabolic enzymes, PUT1 and PUT2. Notably, the Stp2-dependent regulation of PUT1 and PUT2 occurs independent of Put3, the proline-dependent activator. Accordingly, a stp2-/- put3-/- strain is unable to derepress the expression of these enzymes resulting in a severe alkalization defect that nearly phenocopies the abrogated alkalization of a gdh2-/- strain. In wildtype cells, alkalization is tightly dependent on mitochondrial activity and occurs as long as conditions permit respiratory growth. As alkalization proceeds, Gdh2 levels decrease and glutamate is transiently extruded from cells. Together these two processes constitute a rudimentary regulatory system enabling cells to prevent the rapid intracellular build-up of ammonia. Similar to C. albicans, Gdh2-dependent alkalization is dispensable for C. glabrata and C. auris virulence as assessed using a wholeblood infection model. Intriguingly, fungal-dependent alkalization does not influence the growth or proliferation of Lactobacillus crispatus, a potent antagonist of C. albicans that normally resides in the acidic vaginal microenvironment. Our data suggest that it is time to reconsider the idea that pH modulation driven by pathogenic fungi plays a crucial role in shaping the architecture and dynamics of (poly)microbial communities. Other factors are likely to be more critical in contributing to dysbiosis and that favor virulent growth. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.08.03.551799v1?rss=1 Authors: Yao, Y., Lou, X., Du, G., Jin, L., Li, J., Liu, J., Chen, Y., Cheng, S., Zhao, T., Ke, S., Zhang, L., Zhang, P., Xu, Y., He, L., Li, H. Abstract: High-performance biosensors are crucial for elucidating the spatiotemporal regulatory roles and dynamics of membrane lipids, but there is a lack of improvement strategies for biosensors with low sensitivity and low-content substrates detection. Here we developed universal optogenetic strategies to improve a set of membrane biosensors by trapping them into specific region and further reducing the background signal, or by optically-controlled phase separation for membrane lipids detection and tracking. These improved biosensors were superior to typical tools and light simulation would enhance their detection performance and resolution, which might contribute to the design and optimization of other biosensors. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.08.04.552066v1?rss=1 Authors: Charles-Orszag, A., van Wolferen, M., Lord, S. J., Albers, S.-V., Mullins, D. Abstract: Type IV pili are ancient and widespread filamentous organelles found in most bacterial and archaeal phyla where they support a wide range of functions, including substrate adhesion, DNA uptake, self aggregation, and cell motility. In most bacteria, PilT-family ATPases disassemble adhesion pili, causing them to rapidly retract and produce twitching motility, important for surface colonization. As archaea do not possess homologs of PilT, it was thought that archaeal pili cannot retract. Here, we employ live-cell imaging under native conditions (75{degrees}C and pH 2), together with automated single-cell tracking, high-temperature fluorescence imaging, and genetic manipulation to demonstrate that S. acidocaldarius exhibits bona fide twitching motility, and that this behavior depends specifically on retractable adhesion pili. Our results demonstrate that archaeal adhesion pili are capable of retraction in the absence of a PilT retraction ATPase and suggests that the ancestral type IV pilus machinery in the last universal common ancestor (LUCA) relied on such a bifunctional ATPase for both extension and retraction. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.08.02.551712v1?rss=1 Authors: Cai, X., Ng, C. C., Jones, O., Fung, T. S., Ryu, K., Li, D., Thompson, C. B. Abstract: Lactate has long been considered a cellular waste product. However, we found that as extracellular lactate accumulates, it also enters the mitochondrial matrix and stimulates mitochondrial electron transport chain (ETC) activity. The resulting increase in mitochondrial ATP synthesis suppresses glycolysis and increases the utilization of pyruvate and/or alternative respiratory substrates. The ability of lactate to increase oxidative phosphorylation does not depend on its metabolism. Both L- and D-lactate are effective at enhancing ETC activity and suppressing glycolysis. Furthermore, the selective induction of mitochondrial oxidative phosphorylation by unmetabolized D-lactate reversibly suppressed aerobic glycolysis in both cancer cell lines and proliferating primary cells in an ATP-dependent manner and enabled cell growth on respiratory-dependent bioenergetic substrates. In primary T cells, D-lactate enhanced cell proliferation and effector function. Together, these findings demonstrate that lactate is a critical regulator of the ability of mitochondrial oxidative phosphorylation to suppress glucose fermentation. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.08.03.551705v1?rss=1 Authors: Murray, R., Navarrete, N. R. R., Desai, K., Chowdhury, M. R., Chilakapati, S. R., Chong, B., Messana, A., Sobon, H., Rocha, J., Musenge, F., Camblin, A., Ciaramella, G., Sitkovsky, M., Maldini, C., Hatfield, S. Abstract: Biochemical and immunological negative regulators converge to inhibit tumor-reactive Chimeric Antigen Receptor T (CAR-T) cells, which may explain clinical failures of CAR-T cell therapies against solid tumors. Here, we developed a multifaceted approach to genetically engineer allogeneic ("off-the-shelf") CAR-T cells resistant to both biochemical (adenosine) and immunological (PD-L1 and TGF-{beta}) inhibitory signaling. We multiplexed an adenine base editor with a CRISPR-Cas12b nuclease to manufacture a CAR-T cell product comprising six gene edits to evade allorejection (B2M, CIITA), prevent graft-versus-host disease (CD3E) and resist major biochemical (ADORA2A) and immunological (PDCD1, TGFBR2) immunosuppressive barriers in solid tumors. Combinatorial genetic disruption in CAR-T cells enabled superior anti-tumor efficacy leading to improved tumor elimination and survival in humanized mouse models that recapitulated the suppressive features of a human tumor microenvironment (TME). This novel engineering strategy conferred CAR-T cells resistance to a diverse TME, which may unlock the therapeutic potential of CAR-T cells against solid tumors. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.08.04.551933v1?rss=1 Authors: Muller-McNicoll, M., Zarnack, K., McNicoll, F., Keller, M., Sliskovic, I., Okuda, E. K., Riegger, R. J., Arnold, B. Abstract: Nuclear RNA binding proteins (RBPs) are difficult to study because they often belong to large protein families and form extensive networks of auto- and cross-regulation. They are highly abundant and often localize to condensates with a slow turnover, requiring long depletion times or knockouts that cannot distinguish between direct and indirect or compensatory effects. Here, we developed a system that is optimized for the rapid degradation of nuclear RBPs, called hGRAD. It comes as a 'one-fits-all' plasmid, and integration into any cell line that expresses endogenously GFP-tagged proteins allows an inducible, rapid and complete knockdown. We show that the nuclear RBPs SRSF3, SRSF5, SRRM2 and NONO are completely cleared from nuclear speckles and paraspeckles within two hours. hGRAD works in various cell types, is more efficient than other methods and does not require the expression of exogenous ubiquitin ligases. Combining SRSF5 hGRAD degradation with Nascent-seq uncovered highly dynamic transient transcript changes, compensatory mechanisms and that SRSF5 promotes transcript stability. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.08.03.551802v1?rss=1 Authors: Tojima, T., Suda, Y., Jin, N., Kurokawa, K., Nakano, A. Abstract: Cargo traffic through the Golgi apparatus is mediated by cisternal maturation, but it remains largely unclear how the cis-cisternae, the earliest Golgi sub-compartment, is generated. Here, we use high-speed, high-resolution confocal microscopy to analyze the spatiotemporal dynamics of a diverse set of proteins that reside in and around the Golgi in budding yeast. We find many mobile punctate structures that harbor yeast counterparts of mammalian endoplasmic reticulum (ER)-Golgi intermediate compartment (ERGIC) proteins, which we term "yeast ERGIC". It occasionally attaches onto the ER exit sites and gradually matures into the cis-Golgi. Upon treatment with the Golgi-disrupting agent brefeldin A, the ERGIC proteins form larger aggregates corresponding to the Golgi entry core compartment in plants, while cis- and medial-Golgi proteins are absorbed into the ER. We further analyze the dynamics of several late Golgi proteins. Together with our previous studies, we demonstrate a detailed spatiotemporal profile of the cisternal maturation process from ERGIC to Golgi and further to the trans-Golgi network. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.08.01.551420v1?rss=1 Authors: Amoiridis, M., Meaburn, K., Verigos, J., Gittens, W. H., Ye, T., Neale, M. J., Soutoglou, E. Abstract: DNA replication and transcription generate DNA supercoiling, which can cause topological stress and intertwining of daughter chromatin fibers, posing challenges to the completion of DNA replication and chromosome segregation. Type II topoisomerases (Top2s) are enzymes that relieve DNA supercoiling and decatenate braided sister chromatids. How Top2 complexes deal with the topological challenges in different chromatin contexts, and whether all chromosomal contexts are subjected equally to torsional stress and require Top2 activity is unknown. Here we show that catalytic inhibition of the Top2 complex in interphase has a profound effect on the stability of heterochromatin and repetitive DNA elements. Mechanistically, we find that catalytically inactive Top2 is trapped around heterochromatin leading to DNA breaks and unresolved catenates, which necessitate the recruitment of the structure specific endonuclease, Ercc1-XPF, in an Slx4- and SUMO-dependent manner. Our data are consistent with a model in which Top2 complex resolves not only catenates between sister chromatids but also inter-chromosomal catenates between clustered repetitive elements. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.08.01.551382v1?rss=1 Authors: Choi, H., Liao, Y.-C., Yoon, Y. J., Grimm, J. B., Lavis, L., Singer, R. H., Lippincott-Schwartz, J. Abstract: One-third of the mammalian proteome is comprised of transmembrane and secretory proteins that are synthesized on endoplasmic reticulum (ER). Here, we investigate the spatial distribution and regulation of mRNAs encoding these membrane and secretory proteins (termed 'secretome' mRNAs) through live cell, single molecule tracking to directly monitor the position and translation states of secretome mRNAs on ER and their relationship to other organelles. Notably, translation of secretome mRNAs occurred preferentially near lysosomes on ER marked by the ER junction-associated protein, Lunapark. Knockdown of Lunapark reduced the extent of secretome mRNA translation without affecting translation of other mRNAs. Less secretome mRNA translation also occurred when lysosome function was perturbed by raising lysosomal pH or inhibiting lysosomal proteases. Secretome mRNA translation near lysosomes was enhanced during amino acid deprivation. Addition of the integrated stress response inhibitor, ISRIB, reversed the translation inhibition seen in Lunapark knockdown cells, implying an eIF2 dependency. Altogether, these findings uncover a novel coordination between ER and lysosomes, in which local release of amino acids and other factors from ER-associated lysosomes patterns and regulates translation of mRNAs encoding secretory and membrane proteins. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.31.551394v1?rss=1 Authors: Fernandez-Gonzalez, A., Mukhia, A., Nadkarni, J., Willis, G., Reis, M., Zhumka, K., Vitali, S., Liu, X., Galls, A., Mitsialis, S. A., Kourembanas, S. Abstract: Objective: Macrophages play a central role in the onset and progression of vascular disease in pulmonary hypertension (PH) and cell-based immunotherapies aimed at treating vascular remodeling are lacking. This work evaluates the effect of pulmonary administration of macrophages modified to have an anti-inflammatory/pro-resolving phenotype in attenuating early pulmonary inflammation and progression of experimentally induced PH. Approach and Results: Mouse bone marrow derived macrophages (BMDMs) were polarized in vitro to a regulatory (M2reg) phenotype. M2reg profile and anti-inflammatory capacity were assessed in vitro upon lipopolysaccharide (LPS)/interferon-{gamma} (IFN{gamma}) restimulation, before their administration to 8- to 12- week-old mice. M2reg protective effect was tested at early (2 to 4 days) and late (4 weeks) time points during hypoxia (8.5% O2) exposure. Levels of inflammatory markers were quantified in alveolar macrophages and whole lung, while PH development was ascertained by right ventricular systolic pressure (RSVP) and right ventricular hypertrophy (RVH) measurements. Bronchoalveolar lavage (BAL) from M2reg-transplanted hypoxic mice was collected, and its inflammatory potential tested on naive BMDMs. M2reg macrophages demonstrated a stable anti-inflammatory phenotype upon a subsequent pro-inflammatory stimulus by maintaining the expression of specific anti-inflammatory markers (Tgf{beta}, Il10 and Cd206) and downregulating the induction of proinflammatory cytokines and surface molecules (Cd86, Il6 and Tnf). A single dose of M2regs attenuated the hypoxic monocytic recruitment and perivascular inflammation. Early hypoxic lung and alveolar macrophage inflammation leading to PH development was significantly reduced and, importantly, M2regs attenuated RVH, RVSP and vascular remodeling at 4 weeks post treatment. Conclusion: Adoptive transfer of M2regs halts the recruitment of monocytes and modifies the hypoxic lung microenvironment, potentially changing the immunoreactivity of recruited macrophages and restoring normal immune functionality of the lung. These findings provide new mechanistic insights on the diverse role of macrophage phenotype on lung vascular homeostasis that can be explored as novel therapeutic targets. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.08.01.551134v1?rss=1 Authors: Dabsan, S., Zur, G., Gilad, A., Igbaria, A. Abstract: The Endoplasmic Reticulum (ER) is an essential sensing organelle responsible for the folding and secretion of almost one-third of eukaryotic cells' total proteins. The ER contains numerous enzymes and chaperones which assist in oxidative protein folding and other posttranslational modifications. However, environmental, chemical, and genetic insults often lead to protein misfolding in the ER, accumulating misfolded proteins, altering homeostasis, and causing ER stress. Recently, we reported a novel ER surveillance mechanism by which proteins from the secretory pathway are refluxed to the cytosol to relieve the ER of its content during stress. In cancer cells, the refluxed proteins gain new pro-survival functions, thereby increasing cancer cell fitness. We termed this phenomenon ER to Cytosol Signaling (or ERCY). In yeast, ERCYS is regulated by HLJ1 (an ER-resident tail-anchored HSP40 cochaperone). Here, we found that in mammalian cells, HLJ1 has five putative orthologs possessing J-domains facing the cytosol. Among those, DNAJB12 and DNAJB14 appear to be the most significant, as they were shown to mediate retrograde trafficking/entry into the cytosol from the ER of nonenveloped viruses in a mechanism similar to ERCYS. Mechanistically, we found that DNAJB12 and DNAJB14 bind the cytosolic HSC70 and its cochaperone SGTA - through their cytosolically localized J-domains to facilitate ER-protein reflux to the cytosol. Moreover, we found that DNAJB12 is necessary and sufficient to drive this phenomenon to increase AGR2 reflux and inhibit wt-p53 during ER stress. Thus, we concluded that targeting the DNAJB12/14-HSC70/SGTA axis is a promising strategy to inhibit ERCYS and impair cancer cell fitness. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.08.01.551453v1?rss=1 Authors: Chew, Y. H., Spill, F. Abstract: Metabolites have to diffuse within the sub-cellular compartments they occupy to specific locations where enzymes are, so reactions could occur. Conventional flux balance analysis (FBA), a method based on linear programming that is commonly used to model metabolism, implicitly assumes that all enzymatic reactions are not diffusion-limited though that may not always be the case. In this work, we have developed a spatial method that implements FBA on a grid-based system, to enable the exploration of diffusion effects on metabolism. Specifically, the method discretises a living cell into a two-dimensional grid, represents the metabolic reactions in each grid element as well as the diffusion of metabolites to and from neighbouring elements, and simulates the system as a single linear programming problem. We varied the number of rows and columns in the grid to simulate different cell shapes, and the method was able to capture diffusion effects at different shapes. We then used the method to simulate heterogeneous enzyme distribution, which suggested a theoretical effect on variability at the population level. We propose the use of this method, and its future extensions, to explore how spatiotemporal organisation of sub-cellular compartments and the molecules within could affect cell behaviour. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.31.551359v1?rss=1 Authors: Clippinger, A. K., Naismith, T. V., Yoo, W., Jansen, S., Kast, D., Hanson, P. I. Abstract: ESCRTs (Endosomal Sorting Complex Required for Transport) are a modular set of protein complexes with membrane remodeling activities that include the formation and release of intralumenal vesicles (ILVs) to generate multivesicular endosomes. While most of the 12 ESCRT-III proteins are known to play roles in ILV formation, IST1 has been associated with a wider range of endosomal remodeling events. Here, we extend previous studies of IST1 function in endosomal trafficking and establish that IST1, along with its binding partner CHMP1B, contributes to scission of early endosomal carriers. Functionally, depleting IST1 impairs delivery of transferrin receptor from early/sorting endosomes to the endocytic recycling compartment and instead increases its rapid recycling to the plasma membrane via peripheral endosomes enriched in the clathrin adaptor AP-1. IST1 is also important for export of mannose 6-phosphate receptor from early/sorting endosomes. Examination of IST1 binding partners on these endosomes revealed that IST1 interacts both with CHMP1B and with the MIT domain-containing sorting nexin SNX15 previously reported to regulate endosomal recycling. Kinetic and spatial analyses showed that SNX15 and IST1 occupy a clathrin-containing subdomain distinct from those previously connected to cargo retrieval or degradation. Using live-cell microscopy we demonstrate that SNX15 and CHMP1B alternately control recruitment of IST1 to this domain on the endosomal perimeter and the base of endosomal tubules. These findings indicate that IST1 plays an important and regulated role in specific recycling pathways from the early/sorting endosome. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.31.551375v1?rss=1 Authors: Fu, G., Augspurger, K., Sakizadeh, J., Reck, J., Bower, R., Tritschler, D., Gui, L., Nicastro, D., Porter, M. E. Abstract: Ciliary motility requires the spatiotemporal coordination of multiple dynein motors by regulatory complexes located within the 96 nm axoneme repeat. Many organisms can alter ciliary waveforms in response to internal or external stimuli, but little is known about the specific polypeptides and structural organization of complexes that regulate waveforms. In Chlamydomonas, several mutations convert the ciliary waveform from an asymmetric, ciliary-type stroke to a symmetric, flagellar-type stroke. Some of these mutations alter subunits located at the inner junction of the doublet microtubule and others alter interactions between the dynein arms and the radial spokes. These and other axonemal substructures are interconnected by a network of poorly characterized proteins. Here we re-analyze several motility mutants (mbo, fap57, pf12/pacrg) to identify new components in this network. The mbo (move backwards only) mutants are unable to swim forwards with an asymmetric waveform. Proteomics identified more than 19 polypeptides that are missing or reduced in mbo mutants, including one inner dynein arm, IDA b. Several MBO2-associated proteins are also altered in fap57 and pf12/parcg mutants, suggesting overlapping networks. Two subunits are highly conserved, coiled coil proteins found in other species with motile cilia and others contain potential signaling domains. Cryo-electron tomography and epitope tagging revealed that the MBO2 complex is found on specific doublet microtubules and forms a large, L-shaped structure that contacts the base of IDA b that interconnects multiple dynein regulatory complexes and varies in a doublet microtubule specific fashion. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.31.551351v1?rss=1 Authors: Hawkins, L. M., Wang, C., Chaput, D., Batra, M., Marsilia, C., Awshah, D., Suvorova, E. Abstract: Division of apicomplexan parasites differs drastically from the division of their host cells. A fraction of apicomplexans divides in the traditional binary mode, such as Toxoplasma gondii in asexual stages, whereas the vast majority instead divide in a multinuclear fashion. Such variety of replication modes and a dearth of conserved conventional regulators have hindered the progress of apicomplexan cell cycle studies. We previously identified five Cdk-related kinases (Crk) involved in endodyogenic division of T. gondii tachyzoites. The current study investigates the roles of a novel essential cell cycle kinase TgCrk4. We identified this kinase cyclin partner and demonstrated that TgCrk4 regulates processes carried out during conventional G2 phase, such as repression of chromosome re-replication and centrosome re-duplication. Profiles of the G2 phase confirmed a cell cycle stop prior to the TgCrk6-regulated spindle assembly checkpoint. Accumulation of TgCyc4 in the nucleus and on the centrosomes, supported the role of TgCrk4-TgCyc4 complex as a coordinator of chromosome and centrosome cycles in T. gondii. Furthermore, we identified a previously missing DNA replication licensing factor TgCdt1 that was a dominant interactor of the TgCrk4-TgCyc4 complex. T. gondii Cdt1 is highly divergent, but preserved critical signature domains and appears to play a minimal or no role in licensing DNA replication in G1 phase. Functional analyses indicated the primary role of TgCdt1 is in controlling chromosome re-replication and centrosome re-duplication. Global phosphoproteome analyses identified immediate TgCrk4 substrates, such as DNA replication licensing factor TgORC4, gamma-tubulin nucleation factor TgGCP2, and the catalytic subunit of cell cycle phosphatase TgPP2ACA. Importantly, our phylogenetic and structural analyses established that the functional TgCrk4-TgCyc4 complex was encoded in the limited group of apicomplexans that employ binary cell division. Together with the minimal representation of binary division in Apicomplexa phylum, our findings support the novel view of apicomplexans acquiring binary division to repress ancestral multinuclear mechanisms. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.31.551374v1?rss=1 Authors: Ishioka, M., Nihashi, Y., Sunagawa, Y., Umezawa, K., Shimosato, T., Kagami, H., Morimoto, T., Takaya, T. Abstract: An 18-base myogenetic oligodeoxynucleotide (myoDN), iSN04, acts an anti-nucleolin aptamer and induces myogenic differentiation of skeletal muscle myoblasts. This study investigated the effect of iSN04 on murine embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). In the undifferentiated state, iSN04 inhibited the proliferation of ESCs and iPSCs but did not affect the expression of pluripotent markers. In the differentiating condition, iSN04 treatment of ESCs/iPSCs from day 5 onward dramatically induced the differentiation into Nkx2-5+ beating cardiomyocytes with upregulation of Gata4, Isl1, and Nkx2-5, whereas iSN04 treatment from earlier stages completely inhibited cardiomyogenesis. RNA sequencing revealed that iSN04 treatment from day 5 onward contributes to the generation of cardiac progenitors by modulating the Wnt signaling pathway. Immunostaining showed that iSN04 suppressed the cytoplasmic translocation of nucleolin and restricted it to the nucleoli. These results demonstrate that nucleolin inhibition by iSN04 facilitates the terminal differentiation of cardiac mesoderm into cardiomyocytes, but interferes with the differentiation of early mesoderm into the cardiac lineage. This is the first report on the generation of cardiomyocytes from pluripotent stem cells using a DNA aptamer. Since iSN04 did not induce hypertrophic responses in primary-cultured cardiomyocytes, iSN04 would be useful and safe for the regenerative therapy of heart failure using stem cell-derived cardiomyocytes. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.31.551302v1?rss=1 Authors: Ling, Y. H., Ye, J., Yu, C., Liang, C., Park, G., Corden, J., Wu, C. Abstract: Efficient gene expression demands RNA Polymerase II (RNAPII) to find chromatin targets precisely in space and time. How RNAPII manages this complex diffusive search in a 3D nuclear space remains largely unknown. The disordered carboxy-terminal domain (CTD) of RNAPII, which is essential for recruiting transcription-associated proteins, forms phase-separated droplets in vitro, hinting at a potential role in modulating RNAPII dynamics. Here, we use single-molecule tracking and spatiotemporal mapping in living yeast to show that the CTD is responsible for confining RNAPII diffusion within a subnuclear region enriched for active genes, but without apparent phase separation into condensates. Both Mediator and global chromatin organization are required for sustaining RNAPII confinement. Remarkably, truncating the CTD disrupts RNAPII spatial confinement, prolongs target search, diminishes chromatin binding, impairs pre-initiation complex formation, and reduces transcription bursting. This study illuminates the pivotal role of the CTD in driving spatiotemporal confinement of RNAPII for efficient gene expression. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.31.551286v1?rss=1 Authors: Turan, G., Olgun, C. E., Ayten, H., Toker, P., Ashyralyyev, A., Savas, B., Karaca, E., Muyan, M. Abstract: YPEL2 is a member of the evolutionarily conserved YPEL family involved in cellular proliferation, mobility, differentiation as well as senescence and death. However, the mechanism by which YPEL2, or YPEL proteins, mediates its effects is yet unknown. Proteins perform their functions in a network of proteins whose identities, amounts, and compositions change spatiotemporally in a lineage-specific manner in response to internal and external stimuli. We here explored interaction partners of YPEL2 by using dynamic TurboID-coupled mass spectrometry analyses to infer a function for the protein. Our results using inducible transgene expressions in COS7 cells indicate that proximity interaction partners of YPEL2 are largely involved in RNA and mRNA metabolic processes, ribonucleoprotein complex biogenesis, regulation of gene silencing by miRNA, and cellular responses to stress. We showed that YPEL2 interacts with RNA binding protein ELAVL1 and selective autophagy receptor SQSTM1. We also found that YPEL2 participates in events associated with the formation/disassembly of stress granules in response to sodium arsenite an oxidative stress inducer. Establishing a point of departure in the delineation of structural/functional features of YPEL2, our results suggest that YPEL2 may be involved in stress surveillance mechanisms. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.31.551225v1?rss=1 Authors: Baert, L., Rudy, S., Pellisson, M., Doll, T., Rocchetti, R., Kaiser, M., Mäser, P., Müller, M. Abstract: The parasite Leishmania donovani is one of the species causing visceral leishmaniasis in humans, a deadly infection claiming up to 40,000 lives each year. The current drugs for leishmaniasis treatment have severe drawbacks and there is an urgent need to find new anti-leishmanial compounds. However, the search for drug candidates is complicated by the intracellular lifestyle of Leishmania. Here, we investigate the use of human induced pluripotent stem cell (iPS)-derived macrophages (iMACs) as host cells for L. donovani. iMACs obtained through embryoid body differentiation were infected with L. donovani promastigotes, and high-content imaging techniques were used to optimise the iMACs seeding density and multiplicity of infection, allowing us to reach infection rates up to 70% five days after infection. IC50 values obtained for miltefosine and amphotericin B using the infected iMACs or mouse peritoneal macrophages as host cells were comparable and in agreement with the literature, showing the potential of iMACs as an infection model for drug screening. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.31.551242v1?rss=1 Authors: Polina, I., Mishra, J., Cypress, M. W., Landherr, M., Valkov, N., Chaput, I., Nieto, B., Mende, U., Zhang, P., Jhun, B. S., O-Uchi, J. Abstract: MCU is widely recognized as a responsible gene for encoding a pore-forming subunit of highly mitochondrial-specific and Ca2+-selective channel, mitochondrial Ca2+ uniporter complex (mtCUC). Here, we report a novel short variant derived from the MCU gene (termed MCU-S) which lacks mitochondria-targeted sequence and forms a Ca2+-permeable channel outside of mitochondria. MCU-S was ubiquitously expressed in all cell-types/tissues, with particularly high expression in human platelets. MCU-S formed Ca2+ channels at the plasma membrane, which exhibited similar channel properties to those observed in mtCUC. MCU-S channels at the plasma membrane served as an additional Ca2+ influx pathway for platelet activation. Our finding is completely distinct from the originally reported MCU gene function and provides novel insights into the molecular basis of MCU variant-dependent cellular Ca2+ handling. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.08.01.551528v1?rss=1 Authors: Zhang, X., Das, T., Kalab, P., Hayes, L. R. Abstract: The loss of nuclear TDP-43 localization and its accumulation in cytoplasmic aggregates are hallmarks of neurodegeneration and major therapeutic targets. We recently demonstrated that TDP-43 binding to endogenous nuclear GU-rich RNAs sequesters TDP-43 in the nucleus and restricts its passive nuclear export. Here, we tested the feasibility of synthetic RNA oligonucleotide-mediated augmentation of TDP-43 nuclear localization. Using biochemical assays, we compared the ability of GU-rich oligonucleotides to engage in multivalent, RRM-dependent binding with TDP-43 and identified (GU)16 as a strong multivalent binder. When transfected into cells, unlike monovalent oligonucleotides that displaced TDP-43 from the nucleus, (GU)16 preserved steady-state TDP-43 nuclear localization and prevented transcriptional blockade-induced TDP-43 mislocalization. RNA pulldowns from (GU)16-transfected cells confirmed that (GU)16 induced high molecular weight RNP complexes, incorporating TDP-43 and possibly other GU-binding proteins. Transfected (GU)16 caused partial failure of TDP-43 cryptic exon repression, likely because the high-affinity oligonucleotides diverted TDP-43 from endogenous RNAs. Thus, while GU-rich oligonucleotides can attenuate TDP-43 mislocalization, optimization is needed to avoid TDP-43 loss of function. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.08.01.551406v1?rss=1 Authors: Parada, C., Yan, C.-C., Hung, C.-Y., Tu, I.-P., Hsu, C.-P., Shih, Y.-L. Abstract: The Min system contributes to the spatiotemporal regulation of division sites in Escherichia coli. The MinD and MinE proteins of this system self-organize into oscillatory waves in the form of concentration gradients. How the intracellular Min protein concentration gradients are coordinated with cell growth to achieve spatiotemporal accuracy of cell division is unknown. Here, we report that the MinD concentration gradient becomes progressively steeper as cells elongate, suggesting that the division inhibitory activity at the midcell also decreases with cell growth. Interestingly, the oscillation period appears relatively stable across different cell lengths. Similar features were found in cells under carbon stress conditions, but the gradient was even steeper, likely favoring division at shorter cell lengths. The length-dependent variation of the concentration gradient was further examined in silico using a reaction-diffusion model, which not only supported the above features, but also revealed a decrease in the midcell concentration as the shape of the gradient becomes steeper in growing cells. This growth-dependent regulation of the midcell concentration of MinD may be coupled with the FtsZ ring formation through the MinD-interacting protein MinC. We found that the variable concentration gradients occur by coordinating the reaction rates of the recruitment of MinD and MinE to the membrane and the recharging of MinD with ATP in the cytoplasm. In conclusion, this work uncovers the plasticity of MinD concentration gradients during interpolar oscillations throughout cell growth, an intrinsic property integrated during cell division. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.28.550550v1?rss=1 Authors: Andrews, T. S., Nakib, D., Perciani, C., Ma, X. Z., Liu, L., Winter, E., Camat, D., Chung, S., Manuel, J., Mangroo, S., Hansen, B., Arpinder, B., Thoeni, C., Sayed, B., Feld, J., Gehring, A., Gulamhusein, A., Hirschfield, G. M., Rciutto, A., Bader, G. D., McGilvray, I. D., MacParland, S. A. Abstract: Background: Primary sclerosing cholangitis (PSC) is a serious immune-mediated cholestatic liver disease characterized by bile retention, biliary tree destruction, and progressive fibrosis leading to end stage liver disease and transplantation. There is an unmet need to understand the cellular composition of the PSC liver and how it underlies disease pathogenesis. As such, we generated a comprehensive atlas of the PSC liver and a reference healthy liver dataset using multiple multi-omic modalities with functional validation. Methods: In this work, we employed single-cell (12,000 cells), single-nuclei (23,000 nuclei) and spatial transcriptomics (1 sample by 10x Visium and 3 samples with multi-region profiling by Nanostring GeoMx DSP) to profile the cellular ecosystem in 5 patients with PSC. Transcriptomic profiles were compared to 100k single cell transcriptomes and spatial transcriptomics controls from 24 healthy neurologically deceased donor (NDD) livers. Flow cytometry and intracellular cytokine staining was performed to validate PSC-specific differences in immune phenotype and function. Results: PSC explants with cirrhosis of the liver parenchyma and prominent periductal fibrosis were associated with a unique population of hepatocytes which transformed to a cholangiocyte-like phenotype. Those hepatocytes were surrounded by diverse immune cell populations, including monocyte-like macrophages, liver-resident and circulating natural killer (NK) cells. Cytokines released by inflamed cholangiocytes and fibrosis-resident hepatic stellate cells and endothelial cells recruited CD4+T-cells, dendritic cells, and neutrophils to PSC liver tissues. Tissue-resident macrophages, by contrast, were reduced in number and exhibited a dysfunctional inflammatory response to LPS and IFN-{gamma} stimulation. Conclusions: We present the first comprehensive atlas of the PSC liver and demonstrate hyper-activation and exhaustion-like phenotypes of myeloid cells and markers of chronic cytokine expression in late-stage PSC lesions. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.28.550928v1?rss=1 Authors: Guan, L., Eisenmenger, A., Crasta, K., Sandalova, E., Maier, A. B. Abstract: Cellular senescence is a permanent state of cell arrest, regarded as a therapeutic target for ageing and age-related diseases. Natural products hold the translational potential to promote healthy ageing. This systematic review examined dietary interventions and association with senescence in animals and humans to identify senotherapeutic potential. The databases PubMed and Embase were systematically searched. 82 articles consisting of 78 animal studies and 4 human studies aimed to reduce cellular senescence load using dietary interventions. In animal studies, the most-frequently used senescence model was normal ageing (26 studies), followed by D-galactose-induced models (17 studies). Resveratrol (8 studies), vitamin E (4 studies) and soy protein isolate (3 studies) showed positive effects on reducing the level of senescence markers such as p53, p21, p16 and senescence-associated beta-galactosidase in various tissues of physiological systems. Ginsenoside Rg1 had no positive effect on reducing senescence in human muscle tissues after exercise. Resveratrol, vitamin E and soy protein isolate are promising senotherapeutics studied in animals. Studies testing dietary interventions with senotherapeutic potential in humans are limited and translation is highly warranted. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.31.551204v1?rss=1 Authors: Chen, J. K., Liu, T., Cai, S., Ruan, W., Ng, C. T., Shi, J., Surana, U., Gan, L. Abstract: The structure of chromatin at the nucleosome level inside cells is mysterious. Here we present in situ cryo-ET analyses of chromatin in both G1 and metaphase RPE-1 cells. G1 nucleosomes are concentrated in globular chromatin domains and metaphase nucleosomes are concentrated in the chromatids. Classification analysis reveals that canonical mononucleosomes, ordered stacked dinucleosomes, and mononucleosomes with a disordered gyre-proximal density are abundant in both cell-cycle states. Class averages that have more than two stacked nucleosomes or that have side-by-side dinucleosomes are not detected, suggesting that groups of more than two nucleosomes are heterogeneous. Large multi-megadalton structures are abundant in G1 nucleoplasm, but not found in G1 chromatin domains and metaphase chromatin. The macromolecular phenotypes studied here represent a starting point for the comparative analysis of condensation in normal and unhealthy human cells, in other cell-cycle states, other organisms, and in vitro chromatin assemblies. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.31.551267v1?rss=1 Authors: Liu, A., Kage, F., Sapp, G., Aydin, H., Higgs, H. N. Abstract: Mitochondrial fission occurs in many cellular processes, but the regulation of fission is poorly understood. We show that long-chain acyl coenzyme A (LCACA) activates two related mitochondrial fission proteins, MiD49 and MiD51, by inducing their oligomerization, activating their ability to stimulate DRP1 GTPase activity. The 1:1 stoichiometry of LCACA:MiD in the oligomer suggests interaction in the previously identified nucleotide-binding pocket, and a point mutation in this pocket reduces LCACA binding and LCACA-induced oligomerization for MiD51. In cells, this LCACA binding mutant does not assemble into puncta on mitochondria or rescue MiD49/51 knock-down effects on mitochondrial length and DRP1 recruitment. Furthermore, cellular treatment with the fatty acid analogue 2-bromopalmitate, which causes increased acyl-CoA, promotes mitochondrial fission in an MiD49/51-dependent manner. These results suggest that LCACA is an endogenous ligand for MiDs, inducing mitochondrial fission and providing a potential mechanism for fatty acid-induced mitochondrial fragmentation. Finally, MiD49 or MiD51 oligomers synergize with MFF, but not with actin filaments, in DRP1 activation, suggesting distinct pathways for DRP1 activation. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.28.550978v1?rss=1 Authors: GARNIER, O., JEANNERET, F., DURAND, A., FERTIN, A., MARTIN, D., BERNDT, S., CARPENTIER, G., BATTAIL, C., VILGRAIN, I. Abstract: Rationale: Phosphorylation-dephosphorylation are processes involved in the adhesion of endothelial cells (ECs) to maintain vascular integrity in adults. VE-cadherin is a target for Src -mediated Y685 phosphorylation, identified in highly vascularized human glioblastoma where it is involved in the abnormal feature of tumor blood vessels . Objective: We aimed at understanding the molecular mechanisms through which Y685F-VE-cadherin triggers S1PR1 gene expression and stabilizes lung vessels in adult mice. Methods and Results: We compared lung ECs from a knock-in (KI) mouse carrying a point mutation in VE-cadherin (Tyr 685 to Phe) to Wild type . Analysis of EC parameters showed a difference in the migratory rate was between ECs from KI (22.45% +/- 5.207) and WT (13.24% +/- 5.17) (p-value=0.034). The direct adhesion of ECs from KI mice to fibronectin was significantly higher (37.625 +/- 9.23) than that of the WT (26.8 +/- 3.258, p-value=0.012). In the fibrin bead assay, ECs from KI showed a weaker angiogenic response. The transcriptome of mutated ECs showed that 884 genes were dysregulated of which 766 genes were downregulated and 118 genes were upregulated. The Gene Ontology Enrichment showed that most of the genes were related to cell-cell adhesion and angiogenesis. Focusing on angiogenic genes, we found that Sphingosine-1-phosphate-receptor was a gene upregulated in mutated ECs which was confirmed by RT-PCR and westernblotting. Mechanistically, chromatin immunoprecipitation assay (CHIPS) demonstrated that FOXF1 directly bound to the S1pr1 promoter 7 fold greater than WT. As a consequence , VE-cadherin at the membrane was higher in the mutant vs WT (100 +/- 6.52 for WT vs 189.7 +/- 21.06 for KI (p-value 0.0001). Finally, lung morphometric analysis showed less vessels and vascular remodeling with no fibrosis in mutated mice. Conclusions: These data extend our knowledge on pY-VE-cadherin mediated pathological angiogenesis and provide new therapeutic opportunities to vascular normalization through pharmacological inhibition of the Y685-VE-cadherin phosphorylation. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.28.550203v1?rss=1 Authors: Chen, T., Huertas Fernandez-Espartero, C., Illand, A., Tsai, C.-T., Yang, Y., Klapholz, B., Jouchet, P., Fabre, M., Rossier, O., Cui, B., Leveque-Fort, S., Brown, N. H., Giannone, G. Abstract: Morphogenesis requires building stable macromolecular structures from highly dynamic proteins. Muscles are anchored by long-lasting integrin adhesions to resist contractile force. However, the mechanisms governing integrin diffusion, immobilization, and activation within developing tissue remain elusive. Here, we show that actin polymerisation-driven membrane protrusions form nanotopographies resulting in strong adhesions in the Drosophila muscle attachment site. With super-resolution microscopy and single protein tracking, we show that integrins assemble invadosomes-like adhesive belts around Arp2/3-dependent actin protrusions, which promotes enhanced integrin molecular immobilization and confinement in diffusion traps. Actin filaments also display restricted motion and confinement, indicating strong mechanical connection with integrins. Using isolated muscle cells, we show that substrate nanotopography, instead of rigidity, drives adhesion maturation by regulating actin protrusion, integrin diffusion and immobilization. These results point to the existence of a molecular clutch in developing tissue required for the formation of stable adhesions and highlight the importance of geometrical information in cellular and tissue morphogenesis. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.29.551071v1?rss=1 Authors: Bartke, R., Hockemeyer, D. Abstract: Telomeric DNA ends in a 3' single stranded overhang that is implicated in the protective function of telomeres ensuring genomic stability in mammals. Telomere overhang formation relies on the coordinated interplay between DNA synthesis and exonuclease activity. DCLRE1B/hSNM1B/Apollo generates an initial resection at the newly synthesized, blunt-ended leading strand telomere. This resection is thought to be required for further nucleolytic processing at the leading strand telomere. Here, we investigated the functional relevance of Apollo in human pluripotent stem cells (hPSCs) by generating Apollo deficient cells. Leveraging CRISPR/Cas9 technology, we generated locally haploid hPSCs (loHAPs) that lack one allele of Apollo. Subsequently, we mutated the remaining Apollo allele and monitored the resultant allele spectrum over 3 weeks. Surprisingly, cells survived regardless of Apollo status. These results suggest that, in hPSCs, Apollo is not acutely essential for cellular survival. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.30.551178v1?rss=1 Authors: Ma, C.-I. J., Brill, J. A. Abstract: Secretory granules (SGs) are crucial for normal animal physiology due to their role in regulated exocytosis of biologically active molecules. SG membranes are enriched in phosphatidylinositol 4-phosphate (PI4P) and cholesterol, and previous studies suggest lipid composition is important for SG biogenesis and function. Nonetheless, the molecular details of how lipids are regulated during SG biogenesis remain poorly understood. Here, we identify Oxysterol binding protein (Osbp) as a novel regulator of SG biogenesis in a Drosophila model. We show Osbp expression level positively correlates with SG size and that Osbp requires type II phosphatidylinositol 4-kinase (PI4KII) to increase SG size. Moreover, Osbp is needed for proper PI4KII and PI4P distribution, autophagic resolution and formation of cholesterol-rich endosomal tubules that are positive for PI4KII. Feeding larvae food supplemented with sterol leads to partial suppression of SG size and PI4P distribution defects in Osbp mutants. Our results indicate that reciprocal regulation of Osbp and PI4KII drives formation of membrane tubules that mediate SG maturation through elevating PI4P levels on SG membranes. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.28.551049v1?rss=1 Authors: Keshavanarayana, P., Spill, F. Abstract: The inner lining of blood vessels, the endothelium, is made up of endothelial cells. Vascular endothelial (VE)-cadherin protein forms a bond with VE-cadherin from neighbouring cells (homophilic bond) to determine the size of gaps between the cells and thereby regulate the size of particles that can cross the endothelium. Chemical cues such as Thrombin, along with mechanical properties of the cell and extracellular matrix (ECM) are known to affect the permeability of endothelial cells. Abnormal permeability is found in patients suffering from diseases including cardiovascular diseases, cancer, and COVID-19. Even though some of the regulatory mechanisms affecting endothelial permeability are well studied, details of how several mechanical and chemical stimuli acting simultaneously affect endothelial permeability are not yet understood. In this article, we present a continuum-level mechanical modelling framework to study the highly dynamic nature of the VE-cadherin bonds. Taking inspiration from the catch-slip behaviour that VE-cadherin complexes are known to exhibit, we model VE-cadherin homophilic bond as cohesive contact with damage following a traction-separation law. We explicitly model the actin-cytoskeleton, and substrate to study their role in permeability. Our studies show that mechano-chemical coupling is necessary to simulate the influence of the mechanical properties of the substrate on permeability. Simulations show that shear between cells is responsible for the variation in permeability between bicellular and tri-cellular junctions, explaining the phenotypic differences observed in experiments. An increase in the magnitude of traction force that endothelial cells experience results in increased permeability, and it is found that the effect is higher on stiffer ECM. Finally, we show that the cylindrical monolayer exhibits higher permeability than the planar monolayer under unconstrained cases. Thus, we present a contact mechanics-based mechano-chemical model to investigate the variation in permeability of endothelial monolayer due to multiple loads acting simultaneously. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.28.551009v1?rss=1 Authors: Azevedo, L. P., Rios-Santos, F., Branco, C. B., Pressinotti, L. N., Reis, E. d. M., Filho, S. V., Martins, D. T. d. O., de Vasconcelos, L. G., Ferraz, R. H. d. S., Mesquita, F. V., Silva, W. d. A., Junior, P. T. d. S. Abstract: In previous studies, the oil extracted from the visceral fat of Caiman yacare (Daudin, 1802) demonstrated a wound-healing effect on the skin of Wistar rats. To enhance knowledge our about the mechanism underlying this effect, we analysed the oils toxicological potential in vitro. Cytotoxicity, genotoxicity, pro-oxidant, and antioxidant activities were evaluated in a V79-4 cell line. The oil was obtained using the Soxhlet method, and the proportions of the fatty acid profile was previously identified 43.74 % saturated and 34.65 % unsaturated fatty acids. Protocol 487 of the Organisation for Economic Co-operation and Development (OECD) was employed for cell line selection and concentrations. Cytotoxicity was determined using the MTT assay and clonogenic survival. Pro-oxidant and antioxidant activities were analysed using flow cytometry. Genotoxicity was evaluated using comet and micronucleus assays. The oil did not demonstrate cytotoxicity up to a concentration of 500 g/mL. At concentrations of 250 and 500 g/mL, the oil exerted a protective effect against oxidative stress and showed genotoxic effects only at the highest concentration (2000 g/mL). Like other oils of interest for human health, the oil extracted from the visceral fat of C. yacare demonstrated low toxicological potential in vitro. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.28.550940v1?rss=1 Authors: Saladin, L., Breton, V., Lequeu, T., Didier, P., Danglot, L., Collot, M. Abstract: Photomodulable fluorescent probes are drawing an increasing attention due to their applications in advanced bioimaging. Whereas photoconvertible probes can be advantageously used in tracking, photoswitchable probes constitute key tools for single molecule localization microscopy to perform super resolution imaging. Herein we shed light on a red and far-red BODIPY, namely BDP-576 and BDP-650 possessing both properties of conversion and switching. Our study demonstrates that theses pyrrolyl-BODIPYs respectively convert towards typical green- and red-emitting BODIPYs that are perfectly adapted to microscopy. We also showed that these pyrrolyl-BODIPYs undergo Directed Photooxidation Induced Conversion, a photoconversion mechanism that we recently introduced and where the pyrrole moiety plays a central role. These unique features were used to develop targeted photoconvertible probes towards different organelles or subcellular units (plasma membrane, mitochondria, nucleus, actin, Golgi apparatus, etc.) using chemical targeting moieties and Halo tag. We notably showed that BDP-650 could be used to track intracellular vesicles over more than 20 minutes in two color imaging with laser scanning confocal microscopy demonstrating its robustness. The switching properties of these photoconverters were studied at the single molecule level and were then successfully used in live Single Molecule Localization Microscopy in epithelial cells and neurons. Both membrane and mitochondria targeted probes could be used to decipher membrane 3D architecture and mitochondria dynamics at the nanoscale. This study builds a bridge between the photoconversion and photoswitching properties of probes undergoing directed photooxidation and shows the versatility and efficacy of this mechanism in live advanced imaging. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.29.551032v1?rss=1 Authors: Sun, C., Zhou, C., Daneshvar, K., Kratkiewicz, A. J., Ben Saad, A., Hess, A., Chen, J. Y., Pondick, J. V., York, S. R., Li, W., Moran, S. P., Gentile, S., Ur Rahman, R., Li, Z., Sparks, R., Habboub, T., Kim, B.-M., Choi, M., Affo, S., Schwabe, R. F., Popov, Y. V., Mullen, A. C. Abstract: Background & Aims: Fibrosis is the common endpoint for all forms of chronic liver injury, and progression of fibrosis leads to the development of end-stage liver disease. Activation of hepatic stellate cells (HSCs) and their transdifferentiation to myofibroblasts results in the accumulation of the extracellular matrix (ECM) proteins that form the fibrotic scar. Long noncoding (lnc) RNAs regulate the activity of HSCs and may provide targets for fibrotic therapies. Methods: We identified lncRNA TILAM as expressed near COL1A1 in human HSCs and performed loss-of-function studies in human HSCs and liver organoids. Transcriptomic analysis of HSCs isolated from mice defined the murine ortholog of TILAM. We then generated Tilam-deficient GFP reporter mice and quantified fibrotic responses to carbon tetrachloride (CCl4) and choline-deficient L-amino acid defined high fat diet (CDA-HFD). Co-precipitation studies, mass spectrometry, and gene expression analyses identified protein partners of TILAM. Results: TILAM is conserved between human and mouse HSCs and regulates expression of ECM proteins, including collagen. Tilam is selectively induced in murine HSCs during the development of fibrosis in vivo. In both male and female mice, loss of Tilam results in reduced fibrosis in the setting of CCl4 and CDA-HFD injury models. TILAM interacts with promyelocytic leukemia protein (PML) to stabilize PML protein levels and promote the fibrotic activity of HSCs. Conclusion: TILAM is activated in HSCs and interacts with PML to drive the development of liver fibrosis. Depletion of TILAM may serve as a therapeutic approach to combat the development of end stage liver disease. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.28.551038v1?rss=1 Authors: Garcia, S. M., Diaz, A., Lau, J., Chi, H. M., Lizarraga, M., Davies, M. R., Liu, X., Feeley, B. T. Abstract: Fibroadipogenic progenitors (FAPs) maintain healthy skeletal muscle in homeostasis but drive muscle degeneration in chronic injuries by promoting adipogenesis and fibrosis. To uncover how these stem cells switch from a pro-regenerative to pro-degenerative role we perform single-cell mRNA sequencing of human FAPs from healthy and injured muscles across a spectrum of injury. We identify multiple subpopulations with progenitor, adipogenic, or fibrogenic gene signatures. We utilize full spectrum flow cytometry to identify distinct FAP subpopulations based on highly multiplexed protein expression. We uncover that injury severity increases adipogenic commitment of FAP subpopulations and is driven by the downregulation of DLK1. Treatment of FAPs with DLK1 reduces adipogenesis, suggesting that during injury, reduced DLK1 within a subpopulation of FAPs may drive adipogenic degeneration. This work highlights how stem cells perform varied functions depending on tissue context, by dynamically regulating subpopulation fate commitment, which can be targeted improve patient outcomes after injury. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.28.550934v1?rss=1 Authors: Galan, L., Alvarez-Villanueva, D., Maqueda, M., Barrero, M., IGLESIAS, A., Bertran, j., ALVAREZ, D., GARCIA-PRIETO, C., BALLARE, C., RODRIGUEZ-CORTEZ, V., BUENO, C., VIDAL, A., Villanueva, A., Menendez, P., Di Croce, L., Payer, B., Esteller, M., Espinosa, L., Bigas, A. Abstract: Inflammatory signals are key in development and cell differentiation but their orchestration with pluripotency and stemness signals is poorly understood. Our previous work identified a chromatin function of I{kappa}B, the NF-{kappa}B inhibitor, that is crucial for differentiation in different types of somatic stem cells. Here we demonstrate that deficiency of I{kappa}B imposes a profound chromatin rewiring defect that impacts on DNA methylation, histone post-translational modifications and transcriptional regulation, stabilizing mouse embryonic stem cells (ESCs) in a ground state of pluripotency while preventing them from pluripotency exit and differentiation. By engineering separation-of-function mutants of I{kappa}B with specific binding to either NF-{kappa}B or histones, we demonstrate that regulation of pluripotency state by I{kappa}B is independent of NF-{kappa}B but requires the chromatin-related I{kappa}B function. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.28.550973v1?rss=1 Authors: Edwards, F., Fantozzi, G., Simon, A. Y., Morretton, J.-P., Herbette, A., Tijhuis, A. E., Wardenaar, R., Foulane, S., Gemble, S., Spierings, D. C. J., Foijer, F., Mariani, O., Vincent-Salomon, A., Roman-Roman, S., Sastre-Garau, X., Goundiam, O., Basto, R. Abstract: Centrosome amplification is a feature of cancer cells associated with chromosome instability and invasiveness. Enhancing chromosome instability and subsequent cancer cell death via centrosome unclustering and multipolar divisions is an aimed-for therapeutic approach. Here we show that centrosome amplification favors responses to conventional chemotherapy independently of multipolar divisions and chromosome instability. We perform single-cell live imaging of chemotherapy responses in epithelial ovarian cancer cell lines and observe increased cell death when centrosome amplification is induced. By correlating cell fate with mitotic behaviors, we show that enhanced cell death occurs independently of chromosome instability. We identify that cells with centrosome amplification are primed for apoptosis. We show they are dependent on the apoptotic inhibitor BCL-XL, and that this is not a consequence of mitotic stresses associated with centrosome amplification. Given the multiple mechanisms that promote chemotherapy responses in cells with centrosome amplification, we assess such a relationship in an epithelial ovarian cancer patient cohort. We show that high centrosome numbers associate with improved chemotherapy responses and longer overall survival. Our work identifies apoptotic priming as a clinically relevant consequence of centrosome amplification, expanding our understanding of this pleiotropic cancer cell feature. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.28.550943v1?rss=1 Authors: Roet, J. E. G., Mikula, A. M., de Kok, M., Chadick, C. H., Garcia Vallejo, J. J., Roest, H. P., van der Laan, L. J. W., de Winde, C. M., Mebius, R. E. Abstract: Autofluorescence is an intrinsic feature of cells, caused by the natural emission of light by its cellular content, that can complicate analysis of flow cytometry data. Different cell types have different autofluorescence spectra and even within one cell type heterogeneity of autofluorescence spectra can be present, for example as a consequence of activation status or metabolic changes. By using full spectrum flow cytometry, the emission spectrum of a fluorochrome is captured by a set of detectors across a range of wavelengths, creating an unique spectrum for this fluorochrome, that is used to unmix the signal of a full stained sample into the signals of the different fluorochromes. Importantly, this technology can also be used to identify the autofluorescence signal of an unstained sample, which can be used for unmixing purposes and to separate the autofluorescence signal from the fluorophore signals. However, this only works if the sample has one homogeneous autofluorescence spectrum. To analyze samples with a heterogeneous autofluorescence spectral profile, we here setup an unbiased workflow to detect all different autofluorescence spectra present in a sample to take them along as 'autofluorescence signatures' during the unmixing of the full stained samples. First, clusters of cells with similar autofluorescence spectra are identified by unbiased dimensional reduction and clustering. Then, unique autofluorescence clusters are determined and are used to improve the unmixing accuracy of the full stained sample. This unbiased method allows for the identification of all autofluorescence spectra present in a sample, independent of cell types and intensity of the autofluorescence spectra. Furthermore, this method is equally useful for spectral analysis of different biological samples, including tissue cell suspensions, peripheral blood mononuclear cells and in vitro cultures of (primary) cells. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.28.550936v1?rss=1 Authors: Cerina, M., Levers, M., Keller, J. M., Frega, M. Abstract: In patients suffering from cerebral ischemic stroke, there is an urgent need for treatments to protect brain cells. Recently, treatment strategies that induce neuronal activity have been shown to be neuroprotective. However, the biological mechanisms underlying the benefit from neuronal activation are unknown. We hypothesized that neuronal activation might trigger the astrocyte-to-neuron lactate shuttle, whereby lactate is released from astrocytes to support the energy requirements of hypoxic neurons, and this leads to the observed neuroprotection. We tested this by establishing a human cell based in vitro model of the ischemic penumbra. We found that lactate transporters are involved in the neuroprotective effect mediated by neuronal activation, that lactate exogenously administered before hypoxia correlated with neuroprotection, and that stimulation of astrocyte with consequent endogenous production of lactate resulted in neuroprotection. We presented evidence that lactate contributes to neuroprotection during hypoxia providing a potential basis for therapeutic approaches in ischemic stroke. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.28.550974v1?rss=1 Authors: Peterman, E., Quitevis, E. J. A., Goo, C. E. A., Rasmussen, J. P. Abstract: Skin is often the first physical barrier to encounter invading pathogens and physical damage. Damage to the skin must be resolved quickly and efficiently to maintain organ homeostasis. Epidermal-resident immune cells known as Langerhans cells use dendritic protrusions to dynamically surveil the skin microenvironment, which contains epithelial keratinocytes and somatosensory peripheral axons. The mechanisms governing Langerhans cell dendrite dynamics and responses to tissue damage are not well understood. Using skin explants from adult zebrafish, we show that Langerhans cells maintain normal surveillance activity following axonal degeneration and use their dynamic dendrites to engulf small axonal debris. By contrast, a ramified-to-rounded shape transition accommodates the engulfment of larger keratinocyte debris. We find that Langerhans cell dendrites are richly populated with actin and sensitive to a broad spectrum actin inhibitor. We further show that Rho-associated kinase (ROCK) inhibition leads to elongated dendrites, perturbed clearance of large debris, and reduced Langerhans cell migration to tissue-scale wounds. Altogether, our work describes the unique dynamics of Langerhans cells and involvement of the ROCK pathway in immune cell responses to damage of varying magnitude. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.28.548760v1?rss=1 Authors: Furusho, T., Adachi, K., Galbraith-Liss, M., Sairavi, A., Das, R., Nakai, H. Abstract: Despite recent remarkable advancements in adeno-associated virus (AAV) vector technologies, effective gene delivery to the kidney remains a significant challenge. Here we show that AAV vector transduction in proximal tubules and podocytes, the crucial targets for renal gene therapy, can be enhanced remarkably through a meticulous selection of both AAV capsids and route of administration, tailored to the condition of the kidney. In this study, we performed a side-by-side comparison of 47 AAV capsids using AAV Barcode-Seq and identified six AAV capsids, including AAV-KP1, that exhibit remarkable enhancement of renal transduction in mice when delivered locally via the renal vein or the renal pelvis. Individual capsid validation analyses revealed that local delivery of AAV-KP1, but not AAV9, enables remarkably enhanced proximal tubule transduction while minimizing off-target liver transduction. In a mouse model of chronic kidney disease, intravenous administration of AAV9, not AAV-KP1, showed efficient renal tubule and podocyte transduction, which was not observed in the control wild-type mice. We also provide evidence that these contrasting observations between AAV-KP1 and AAV9 are attributed to their distinct pharmacokinetic profiles. Thus, this study highlights the importance of context-dependent capsid selection and engineering for successful renal gene therapy. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.28.551061v1?rss=1 Authors: Keyport Kik, S., Christopher, D., Glauninger, H., Wong Hickernell, C., Bard, J. A. M., Ford, M., Sosnick, T. R., Drummond, D. A. Abstract: Cells must sense and respond to sudden maladaptive environmental changes--stresses--to survive and thrive. Across eukaryotes, stresses such as heat shock trigger conserved responses: growth arrest, a specific transcriptional response, and biomolecular condensation of protein and mRNA into structures known as stress granules under severe stress. The composition, formation mechanism, adaptive significance, and even evolutionary conservation of these condensed structures remain enigmatic. Here we provide an unprecedented view into stress-triggered condensation, its evolutionary conservation and tuning, and its integration into other well-studied aspects of the stress response. Using three morphologically near-identical budding yeast species adapted to different thermal environments and diverged by up to 100 million years, we show that proteome-scale biomolecular condensation is tuned to species-specific thermal niches, closely tracking corresponding growth and transcriptional responses. In each species, poly(A)-binding protein--a core marker of stress granules--condenses in isolation at species-specific temperatures, with conserved molecular features and conformational changes modulating condensation. From the ecological to the molecular scale, our results reveal previously unappreciated levels of evolutionary selection in the eukaryotic stress response, while establishing a rich, tractable system for further inquiry. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.27.549915v1?rss=1 Authors: Glover, H., Mendes, M., Gomes-Neto, J., Ruscilowicz-Jones, E. V., Rigden, D., Dittmar, G., Urbe, S., Clague, M. J. Abstract: The microtubule network is formed from polymerised tubulin subunits and associating proteins, which govern microtubule dynamics and a diverse array of functions. To identify novel microtubule binding proteins, we have developed an unbiased biochemical assay which relies on the selective extraction of cytosolic proteins from cells, whilst leaving behind the microtubule network. Candidate proteins are linked to microtubules by their sensitivities to the depolymerising drug nocodazole or the microtubule stabilising drug, taxol, which are then quantitated in a triplexed mass spectrometry experiment. Our approach is benchmarked by co-segregation of tubulin and previously established microtubule-binding proteins. We then identify several novel candidate microtubule binding proteins. Amongst these, we have selected the ubiquitin E3 ligase TRIM3 (Tripartite motif-containing protein 3) for further characterisation. TRIM3 binding to microtubules is mapped to its C-terminal NHL-repeat region. We show that TRIM3 is required for the rapid accumulation of acetylated tubulin, following treatment with the microtubule stabilising drug taxol. Furthermore, loss of TRIM3, partially recapitulates the reduction in nocodozole-resistant microtubules characteristic of Alpha Tubulin Acetyltransferase 1 (ATAT1) depletion. These results can be explained by a decrease in ATAT1 that follows depletion of TRIM3 that is independent of transcription. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.28.550915v1?rss=1 Authors: Kravec, M., Sedo, O., Nedvedova, J., Micka, M., Sulcova, M., Gomoryova, K., Potesil, D., Ganji, R. S., Cervenka, I., Zdrahal, Z., Harnos, J., Tripsianes, K., Janke, C., Barinka, C., Bryja, V. Abstract: Polyglutamylation is a reversible post-translational modification that is catalyzed by enzymes from the tubulin tyrosine ligase-like (TTLL) family. Here, we found that TTLL11 generates a previously unknown type of polyglutamylation initiated by the addition of a glutamate residue to the free C-terminal carboxyl group of a substrate protein. TTLL11 efficiently polyglutamylates the Wnt signaling protein Disheveled 3 (DVL3), thereby changing the interactome of DVL3, as well as increases its capacity to get phosphorylated, to undergo liquid-liquid phase separation (LLPS) and to act in the non-canonical Wnt pathway. Both carboxyterminal polyglutamylation and the resulting reduction in LLPS capacity of DVL3 were reverted by the deglutamylating enzyme CCP6, which demonstrates the causal relationship between TTLL11-mediated polyglutamylation and LLPS. We thus discovered a novel type of posttranslational modification, which significantly broadens the range of proteins that can be modified by polyglutamylation and provide first evidence that polyglutamylation can act as a regulator of protein LLPS. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.27.550898v1?rss=1 Authors: Rosario, A. A. A., McInally, S. G., Jelenkovic, P. R., Goode, B. L., Kondev, J. Abstract: Actin is a key cytoskeletal protein that forms filaments that bundle into linear structures in vivo, which are involved in motility, signaling, and cell division. Despite the rapid turnover of individual actin monomers, these structures are often maintained at a specific length, which is important for their function. Length control is commonly attributed to length-dependent assembly or disassembly of the structure, whereby a stable length is achieved when the two opposing processes are balanced. Here we show that regardless of the nature of the length-dependent feedback, such balance point models predict a Gaussian distribution of lengths with a variance that is proportional to the steady state length. Contrary to this prediction, a reexamination of experimental measurements on the lengths of stereocilia, microvilli, actin cables, and filopodia reveals that the variance scales with the square of the steady state length. We propose a model in which the individual filaments in bundles undergo independent assembly dynamics, and the length of the bundle is set by the length of the longest filament. This model predicts a non-Gaussian distribution of bundle lengths with a variance that scales with the square of the steady state length. Our theory underscores the importance of crosslinking filaments into networks for size control of cytoskeleton structures. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.27.550771v1?rss=1 Authors: Zhao, H., Wu, K., Chen, Z.-J., Zhang, Y., Zhao, R., Yang, C., Song, J., Liu, P., Li, Y., Liu, B., Li, T., Yin, C., Lu, M., Hou, Z., Zhang, C. Abstract: Human endometrial receptivity is a critical determinant of pregnancy success; however, in vivo studies of its features and regulation are particularly challenging due to ethical restriction. Recently,the development of human endometrial organoids has provided a powerful model to examine this intricate biological process. In this study, we established a specialized human window-of-implantation (WOI) endometrial organoid system that mimics the in vivo receptive endometrium.The endometrial organoid recapitulates not only the structural attributes of glands and stroma, but also the in vivo endometrium's cell types at the single-cell level. Furthermore, the WOI endometrial organoid exhibits the features of in vivo implantation endometrium such as hormone responsiveness, secretory functions, and distinctive implantation window hallmarks, including decidualization, extracellular matrix (ECM) remodeling, pinopode formation, cilia generation and metabolism. This in vitro model also effectively demonstrates the process of proliferation-secretory transformation, ciliary epithelium differentiation, and epithelial-mesenchymal transition (EMT). As such, WOI organoids hold great promise as a robust platform to unravel the intricate mechanisms governing endometrial receptivity regulation, maternal-fetal interactions, and associated diseases, ultimately driving impactful advancements in the field. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.26.550187v1?rss=1 Authors: Sadeh, O., Haddad, R., Ziv, T., Haimovich-Caspi, L., Shemesh, A., Kehat, I. Abstract: Cardiomyocyte sarcomeres contain localized ribosomes, but the factors responsible for their localization and the significance of localized translation are unknown. Using proximity labeling, we identified Ribosomal Protein SA (RPSA) as a Z-line protein. In cultured cardiomyocytes, the loss of RPSA led to impaired local protein translation and reduced sarcomere integrity. By employing CAS9 expressing mice along with adeno-associated viruses expressing CRE recombinase and single-guide RNAs targeting Rpsa, we knocked out RPSA in vivo and observed mis-localization of ribosomes and diminished local translation. These genetic mosaic mice with RPSA knockout in a subset of cardiomyocytes developed dilated cardiomyopathy, featuring atrophy of RPSA-deficient cardiomyocytes, compensatory hypertrophy of unaffected cardiomyocytes, left ventricular dilation, and impaired contractile function. We demonstrate that RPSA C-terminal domain is sufficient for localization to the Z-lines. These findings highlight RPSA as a ribosomal factor responsible for ribosome localization to the Z-line, facilitating local translation and sarcomere maintenance. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.27.550790v1?rss=1 Authors: Bueno, C., Garcia-Bernal, D., Martinez, S., Blanquer, M., Moraleda, J. M. Abstract: Despite a considerable interest in understanding the mechanisms regulating nucleus structure, chromatin organization and nuclear positioning over decades, the exact significance of the variety of morphologies and positioning that cell nuclei can adopt and their relationship in cellular function is still far from being clearly understood. In this study, we examined the functional significance of the variety of morphologies and positioning that cell nuclei of human bone marrow-derived mesenchymal stem cells can adopt during neural-like differentiation. Here, we show that after neural induction, human bone marrow-derived mesenchymal stem cells an intermediate cellular state in which the nuclei are observed to be able to move within the cells, switching shapes and positioning and even generating cellular protrusions as they attempt to contact the cells around them. These findings suggest that changes in nuclear positioning are due to the fact that human cell nuclei are somehow sensing their surroundings. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.27.550674v1?rss=1 Authors: Verdys, P., Rey Barroso, J., Vermeil, J., Bergert, M., Sanchez, T., Metais, A., Mangeat, T., Bellard, E., Bigot, C., Girard, J.-P., Maridonneau-Parini, I., Verollet, C., Lagarrigue, F., Diz-Munoz, A., Heuvingh, J., Piel, M., Du Roure, O., Le Cabec, V., Carreno, S., Poincloux, R. Abstract: The cellular actin cortex provides crucial mechanical support and plays critical roles in numerous functions, including cell division and migration. The proteins of the ERM family, ezrin, radixin, and moesin, are central to these processes by linking the plasma membrane to the actin cytoskeleton. To investigate the individual contributions of these three proteins to leukocyte migration, we generated single and triple ERM knock-out macrophages. Surprisingly, we found that even in the absence of ERMs, macrophages can still form the different actin structures promoting cell migration, such as filopodia, lamellipodia, podosomes, and ruffles. Furthermore we discovered that, unlike every other cell type previously investigated, the single or triple knock-out of ERMs does not affect macrophage migration in a large diversity of contexts. Finally, we demonstrated that the loss of ERMs in macrophages does not affect the mechanics of their actin cortex. These findings challenge the notion that ERMs are universally essential for cortex mechanics and cell migration and support the notion that the macrophage cortex may have diverged from that of other cells to allow for their adaptive cortical plasticity. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.26.550629v1?rss=1 Authors: Li, W., Tu, J., Zheng, J., Das, A., Yan, Q., Jiang, X., Ding, W., Bai, X., Lai, K., Yang, S., Yang, C., Zou, J., Diwan, A. D., Zheng, Z. Abstract: Chronic low back pain (LBP) is the leading cause of global disability. Vertebral bone marrow lesions (BMLs), one etiological factor for chronic LBP, are MRI signal changes in the vertebral bone marrow that extend from the disc endplate. The adipogenesis of bone marrow mesenchymal stem cells (BM-MSCs) could explain fatty replacement (FR) in normal bone marrow. FR is the most common type of BMLs. Here we show how the gut microbiome and serum metabolome change and how they interact in LBP patients with or without FR. The serum metabolome of chronic LBP patients with FR is characterized by decreased levels of branched-chain amino acids (BCAAs), which correlate with a gut microbiome that has important capability to regulate BCAA degradation pathway. Ruminococcus gnavus, Roseburia hominis and Lachnospiraceae bacterium 8 1 57FAA are identified as the main species driving the association between biosynthesis of BCAAs and BM-MSCs metabolism in LBP with FR individuals. In vitro work demonstrates that BCAAs can induce the adipogenesis of BM-MSCs by activating the SIRT4 pathway. Our findings provide a deep insight into understanding the role of the disturbed gut ecosystem in FR and LBP. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.26.550666v1?rss=1 Authors: Zhang, G., Zhang, Y., Young, R., Garvanska, D., Song, C., Zhai, Y., Wang, Y., Jiang, H., Fang, J., Nilsson, J., Alfieri, C. Abstract: Accurate chromosome segregation is coordinated by the spindle assembly checkpoint (SAC) through its effector the mitotic checkpoint complex (MCC), to inhibit the anaphase-promoting complex or cyclosome (APC/C). Cdc20 is an essential mitotic regulator since it promotes mitotic exit through activating the APC/C and monitors kinetochore-microtubule attachment through activating the SAC. The proper functioning of Cdc20 requires multiple interactions with APC/C and MCC subunits. To functionally assess each of these interactions within cells requires efficient depletion of endogenous Cdc20, which is highly difficult to achieve by RNAi. Here we generated Cdc20 RNAi sensitive cell lines by CRISPR/Cas9 which display a penetrant metaphase arrest phenotype by a single RNAi treatment. In this null background, we accurately measured the contribution of each known motif of Cdc20 on APC/C and SAC activation. The CRY box, a previously identified degron was found to be critical for the SAC by promoting the MCC formation and stabilizing the interaction between the MCC and APC/C. These data reveal additional regulatory components within the SAC and establish a novel method to interrogate Cdc20 function. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.26.550704v1?rss=1 Authors: Uhrig, M. E., Sharma, N., Maxwell, P., Selemenakis, P., Wiese, C. Abstract: RAD54L is a DNA motor protein with critical roles in homologous recombination DNA repair (HR). In vitro, RAD54L was also shown to catalyze the reversal and restoration of model replication forks. Little, however, is known about the role of RAD54L in regulating the dynamics of DNA replication in cells. Here, we show that RAD54L functions as a fork remodeler and restrains the progression of replication forks in human cells. Analogous to HLTF and FBH1, and consistent with a role in fork reversal, RAD54L catalyzes the slowing of fork progression in response to replication stress. In BRCA1/2-deficient cells, RAD54L activity leads to nascent strand DNA degradation, and loss of RAD54L reduces DNA double-strand break formation. Using a separation-of-function mutation, we show that RAD54L-mediated fork restraint depends on its ability to catalyze branch migration. Our results reveal a new role for RAD54L in regulating the dynamics of replication forks in cells and highlight the impact of RAD54L function on the treatment of patients with BRCA1/2-deficient tumors. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.26.550730v1?rss=1 Authors: Faiz, M., Kalev-Zylinska, M. L., Singleton, D. C., Hay, M. P., Ledgerwood, E. C. Abstract: Endoplasmic reticulum stress triggers the unfolded protein response (UPR) to promote cell survival or apoptosis. Transient endoplasmic reticulum stress activation has been reported to trigger megakaryocyte production, and UPR activation has been reported as a feature of megakaryocytic cancers. However, the role of UPR signaling in megakaryocyte biology is not fully understood. We studied the involvement of UPR in human megakaryocytic differentiation using PMA (phorbol 12-myristate 13-acetate)-induced maturation of megakaryoblastic cell lines and thrombopoietin-induced differentiation of human peripheral blood-derived hematopoietic stem cells. Our results demonstrate that an adaptive UPR mediated by IRE1 (inositol-requiring enzyme 1) endonuclease activity is required for megakaryocyte differentiation. Differentiation did not alter the response to the canonical endoplasmic reticulum stressors DTT or thapsigargin. However, thapsigargin, but not DTT, inhibited differentiation, consistent with the involvement of Ca2+ signaling in megakaryocyte differentiation. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.26.550758v1?rss=1 Authors: Luo, Y., Hao, H., Wang, Z., Ong, C., Dutcher, R., Xu, Y., Liu, J., Pedersen, L. C., Xu, D. Abstract: TRAIL (TNF-related apoptosis-inducing ligand) is a potent inducer of tumor cell apoptosis through TRAIL receptors. While it has been previously pursued as a potential anti-tumor therapy, the enthusiasm subsided due to unsuccessful clinical trials and the fact that many tumors are resistant to TRAIL. In this report we identified heparan sulfate (HS) as an important regulator of TRAIL-induced apoptosis. TRAIL binds HS with high affinity (73 nM) and HS induces TRAIL to form higher-order oligomers. The HS-binding site of TRAIL is located at the N-terminus of soluble TRAIL, which includes three basic residues. Binding to cell surface HS plays an essential role in promoting the apoptotic activity of TRAIL in both breast cancer and myeloma cells, and this promoting effect can be blocked by heparin, which is commonly administered to cancer patients. We also quantified HS content in several lines of myeloma cells and found that the cell line showing the most resistance to TRAIL has the least expression of HS, which suggests that HS expression in tumor cells could play a role in regulating sensitivity towards TRAIL. We also discovered that death receptor 5 (DR5), TRAIL and HS can form a ternary complex and that cell surface HS plays an active role in promoting TRAIL-induced cellular internalization of DR5. Combined, our study suggests that TRAIL-HS interactions could play multiple roles in regulating the apoptotic potency of TRAIL and might be an important point of consideration when designing future TRAIL-based anti-tumor therapy. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.26.550695v1?rss=1 Authors: Hood, E. M., Lipinski, R. A. J., Lipinski, D. M. Abstract: PURPOSE ARPE19 cells are a commonly used cell culture model for the study of retinal pigment epithelial cell biology and pathologies. However, numerous studies have demonstrated that ARPE19 undergo morphologic, transcriptomic and genomic alterations over time and with increasing passage number. Herein, we explore the mechanisms underlying increased resistance to the delivery of exogenous genetic material via transfection in ARPE19 cells using mass spectrometry. METHODS ARPE19 cells (N=5 wells/reagent) were seeded in 6-well plates at passages 24 through 30. At 70% confluency an mCherry reporter construct was delivered via transfection using Lipofectamine 3000, Lipofectamine LTX, Lipofectamine Stem, or PEI (polyethylenimine) reagents. After 72 hours, transfection efficiency was quantified by fluorescence microscopy and flow cytometry. Mass spectrometry and immunofluorescence of ARPE19 cells were performed at passages 24 and 30 to evaluate altered protein synthesis and localization between passage numbers. RESULTS ARPE19 transfection showed a maximum transfection efficiency of 32.4% at P26 using Lipofectamine 3000 reagent. All lipofectamine based reagents demonstrated statistically significant decreases in transfection efficiency between passages 24 and 30. Mass spectrometry analysis revealed 18 differentially expressed proteins, including down-regulation of clathrin light chain B (CLTB) and legumain (LGMN) that was confirmed via immunofluorescence imaging, which indicated altered intracellular localization. CONCLUSIONS ARPE19 cells demonstrate passage number dependent changes in lipofectamine-based transfection efficiency. Mass spectrometry and immunofluorescence indicates the observed decrease in transfection efficiency involves the dysregulation of endocytosis and intracellular endolysosomal trafficking at later passages. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.26.550681v1?rss=1 Authors: Chelaghma, S., Ke, H., Barylyuk, K., Krueger, T., Koreny, L., Waller, R. F. Abstract: Apicomplexans are ubiquitous intracellular parasites of animals. These parasites use a programmed sequence of secretory events to find, invade, and then reengineer their host cells to enable parasite growth and proliferation. The secretory organelles micronemes and rhoptries mediate the first steps of invasion. Both secrete their contents through the apical complex which provides an apical opening in the parasite's elaborate inner membrane complex (IMC) -- an extensive subpellicular system of flattened membrane cisternae and proteinaceous meshwork that otherwise limits access of the cytoplasm to the plasma membrane for material exchange with the cell exterior. After invasion, a second secretion programme drives host cell remodelling and occurs from dense granules. The site(s) of dense granule exocytosis, however, has been unknown. In Toxoplasma gondii, small subapical annular structures that are embedded in the IMC have been observed, but the role or significance of these apical annuli to plasma membrane function has also been unknown. Here, we determined that integral membrane proteins of the plasma membrane occur specifically at these apical annular sites, that these proteins include SNARE proteins, and that the apical annuli are sites of vesicle fusion and exocytosis. Specifically, we show that dense granules require these structures for the secretion of their cargo proteins. When secretion is perturbed at the apical annuli, parasite growth is arrested. The apical annuli, therefore, represent a second structure in the IMC specialised for protein secretion, and reveal that in Toxoplasma there is a physical separation of the processes of pre- and post-invasion secretion that mediate host-parasite interactions. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.27.550830v1?rss=1 Authors: Schmutz, S., Commere, P.-H., Montcuquet, N., Cumano, A., Ait-Mansour, C., Novault, S., Hasan, M. Abstract: The analytical capability of flow cytometry is crucial for differentiating the growing number of cell subsets found in human blood. This is important for accurate immunophenotyping of patients with few cells and a large number of parameters to monitor. Here, we present a 43-parameter panel to analyze peripheral blood mononuclear cells from healthy individuals using 41 fluorescence-labelled monoclonal antibodies, an autofluorescent channel, and a viability dye. We demonstrate minimal population distortions that lead to optimized population identification and reproducible results. We have applied an advanced approach in panel design, in selection of sample acquisition parameters and in data analysis. Appropriate autofluorescence identification and integration in the unmixing matrix, allowed for resolution of unspecific signals and increased dimensionality. Addition of one laser without assigned fluorochrome resulted in decreased fluorescence spill over and improved discrimination of cell subsets. It also increased staining index when autofluorescence was integrated in the matrix. We conclude that spectral flow cytometry is highly valuable tool for high-end immunophenotyping, and that fine-tuning of major experimental steps is key for taking advantage of its full capacity. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.27.550888v1?rss=1 Authors: Brunetta, H. S., Jung, A. S., Francisco, A., Castilho, R. F., Mori, M. A., Bartelt, A. Abstract: In thermogenic adipocytes, uncoupling protein-1 (UCP1) is a key mediator of non-shivering thermogenesis (NST) by uncoupling the electron transport chain from FoF1-ATP synthase-mediated ATP production. While regulatory mechanisms of UCP1 are important for NST, it is unknown whether also the activity of ATP synthase is modulated during NST. Here, we show a critical role of Inhibitory Factor 1 (IF1), an inhibitor of ATP synthase, for brown adipocyte energy metabolism. In mice, IF1 protein content is diminished in brown adipose tissue of mice after 5 days of cold exposure. Additionally, the capacity of ATP synthase to generate mitochondrial membrane potential (MMP) through ATP hydrolysis (the so-called reverse mode) was higher in mitochondria isolated from cold-adapted mice compared to mice housed at room temperature. While silencing of IF1 in cultured brown adipocytes did not affect MMP, IF1 overexpression resulted in an inability of mitochondria to sustain MMP upon adrenergic stimulation. The effects of IF1 overexpression on MMP were blunted when UCP1 was silenced or when a mutant IF1, incapable of binding to ATP synthase, was used. In brown adipocytes, IF1 ablation was sufficient to increase mitochondrial lipid oxidation and the cellular dependency on glycolysis to produce ATP. Conversely, IF1 overexpression blunted mitochondrial respiration without causing energetic stress, leading to a quiescent-like phenotype in brown adipocytes. In conclusion, our data show that the cold-induced downregulation of IF1 facilitates the reverse mode of ATP synthase and enables proper bioenergetic adaptation of brown adipose tissue to effectively support NST. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.25.550461v1?rss=1 Authors: Paul, R., Chakrabarty, A., Dey, S., Samanta, S., Pandey, R., Maji, S., Pezacki, A. T., Chang, C. J., Datta, R., Gupta, A. Abstract: Copper plays a key role in host-pathogen interaction. We found that during Leishmania major infection, the parasite-harboring macrophage regulates its copper homeostasis pathway in a way to facilitate copper-mediated neutralization of the pathogen. Copper-ATPase ATP7A transports copper to amastigote-harboring phagolysosomes to induce stress on parasites. Leishmania in order to evade the copper stress, utilizes a variety of manipulative measures to lower the host-induced copper stress. It induces deglycosylation and degradation of host-ATP7A and downregulation of copper importer, CTR1 by cysteine oxidation. Additionally, Leishmania induces CTR1 endocytosis that arrests copper uptake. In mouse model of infection, we report an increase in systemic bioavailable copper in infected animals. Heart acts as the major organ for diverting its copper reserves to systemic circulation to fight-off infection by downregulating its CTR1. Our study explores reciprocal mechanism of manipulation of host copper homeostasis pathway by macrophage and Leishmania to gain respective advantages in host-pathogen interaction. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.24.550385v1?rss=1 Authors: Bramey, N., Melo-Narvaez, M. C., See, F., Ballester-Llobell, B., Steinchen, C., Jain, E., Hafner, K., Yildirim, A. O., Koenigshoff, M., Lehmann, M. Abstract: Aging is the main risk factor for chronic lung diseases including idiopathic pulmonary fibrosis (IPF) and chronic obstructive pulmonary disease (COPD). Accordingly, hallmarks of aging such as cellular senescence are increased in different cell types such as fibroblasts in the lungs of patients. However, whether the senescent phenotype of fibroblasts derived from IPF or COPD differs is still unknown. Therefore, we characterized senescence at baseline and after exposure to disease-relevant insults (H2O2, bleomycin, and TGF-{beta}1) in primary human lung fibroblasts (phLF) from control donors, IPF, and COPD patients. We found that phLF from different disease origins have a low baseline senescence. Moreover, senescence trigger and not disease origin defines their senescence phenotype. Finally, senescent fibroblasts interfere with the stem cell capacity of alveolar progenitors in vitro. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.26.549647v1?rss=1 Authors: Carmona, B., Camelo, C., Mehraz, M., Lemullois, M., Lince-Faria, M., Coyaud, E., Marinho, H. S., Goncalves, J., Nolasco, S., Pinto, F., Raught, B., Tassin, A.-M., Koll, F., Soares, H. Abstract: Centrosomes are organelles consisting of two structurally and functionally distinct centrioles, with the mother centriole having complex distal (DA) and subdistal appendages (SDA). Despite their importance, how appendages are assembled and maintained remains unclear. This study investigated human TBCCD1, a centrosomal protein essential for centrosome positioning, to uncover its localization and role at centrioles. We found that TBCCD1 localizes at both proximal and distal regions of the two centrioles, forming a complex structure spanning from SDA to DA and extending inside and outside the centriole lumen. TBCCD1 depletion caused centrosome mispositioning, which was partially rescued by taxol, and the loss of microtubules (MTs) anchored to centrosomes. TBCCD1 depletion also reduced levels of SDA proteins involved in MT anchoring such as Centriolin/CEP110, Ninein, and CEP170. Additionally, TBCCD1 was essential for the correct positioning of motile cilia basal bodies and associated structures in Paramecium. This study reveals that TBCCD1 is an evolutionarily conserved protein essential for centriole and basal body localization and appendage assembly and maintenance. A BioID screening also linked TBCCD1 to ciliopathy-associated protein networks. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.24.550259v1?rss=1 Authors: Howell, J., Omwenga, S., Jimenez, M., Hammarton, T. C. Abstract: Promastigote Leishmania mexicana have a complex cell division cycle characterised by the ordered replication of several single-copy organelles, a prolonged S phase and very rapid G2 and cytokinesis phases, accompanied by cell cycle stage-associated morphological changes. Here we exploit these morphological changes to develop a high-throughput and semi-automated imaging flow cytometry (IFC) pipeline to analyse the cell cycle of L. mexicana in live cells. Firstly, we demonstrate that, unlike several other DNA stains, Vybrant DyeCycle Orange (DCO) is non-toxic and enables quantitative DNA imaging in live L. mexicana promastigotes. Secondly, by tagging the orphan spindle kinesin, KINF, with mNeonGreen, we describe KINFs cell cycle-dependent expression and localisation. Then, by combining manual gating of DCO DNA intensity profiles with automated masking and morphological measurements of parasite images, visual determination of the number of flagella per cell, and automated masking and analysis of mNG:KINF fluorescence, we provide a newly detailed description of L. mexicana promastigote cell cycle events that, for the first time, includes the durations of individual G2, mitosis and post-mitosis phases. By applying IFC in this way, we were able, in minutes, to capture tens of thousands of high quality brightfield and fluorescent images of live L. mexicana cells in solution, and to acquire quantitative data across multiple parameters for every image captured. Our custom-developed masking and gating scheme, allowed us to identify elusive G2 cells, show that cytokinesis commences during early mitosis and continues after mitosis is complete, and identify newly divided cells that were within the first 12 minutes of the new cell cycle. Our new pipeline offers many advantages over traditional methods of cell cycle analysis such as fluorescence microscopy and flow cytometry and paves the way for novel high-throughput analysis of Leishmania cell division. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.25.550535v1?rss=1 Authors: Sheridan, J., Grata, A., Suva, E. E., Bresteau, E., Mitchell, L. R., Mitchell, B. Abstract: The skin of Xenopus embryos contains numerous multiciliated cells (MCCs), which collectively generate a directed fluid flow across the epithelial surface essential for distributing the overlaying mucous. MCCs develop into highly specialized cells to generate this flow, containing approximately 150 evenly spaced centrioles that give rise to motile cilia. MCC-driven fluid flow can be impaired when ciliary dysfunction occurs, resulting in primary ciliary dyskinesia (PCD) in humans. Mutations in a large number of genes (~50) have been found to be causative to PCD. Recently, studies have linked low levels of Adenylate Kinase 7 (AK7) gene expression to patients with PCD; however, the mechanism for this link remains unclear. Additionally, AK7 mutations have been linked to multiple PCD patients. Adenylate kinases modulate ATP production and consumption, with AK7 explicitly associated with motile cilia. Here we reproduce an AK7 PCD-like phenotype in Xenopus and describe the cellular consequences that occur with manipulation of AK7 levels. We show that AK7 localizes throughout the cilia in a DPY30 domain-dependent manner, suggesting a ciliary function. Additionally, we find that AK7 overexpression increases centriole number, suggesting a role in regulating centriole biogenesis. We find that in AK7-depleted embryos, cilia number, length, and beat frequency are all reduced, which in turn, significantly decreases the tissue-wide mucociliary flow. Additionally, we find a decrease in centriole number and an increase in sub-apical centrioles, implying that AK7 influences both centriole biogenesis and docking, which we propose underlie its defect in ciliogenesis. We propose that AK7 plays a role in PCD by impacting centriole biogenesis and apical docking, ultimately leading to ciliogenesis defects that impair mucociliary clearance. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.25.550451v1?rss=1 Authors: Körner, M., Meyer, S., Marincola, G., Kern, M., Grimm, C., Schülein-Völk, C., Fischer, U., Hofmann, K., Buchberger, A. Abstract: The ATPase p97 (also known as VCP, Cdc48) has crucial functions in a variety of important cellular processes such as protein quality control, organellar homeostasis and DNA damage repair, and its de-regulation is linked to neuro-muscular diseases and cancer. p97 is tightly controlled by numerous regulatory cofactors, but the full range and function of the p97-cofactor network is unknown. Here, we identify the hitherto uncharacterized FAM104 proteins as a conserved family of p97 interactors. FAM104 proteins bind p97 directly via a novel, alpha-helical motif and associate with the p97- UFD1-NPL4 complex in cells. FAM104 proteins localize to the nucleus and promote both the nuclear import and chromatin binding of p97. Loss of FAM104 proteins results in slow growth and hypersensitivity to p97 inhibition in the absence and presence of DNA damage, suggesting that FAM104 proteins are critical regulators of nuclear p97 functions. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.25.550562v1?rss=1 Authors: Phillips, J. E., Pan, D. Abstract: The genomes of close unicellular relatives of animals encode orthologs of many genes that regulate animal development. However, little is known about the function of such genes in unicellular organisms or the evolutionary process by which these genes came to function in multicellular development. The Hippo pathway, which regulates cell proliferation and tissue size in animals, is present in some of the closest unicellular relatives of animals, including the amoeboid organism Capsaspora owczarzaki. We previously showed that the Capsaspora ortholog of the Hippo pathway nuclear effector Yorkie/YAP/TAZ (coYki) regulates actin dynamics and the three-dimensional morphology of Capsaspora cell aggregates, but is dispensable for cell proliferation control (Phillips et al., 2022). However, the function of upstream Hippo pathway components, and whether and how they regulate coYki in Capsaspora, remained unknown. Here, we analyze the function of the upstream Hippo pathway kinases coHpo and coWts in Capsaspora by generating mutant lines for each gene. Loss of either kinase results in increased nuclear localization of coYki, indicating an ancient, premetazoan origin of this Hippo pathway regulatory mechanism. Strikingly, we find that loss of either kinase causes a contractile cell behavior and increased density of cell packing within Capsaspora aggregates. We further show that this increased cell density is not due to differences in proliferation, but rather actomyosin-dependent changes in the multicellular architecture of aggregates. Given its well-established role in cell density-regulated proliferation in animals, the increased density of cell packing in coHpo and coWts mutants suggests a shared and possibly ancient and conserved function of the Hippo pathway in cell density control. Together, these results implicate cytoskeletal regulation but not proliferation as an ancestral function of the Hippo pathway and uncover a novel role for Hippo signaling in regulating cell density in a proliferation-independent manner. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.25.550521v1?rss=1 Authors: Dou, D., Aiken, J., Holzbaur, E. L. F. Abstract: Gain-of-function mutations in the LRRK2 gene cause Parkinson's disease (PD), characterized by debilitating motor and non-motor symptoms. Increased phosphorylation of a subset of RAB GTPases by LRRK2 is implicated in PD pathogenesis. We find that increased phosphorylation of RAB3A, a cardinal synaptic vesicle precursor (SVP) protein, disrupts anterograde axonal transport of SVPs in iPSC-derived human neurons (iNeurons) expressing hyperactive LRRK2-p.R1441H. Knockout of the opposing protein phosphatase 1H (PPM1H) in iNeurons phenocopies this effect. In these models, the compartmental distribution of synaptic proteins is altered; synaptophysin and synaptobrevin-2 become sequestered in the neuronal soma with decreased delivery to presynaptic sites along the axon. We find that RAB3A phosphorylation disrupts binding to the motor adapter MADD, potentially preventing formation of the RAB3A-MADD-KIF1A/1B{beta} complex driving anterograde SVP transport. RAB3A hyperphosphorylation also disrupts interactions with RAB3GAP and RAB-GDI1. Our results reveal a mechanism by which pathogenic hyperactive LRRK2 may contribute to the altered synaptic homeostasis associated with characteristic non-motor and cognitive manifestations of PD. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.25.550533v1?rss=1 Authors: Ceglowski, J., Hoffman, H. K., Hoff, K. J., McCurdy, B. L., Moore, J., Prekeris, R. Abstract: The primary cilium is a critical sensory organelle that is built of axonemal microtubules ensheathed by a ciliary membrane. In polarized epithelial cells, primary cilia reside on the apical surface and must extend these microtubules directly into the extracellular space and remain a stable structure. However, the factors regulating cross-talk between ciliation and cell polarization, as well as, axonemal microtubule growth and stabilization in polarized epithelia are not fully understood. In this study, we find TTLL12, a previously uncharacterized member of the Tubulin Tyrosine Ligase-Like (TTLL) family, localizes to the base of primary cilia and is required for cilia formation in polarized renal epithelial cells. We also show that TTLL12 directly binds to the tubulin heterodimer in vitro and regulates microtubule dynamics, stability, and post-translational modifications (PTMs). While all other TTLLs catalyze the addition of glutamate or glycine to microtubule C-terminal tails, TTLL12 uniquely affects tubulin PTMs by promoting both microtubule lysine acetylation and arginine methylation. Together, this work identifies a novel microtubule regulator and provides insight into the requirements for apical extracellular axoneme formation. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.25.550499v1?rss=1 Authors: O'Shaughnessy, E. C., Lam, M., Ryken, S. E., Wiesner, T., Lukasik, K., Zuchero, J. B., Leterrier, C., Adalsteinsson, D., Gupton, S. L. Abstract: Exocytosis is a fundamental process used by all eukaryotic cells to regulate the composition of the plasma membrane and facilitate cell-cell communication. To investigate the role exocytosis plays in neuronal morphogenesis, previously we developed computational tools with a graphical user interface (GUI) to enable the automatic detection and analysis of exocytic events (ADAE GUI) from fluorescence timelapse images. Though these tools have proven useful, we found that the code was brittle and not easily adapted to different experimental conditions. Here, we developed and validated a robust and versatile toolkit, we have named pHusion, for the analysis of exocytosis written in ImageTank, a graphical programming language that combines image visualization and numerical methods. We tested this method using a variety of imaging modalities and pH-sensitive fluorophores, diverse cell types, and various exocytic markers to generate a flexible and intuitive package. Using pHusion, we show that VAMP3-mediated exocytosis occurs 30-times more frequently in melanoma cells compared with primary oligodendrocytes, that VAMP2-mediated fusion events in mature rat hippocampal neurons are much longer lasting than those in immature murine cortical neurons, and that clustering of exocytic events occurs across cell types. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.24.550444v1?rss=1 Authors: Torrino, S., Oldham, W., Tejedor, A. R., Sanchez-Burgos, I., Rachedi, N., Fraissard, K., Chauvet, C., Sbai, C., O'Hara, B. P., Abelanet, S., Brau, F., Clavel, S., Collepardo-Guevara, R., Rene Espinosa, J., ben-sahra, I., BERTERO, T. Abstract: Biomolecular condensates regulate a wide range of cellular functions from signaling to RNA metabolism1,2, yet, the physiologic conditions regulating their formation remain largely unexplored. Biomolecular condensate assembly is tightly regulated by the intracellular environment. Changes in the chemical or physical conditions inside cells can stimulate or inhibit condensate formation3,5. However, whether and how the external environment of cells can also regulate biomolecular condensation remain poorly understood. Increasing our understanding of these mechanisms is paramount as failure to control condensate formation and dynamics can lead to many diseases6,7. Here, we provide evidence that matrix stiffening promotes biomolecular condensation in vivo. We demonstrate that the extracellular matrix links mechanical cues with the control of glucose metabolism to sorbitol. In turn, sorbitol acts as a natural crowding agent to promote biomolecular condensation. Using in silico simulations and in vitro assays, we establish that variations in the physiological range of sorbitol, but not glucose, concentrations, are sufficient to regulate biomolecular condensates. Accordingly, pharmacologic and genetic manipulation of intracellular sorbitol concentration modulates biomolecular condensates in breast cancer, a mechano-dependent disease. We propose that sorbitol is a mechanosensitive metabolite enabling protein condensation to control mechano-regulated cellular functions. Altogether, we uncover molecular driving forces underlying protein phase transition and provide critical insights to understand the biological function and dysfunction of protein phase separation. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.25.550329v1?rss=1 Authors: Tong, C. S., Su, M., Sun, H., Chua, X. L., Guo, S., Ramaraj, R. S., Lee, A. G., Ong, N. W. P., Miao, Y., Wu, M. Abstract: The coordination between actin and microtubule network is crucial, yet our understanding of the underlying mechanisms remains limited. In this study, we used travelling waves in the cell cortex to characterize the collective dynamics of cytoskeletal networks. Our findings show that Cdc42 and F-BAR-dependent actin waves in mast cells are mainly driven by formin-mediated actin polymerization, with the microtubule-binding formin FH2 domain-containing protein 1 (FHDC1) identified as an early regulator. The depolymerization of microtubules coincides with the nucleation of actin waves, and the concurrent release of FHDC1 from microtubule is required for actin waves. Lastly, we show the importance of the actin-microtubule linkage mediated by FHDC1 in crucial cellular processes such as cell division and migration. Our data provided molecular insights into the nucleation mechanisms of actin waves and uncover an antagonistic interplay between microtubule and actin polymerization in their collective dynamics. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.23.550229v1?rss=1 Authors: Manzer, K. M., Fromme, J. C. Abstract: Arf GTPases are central regulators of the Golgi complex, which serves as the nexus of membrane trafficking pathways in eukaryotic cells. Arf proteins recruit dozens of effectors to modify membranes, sort cargos, and create and tether transport vesicles, and are therefore essential for orchestrating Golgi trafficking. The regulation of Arf activity is controlled by the action of Arf-GEFs, which activate via nucleotide exchange, and Arf-GAPs, which inactivate via nucleotide hydrolysis. The localization dynamics of Arf GTPases and their Arf-GAPs during Golgi maturation have not been reported. Here we use the budding yeast model to examine the temporal localization of the Golgi Arf-GAPs. We also determine the mechanisms used by the Arf-GAP Age2 to localize to the Golgi. We find that the catalytic activity of Age2 and a conserved sequence in the unstructured C-terminal domain of Age2 are both required for Golgi localization. This sequence is predicted to form an amphipathic helix and mediates direct binding of Age2 to membranes in vitro. We also report the development of a probe for sensing active Arf1 in living cells and use this probe to characterize the temporal dynamics of Arf1 during Golgi maturation. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.24.550360v1?rss=1 Authors: del Pino, R. C., Zoltner, M., Yamada, K., Butterfield, E. R., Field, M. Abstract: Post-translational modifications (PTMs) modulate protein function, with ubiquitylation a pre-eminent example with major roles in protein turnover. Ubiquitylation utilises a ligase enzyme cascade for conjugation of ubiquitin to client proteins and cullin-RING ligases are amongst the most complex known. We reconstructed evolution of cullin-RING E3 ubiquitin ligases across eukaryotes and experimentally characterised two cullin complexes in trypanosomatids, a taxon highly divergent from animals and fungi. We find considerable diversity within cullins and, in particular, trypanosomatids share only a minority of cullins with other lineages. Furthermore, we identify expansions in cullin client adaptor protein families, novel client adaptors and demonstrate client specificity. Finally we show that ornithine decarboxylase (TbODC), an important target of the drug trypanosome eflornithine, is a substrate for TbCul-A and overturn earlier models for eflornithine specificity. These studies highlight lineage-specific roles for cullin E3s and their contributions towards eukaryotic complexity. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.24.550336v1?rss=1 Authors: Kumar, R., Tatiya, P., Dey, D., Ratra, Y., Mian, S. Y., Chaudhary, S., Suhag, K., Basak, S., Banerjee, M. Abstract: Downregulation of the host cell pro-apoptotic pathways confers a selective advantage to viral pathogens, and many viruses interfere with such regulatory mechanisms in order to enhance their propagation in infected cells. The Scribble cell polarity complex, which is composed of multiple adapter proteins including human Scribble, Dlg and MAGI, controls a variety of host cell functionalities including apoptosis, morphology, polarity, signaling and migration. In the recent years, components from several viral pathogens, including oncogenic viruses, have been found to associate with and modulate the activities of this complex, particularly that of the pro-apoptotic protein Scribble. Here, we establish a critical role for the downregulation of Scribble expression for the potentiation of Chikungunya Virus (CHIKV) infection. CHIKV is a well-known mosquito-borne alphavirus, which has caused outbreaks in more than 100 countries and constitutes a global health hazard. While the majority of the CHIKV proteome is well characterized, the role of a small structural protein 6K, and its transframe variant, TF, which is generated from the 6K sequence by ribosomal slippage and frameshifting, is not established as yet. We show that TF triggers the punctation, ubiquitination and degradation of Scribble during CHIKV infection, and that the downregulation of Scribble during CHIKV infection. We show that the association of TF with the PDZ domains of Scribble is mediated through a PDZ-domain binding motif at the C-terminus of TF. Thus, our work establishes a role for the downregulation of Scribble in alphavirus infections for the first time, while allocating a novel role in host modulation to the mysterious 6K/TF component of alphaviruses. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.24.550375v1?rss=1 Authors: Willet, A. H., Turner, L. A., Park, J. S., Ren, L., Snider, C. E., Gould, K. L. Abstract: Phosphatidylinositol (PI)-4-phosphate (PI4P) is a lipid found at the plasma membrane (PM) and Golgi in cells from yeast to humans. PI4P is generated from PI by PI4-kinases and can be converted to PI-4,5-bisphosphate [PI(4,5)P2]. Schizosaccharomyces pombe have 2 essential PI4-kinases: Stt4 and Pik1. Stt4 localizes to the PM and its loss from the PM results in a decrease of PM PI4P and PI(4,5)P2. As a result, cells divide non-medially due to disrupted cytokinetic ring-PM anchoring. However, the localization and function of S. pombe Pik1 has not been thoroughly examined. Here, we found that Pik1 localizes exclusively to the trans-Golgi and is required for Golgi PI4P production. We determined that Ncs1 regulates Pik1, but unlike in other organisms, it is not required for Pik1 Golgi localization. When Pik1 function was disrupted, PM PI4P but not PI(4,5)P2 levels were reduced, a major difference with Stt4. We conclude that Stt4 is the chief enzyme responsible for producing the PI4P that generates PI(4,5)P2. Also, that cells with disrupted Pik1 do not divide asymmetrically highlights the specific importance of PM PI(4,5)P2 for cytokinetic ring-PM anchoring. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.23.550233v1?rss=1 Authors: Ikawa, K., Hiro, S., Kondo, S., Ohsawa, S., Sugimura, K. Abstract: Directional cell rearrangement is a critical process underlying correct tissue deformation during morphogenesis. Although the involvement of F-actin regulation in cell rearrangement has been established, the role and regulation of actin binding proteins (ABPs) in this process are not well understood. In this study, we investigated the function of Coronin-1, a WD-repeat actin-binding protein, in controlling directional cell rearrangement in the Drosophila pupal wing. Transgenic flies expressing Coronin-1-EGFP were generated using CRISPR-Cas9. We observed that Coronin-1 localizes at the reconnecting junction during cell rearrangement, which is dependent on actin interacting protein 1 (AIP1), an actin disassembler and a known regulator of wing cell rearrangement. Loss of Coronin-1 function reduces cell rearrangement directionality and hexagonal cell fraction. These results suggest that Coronin-1 promotes directional cell rearrangement via its interaction with AIP1, highlighting the role of ABPs in the complex process of morphogenesis. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.24.550263v1?rss=1 Authors: Yang, J., Zou, S., Qiu, Z., Lai, M., Long, Q., Chen, H., Zhang, S., Xie, X., Gong, Y., Liu, A., Li, M., Bai, X. Abstract: Quiescence (G0) maintenance and exit are crucial for tissue homeostasis and regeneration in mammals. Here, we show that methyl-CpG binding protein 2 (Mecp2) expression is cell cycle-dependent and negatively regulates quiescence exit in cultured cells and in an injury-induced liver regeneration mouse model. Specifically, acute reduction of Mecp2 is required for efficient quiescence exit, as deletion of Mecp2 accelerates, while overexpression of Mecp2 delays quiescence exit, and forced expression of Mecp2 after Mecp2 conditional knockout rescues cell cycle reentry. The E3 ligase Nedd4 mediates the ubiquitination and degradation of Mecp2, and thus facilitates quiescence exit. Genome-wide study uncovered the dual role of Mecp2 in preventing quiescence exit by transcriptionally activating metabolic genes while repressing proliferation-associated genes. Particularly, disruption of two nuclear receptors (NRs), Rara or Nr1h3, accelerates quiescence exit, mimicking the Mecp2 depletion phenotype. Our studies unravel a previously unrecognized role for Mecp2 as an essential regulator of quiescence exit and tissue regeneration. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.24.549756v1?rss=1 Authors: Shah, D. V., Leonardi, D., Waldorn, A. Abstract: Cellular aging, also known as senescence, is a form of proliferation arrest that occurs in cells with age. In individuals, this process occurs due to telomere degradation and consequent dysfunction. In the nervous system, senescence of astrocytes, the most common neuronal support cells, has been associated with inflammation and forms of neurodegeneration associated with various disease pathologies. Thus, studying senescence may be a unique approach to understanding brain and aging and consequential diseases as well. Studies have shown that transcription downregulation of SCN9A led to reversal of senescence in epithelial cells. So, this study explores the role of SCN9A in astrocytes. In silico patient gene analysis reveals multiple significant pathways associated with neuronal aging including ion channel regulation. Subsequent analysis shows the downregulation of SCN9A is associated with genes in the neuronal senescence pathway. In vitro studies showed that astrocytes with knockdown SCN9A did not undergo senescence as significantly as regular astrocytes. Furthermore, protein analysis presented a novel role for SCN9A in the astrocyte senescence pathway and an association with preventing DNA leakage. This study suggests SCN9A plays a large role in neurodegeneration and it should be studied in reversing senescence and even treatment plans for brain aging. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.23.550157v1?rss=1 Authors: Liu, M., Jin, S., Agabiti, S. S., Jensen, T. B., Yang, T., Radda, J. S. D., Ruiz, C. F., Baldissera, G., Muzumdar, M. D., Wang, S. Abstract: Alterations in three-dimensional (3D) genome structures are associated with cancer. However, how genome folding evolves and diversifies during subclonal cancer progression in the native tissue environment remains unknown. Here, we leveraged a genome-wide chromatin tracing technology to directly visualize 3D genome folding in situ in a faithful Kras-driven mouse model of lung adenocarcinoma (LUAD), generating the first single-cell 3D genome atlas of any cancer. We discovered stereotypical 3D genome alterations during cancer development, including a striking structural bottleneck in preinvasive adenomas prior to progression to LUAD, indicating a stringent selection on the 3D genome early in cancer progression. We further showed that the 3D genome precisely encodes cancer states in single cells, despite considerable cell-to-cell heterogeneity. Finally, evolutionary changes in 3D genome compartmentalization -- partially regulated by polycomb group protein Rnf2 through its ubiquitin ligase-independent activity -- reveal novel genetic drivers and suppressors of LUAD progression. Our results demonstrate the importance of mapping the single-cell cancer 3D genome and the potential to identify new diagnostic and therapeutic biomarkers from 3D genomic architectures. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.24.550339v1?rss=1 Authors: Shaughnessy, R., Serres, M., Escot, S., Hammich, H., Cuvelier, F., Salles, A., Rocancourt, M., Verdon, Q., Gaffuri, A.-L., Sourigues, Y., Malherbe, G., Velikovsky, L., Chardon, F., Tinevez, J.-Y., Callebaut, I., Formstecher, E., Houdusse, A., David, N., Pylypenko, O., Echard, A. Abstract: Cilia protrude from the cell surface and play critical roles in intracellular signaling, environmental sensing and development. Actin-dependent contractility and intracellular trafficking are both required for ciliogenesis, but little is known about how these processes are coordinated. Here, we identified a Rac1- and Rab35-binding protein with a truncated BAR domain that we named MiniBAR (aka KIAA0355/GARRE) which plays a key role in ciliogenesis. MiniBAR colocalizes with Rac1 and Rab35 at the plasma membrane and on intracellular vesicles trafficking to the ciliary base and exhibits remarkable fast pulses at the ciliary membrane. MiniBAR depletion leads to short cilia resulting from abnormal Rac-GTP/Rho-GTP levels, increased acto-myosin-II-dependent contractility together with defective trafficking of IFT88 and ARL13B into cilia. MiniBAR-depleted zebrafish embryos display dysfunctional short cilia and hallmarks of ciliopathies including left-right asymmetry defects. Thus, MiniBAR is a unique dual Rac and Rab effector that controls both actin cytoskeleton and membrane trafficking for ciliogenesis. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.22.550177v1?rss=1 Authors: Knapp, B., Willis, L., Gonzalez, C., Vashistha, H., Touma, J. J., Tikhonov, M., Ram, J., Salman, H., Elias, J. E., Huang, K. C. Abstract: The impact of temperature on growth is typically considered under heat- or cold-shock conditions that elicit specific regulation. In between, cellular growth rate varies according to the Arrhenius law of thermodynamics. Here, we use growth-rate dynamics during transitions between temperatures to discover how this behavior arises and what determines the temperature sensitivity of growth. Using a device that enables single-cell tracking across a wide range of temperatures, we show that bacteria exhibit a highly conserved, slow response to temperatures upshifts with a time scale of ~1.5 doublings at the higher temperature, regardless of initial/final temperature or nutrient source. We rule out transcriptional, translational, and membrane reconfiguration as potential mechanisms. Instead, we demonstrate that an autocatalytic enzyme network incorporating temperature-sensitive Michaelis-Menten kinetics recapitulates all temperature-shift dynamics, reveals that import dictates steady-state Arrhenius growth behavior, and successfully predicts alterations in the upshift response observed under simple-sugar or low-nutrient conditions or in fungi. These findings indicate that metabolome rearrangement dictates how temperature affects microbial growth. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.23.550235v1?rss=1 Authors: Tsutsumi, R., Ueberheide, B., Liang, F.-X., Neel, B. G., Sakai, R., Saito, Y. Abstract: Glycolysis is a fundamental cellular process, yet its regulatory mechanisms remain incompletely understood. Here, we show that a subset of glucose transporter 1 (GLUT1/SLC2A1) co-endocytoses with platelet-derived growth factor (PDGF) receptor (PDGFR) upon PDGF-stimulation. Furthermore, multiple glycolytic enzymes localize to these endocytosed PDGFR/GLUT1-containing vesicles adjacent to mitochondria. Contrary to current models, which emphasize the importance of glucose transporters on the cell surface, we find that PDGF-stimulated glucose uptake depends on receptor/transporter endocytosis. Our results suggest that growth factors generate glucose-loaded endocytic vesicles that deliver glucose to the glycolytic machinery in proximity to mitochondria, and argue for a new layer of regulation for glycolytic control governed by cellular membrane dynamics. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.21.550077v1?rss=1 Authors: Santos, F. M., Melo, V. C., Araujo, S., Sousa, C. D. F., Moreira, T. P., Goncalves, M. R., Santos, A. C., Seabra, H. A., Costa, P. A. C., Barrioni, B. R., Souza, P. B., Pereira, M. d. M., Nogueira, M. L., Souza, D. G., Guimaraes, P. P., Texeira, M. M., Queiroz-Junior, C. M., Costa, V. V. Abstract: Mayaro virus (MAYV) is an emerging arbovirus member of the Togaviridae family and Alphavirus genus. MAYV infection causes an acute febrile illness accompanied by persistent polyarthralgia and myalgia. Understanding the mechanisms involved in arthritis caused by alphaviruses is necessary to develop specific therapies. In this work, we investigated the role of the CCL2/CCR2 axis in the pathogenesis of MAYV-induced disease. For this, WT C57BL/6J and CCR2-/- mice were infected with MAYV subcutaneously and evaluated for disease development. MAYV infection induced an acute inflammatory disease in WT mice. The immune response profile was characterized by an increase in the production of inflammatory mediators, such as IL-6, TNF and CCL2. Higher levels of CCL2 at the local and systemic levels, was followed by significant recruitment of CCR2+ macrophages and a cellular response orchestrated by these cells. CCR2-/- mice showed an increase in CXCL-1 levels, followed by a replacement of the macrophage inflammatory infiltrate by neutrophils. Additionally, absence of the CCR2 receptor protected mice from bone loss induced by MAYV. Accordingly, the silencing of CCL2 chemokine expression in vivo and the pharmacological blockade of CCR2 promoted a partial improvement in disease. Cell culture data support the mechanism underlying MAYV's bone pathology in which: i) MAYV infection promoted a pro-osteoclastogenic microenvironment mediated by IL-6, TNF and CCL2 and ii) migration of osteoclast precursors was dependent on the CCR2/CCL2 axis. Overall, these data contribute to the understanding of the pathophysiology of MAYV infection and to the identification future of specific therapeutic targets in MAYV-induced disease. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.22.550175v1?rss=1 Authors: Trewin, A. J., Weeks, K. L., Wadley, G. D., Lamon, S. Abstract: Cardiomyocyte calcium homeostasis is a tightly regulated process. The mitochondrial calcium uniporter (MCU) complex can buffer elevated cytosolic Ca2+ levels and consists of pore-forming proteins including MCU, and various regulatory proteins such as mitochondrial calcium uptake proteins 1 and 2 (MICU1/2). The stoichiometry of these proteins influences the sensitivity to Ca2+ and activity of the complex. However, the factors that regulate their gene expression remain incompletely understood. Long non-coding RNAs (lncRNAs) regulate gene expression through various mechanisms, and we recently found that the lncRNA Tug1 increased the expression of Mcu and associated genes. To further explore this, we performed antisense LNA knockdown of Tug1 (Tug1 KD) in H9c2 rat cardiomyocytes. Tug1 KD increased MCU protein expression, yet pyruvate dehydrogenase dephosphorylation, which is indicative of mitochondrial Ca2+ uptake was not enhanced. However, RNA-seq revealed that Tug1 KD increased Mcu along with differential expression of greater than 1000 genes including many related to Ca2+ regulation pathways in the heart. To understand the effect of this on Ca2+ signalling, we measured phosphorylation of Ca2+/calmodulin-dependent protein kinase II (CaMKII) and its downstream target cAMP Response Element-Binding protein (CREB), a transcription factor known to drive Mcu gene expression. In response a Ca2+ stimulus, the increase in CaMKII and CREB phosphorylation was attenuated by Tug1 KD. Inhibition of CaMKII, but not CREB, partially prevented the Tug1 KD-mediated increase in Mcu. Together, these data suggest that Tug1 modulates MCU expression via a mechanism involving CaMKII and regulates cardiomyocyte Ca2+ signalling which could have important implications for cardiac function. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.24.550435v1?rss=1 Authors: Bradley, R. A., Wolff, I. D., Cohen, P. E., Gray, S. Abstract: During prophase I of meiosis, DNA double-strand breaks form throughout the genome, with a subset repairing as crossover events, enabling the accurate segregation of homologous chromosomes during the first meiotic division. The mechanism by which DSBs become selected to repair as crossovers is unknown, although the crossover positioning and levels in each cell indicate it is a highly regulated process. One of the proteins that localises to crossover sites is the serine/threonine cyclin-dependent kinase CDK2. Regulation of CDK2 occurs via phosphorylation at tyrosine 15 (Y15) and threonine 160 (T160) inhibiting and activating the kinase, respectively. In this study we use a combination of immunofluorescence staining on spread spermatocytes and fixed testis sections, and STA-PUT gravitational sedimentation to isolate cells at different developmental stages to further investigate the temporal phospho regulation of CDK2 during prophase I. Western blotting reveals differential levels of the two CDK2 isoforms (CDK233kDa and CDK239kDa) throughout prophase I, with inhibitory phosphorylation of CDK2 at Y15 occurring early in prophase I, localising to telomeres and diminishing as cells enter pachynema. Conversely, the activatory phosphorylation on T160 occurs later, specifically the CDK233kDa isoform, and T160 signal is detected in spermatogonia and pachytene spermatocytes, where it co-localises with the Class I crossover protein MLH3. Taken together, our data reveals intricate control of CDK2 both with regards to levels of the two CDK2 isoforms, and differential regulation via inhibitory and activatory phosphorylation. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.24.550422v1?rss=1 Authors: Maxian, O., Mogilner, A. Abstract: Chiral actin bundles have been shown to play an important role in cell dynamics, but our understanding of the molecular mechanisms which combine to generate chirality remains incomplete. We numerically simulate a crosslinked filopodial bundle under the actions of helical myosin motors and/or formins and examine the collective buckling and twisting of the actin bundle. We find that the myosin spinning action effectively braids the bundle, compacting it, generating buckling, and enhancing crosslinking. Stochastic fluctuations of actin polymerization rates also contribute to filament buckling and bending of the bundle. Faster turnover of transient crosslinks attenuates the buckling and enhances coiling and compaction of the bundle. Formin twisting action by itself is not effective in inducing filopodial coiling and compaction, but co-rotating formins synergize with helical motors to coil and compact the actin bundle. We discuss implications of our findings for mechanisms of cytoskeletal chirality. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.24.550382v1?rss=1 Authors: Zhao, B., Mertz, J. Abstract: Improving the spatial resolution of a fluorescence microscope has been an ongoing challenge in the imaging community. To address this challenge, a variety of approaches have been taken, ranging from instrumentation development to image post-processing. An example of the latter is deconvolution, where images are numerically deblurred based on a knowledge of the microscope point spread function. However, deconvolution can easily lead to noise-amplification artifacts. Deblurring by post-processing can also lead to negativities or fail to conserve local linearity between sample and image. We describe here a simple image deblurring algorithm based on pixel reassignment that inherently avoids such artifacts and can be applied to general microscope modalities and fluorophore types. Our algorithm helps distinguish nearby fluorophores even when these are separated by distances smaller than the conventional resolution limit, helping facilitate, for example, the application of single-molecule localization microscopy in dense samples. We demonstrate the versatility and performance of our algorithm under a variety of imaging conditions. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.24.550379v1?rss=1 Authors: Stephens, E. B., Henke, W., Kalamvoki, M. Abstract: The open reading frame 3a (ORF3a) is an accessory transmembrane protein that is important to the pathogenicity of SARS-CoV-2. The cytoplasmic domain of ORF3a has three canonical tyrosine-based sorting signals (Yxx{Phi}; where x is any amino acid and {Phi} is a hydrophobic amino acid with a bulky -R group). They have been implicated in the trafficking of membrane proteins to the cell plasma membrane and to intracellular organelles. Previous studies have indicated that mutation of the 160YSNV163 motif abrogated plasma membrane expression and inhibited ORF3a-induced apoptosis. However, two additional canonical tyrosine-based sorting motifs (211YYQL213, 233YNKI236) exist in the cytoplasmic domain of ORF3a that have not been assessed. We removed all three potential tyrosine-based motifs and systematically restored them to assess the importance of each motif or combination of motifs that restored efficient trafficking to the cell surface and lysosomes. Our results indicate that the Yxx{Phi} motif at position 160 was insufficient for the trafficking of ORF3a to the cell surface. Our studies also showed that ORF3a proteins with an intact Yxx{Phi} at position 211 or at 160 and 211 were most important. We found that ORF3a cell surface expression correlated with the co-localization of ORF3a with LAMP-1 near the cell surface. These results suggest that Yxx{Phi} motifs within the cytoplasmic domain may act cooperatively in ORF3a transport to the plasma membrane and endocytosis to lysosomes. Further, our results indicate that certain tyrosine mutants failed to activate caspase 3 and did not correlate with autophagy functions associated with this protein. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.22.550032v1?rss=1 Authors: Al-Refaie, N., Padovani, F., Binando, F., Hornung, J., Zhao, Q., Towbin, B. D., Sarinay Cenik, E., Stroustrup, N., Schmoller, K. M., Cabianca, D. S. Abstract: Chromatin architecture is a fundamental mediator of genome function. Fasting is a major environmental cue across the animal kingdom. Yet, how it impacts on 3D genome organization is unknown. Here, we show that fasting induces a tissue-specific, reversible and large-scale spatial reorganization of chromatin in C. elegans. This fasting-induced 3D genome reorganization requires inhibition of the nutrient-sensing mTOR pathway, a major regulator of ribosome biogenesis. Remarkably, loss of transcription by RNA Pol I, but not RNA Pol II nor Pol III, induces a similar 3D genome reorganization in fed animals, and prevents the restoration of the fed-state architecture upon restoring nutrients to fasted animals. Our work documents the first large-scale chromatin reorganization triggered by fasting and reveals that mTOR and RNA Pol I shape genome architecture in response to nutrients. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.22.550126v1?rss=1 Authors: Pan, S., Li, X., Pan, C., Li, J., Fan, S., Zhang, L., Du, K., Du, Z., Zhang, J., Huang, H., Li, J., Zhang, H., Huang, J., Qin, Z. Abstract: Plants and their associated microbes live in complicated, changeable, and unpredictable environments. They usually interact with each other in many ways by proceeding in multidimensional, multi-scale and multi-level coupling manners, leading to challenges of the co-existence of randomness and determinism, or continuity and discreteness. Gaining a deeper understanding of these diverse interaction mechanisms can facilitate the development of new data mining theories and methods for complex systems, new coupled modelling for the system with different spatiotemporal scales and functional properties, or even universal theory of information and information interactions. In this study, we use a close-loop model to present a plant-microbe interaction system and describe the probable functions from the microbial natural products. Specifically, we report a rhizosphere species, Streptomyces ginsengnesis G7, which produces polyketide lydicamycins and other active metabolites. Interestingly, these distinct molecules have the potential to function both as antibiotics and herbicides for crop protection. Detailed laboratory experiments combined with comprehensive bioinformatics analysis allow us to rationalise a model for this specific plant-microbe interaction process. Our work reveals the benefits of exploring otherwise neglectable resources for the identification of novel functional molecules and provides a good reference to better understand the system biology in the complex ecosystems. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.21.549904v1?rss=1 Authors: Clancy, A., Rusilowicz-Jones, E. V., Wallace, I., Swatek, K. N., Urbe, S., Clague, M. J. Abstract: Type 1 interferon stimulation highly up-regulates all elements of a ubiquitin-like conjugation system that leads to ISGylation of target proteins. An ISG15-specific member of the deubiquitylase family, USP18, is up-regulated in a co-ordinated manner. USP18 can also provide a negative feedback by inhibiting JAK-STAT signaling through protein interactions independently of DUB activity. Here, we provide an acute example of this phenomenon, whereby the early expression of USP18, post-interferon treatment of HCT116 colon cancer cells is sufficient to fully suppress the expression of the ISG15 E1 enzyme, UBA7. Stimulation of lung adenocarcinoma A549 cells with interferon reduces their growth rate but they remain viable. In contrast, A549 USP18 knock-out cells show similar growth characteristics under basal conditions, but upon interferon stimulation a profound inhibition of cell growth is observed. We show that this contingency on USP18 is independent of ISGylation, suggesting non catalytic functions are required for viability. We also demonstrate that global deISGylation kinetics are very slow compared with deubiquitylation. This is not influenced by USP18 expression, suggesting that enhanced ISGylation in USP18 KO cells reflects increased conjugating activity. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.21.550059v1?rss=1 Authors: Muhammad, Z., Brown, P. W., Babazau, L., Alkhamis, A. I., Goni, B. W., Nggada, H. A., Mbaya, K. M., Wray, S., Marte, I. H., Karch, C., Serpell, L., Maina, M. B. Abstract: Genetic backgrounds contribute to cellular phenotypes, drug responsiveness, and health outcomes. However, the majority of human induced pluripotent stem cell (iPSC) lines are derived from individuals of European descent. Thus, there is a major, unmet need in the generation, characterisation, and distribution of iPSCs from diverse ancestries. To begin to address this need, we have generated iPSCs from dermal fibroblasts isolated from a healthy 60-year-old indigenous Nigerian male belonging to the Babur ethnic group. The iPSCs were generated using Sendai virus, and copy number variation (CNV) analysis revealed no new major abnormalities compared to the parental fibroblasts. The iPSCs have been characterised for pluripotency markers and morphology and successfully differentiated into neural progenitor cells and astrocytes. This iPSC line could serve as a healthy control in comparative studies and can be used in disease modelling, toxicity assessments, genetic analyses, and drug discovery processes within an African genetic background. To bolster the inclusion of African models in biomedical research, this iPSC line will be made available to the broader scientific community. Ongoing efforts focus on generating more lines from diverse indigenous populations towards creating a dedicated open-access African iPSC biobank. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.21.550021v1?rss=1 Authors: Georgiou, X., Dimou, S., Diallinas, G., Samiotaki, M. Abstract: Neosynthesized plasma membrane (PM) proteins co-translationally translocate to the ER, concentrate at regions called ER-exit sites (ERes) and pack into COPII secretory vesicles which are sorted to the early-Golgi through membrane fusion. Following Golgi maturation, membrane cargoes reach the late-Golgi, from where they exit in clathrin-coated vesicles destined to the PM, directly or through endosomes. Post-Golgi membrane cargo trafficking also involves the cytoskeleton and the exocyst. The Golgi-dependent secretory pathway is thought to be responsible for the trafficking of all major membrane proteins. However, our recent findings in Aspergillus nidulans showed that several plasma membrane cargoes, such as transporters and receptors, follow a sorting route that seems to bypass Golgi functioning. To gain insight on membrane trafficking and specifically Golgi-bypass, here we used proximity dependent biotinylation (PDB) coupled with data-independent acquisition mass spectrometry (DIA-MS) for identifying transient interactors of the UapA transporter. Our assays, which included proteomes of wild-type and mutant strains affecting ER-exit or endocytosis, identified both expected and novel interactions that might be physiologically relevant to UapA trafficking. Among those, we validated, using reverse genetics and fluorescence microscopy, that COPI coatomer is essential for ER-exit and anterograde trafficking of UapA and other membrane cargoes. We also showed that ArfAArf1 GTPase activating protein (GAP) Glo3 contributes to UapA trafficking at increased temperature. This is the first report addressing the identification of transient interactions during membrane cargo biogenesis using PDB and proteomics coupled with fungal genetics. Our work provides a basis for dissecting dynamic membrane cargo trafficking via PDB assays. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.21.550090v1?rss=1 Authors: Ackerman, J. E., Adjei-Sowah, E., Korcari, A., Muscat, S. N., Nichols, A. E., Buckley, M. R., Loiselle, A. E. Abstract: Tendon injuries are a major clinical problem, with poor patient outcomes caused by abundant scar tissue deposition during healing. Myofibroblasts play a critical role in the initial restoration of structural integrity after injury. However, persistent myofibroblast activity drives the transition to fibrotic scar tissue formation. As such, disrupting myofibroblast persistence is a key therapeutic target. While myofibroblasts are typically defined by the presence of SMA+ stress fibers, SMA is expressed in other cell types including the vasculature. As such, modulation of myofibroblast dynamics via disruption of SMA expression is not a translationally tenable approach. Recent work has demonstrated that Periostin-lineage (PostnLin) cells are a precursor for cardiac fibrosis-associated myofibroblasts. In contrast to this, here we show that PostnLin cells contribute to a transient SMA+ myofibroblast population that is required for functional tendon healing, and that Periostin forms a supportive matrix niche that facilitates myofibroblast differentiation and persistence. Collectively, these data identify the Periostin matrix niche as a critical regulator of myofibroblast fate and persistence that could be targeted for therapeutic manipulation to facilitate regenerative tendon healing. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.21.550042v1?rss=1 Authors: Rutkowski, D. M., Vincenzetti, V., Vavylonis, D., Martin, S. G. Abstract: Local Cdc42 GTPase activation promotes polarized exocytosis, resulting in membrane flows that deplete low-mobility membrane-associated proteins from the growth region. To investigate the self-organizing properties of the Cdc42 secretion-polarization system under membrane flow, we developed a reaction-diffusion particle model. The model includes positive feedback activation of Cdc42, hydrolysis by GTPase-activating proteins (GAPs), and flow-induced displacement by exo/endocytosis. Simulations show how polarization relies on flow-induced depletion of low mobility GAPs. To probe the role of Cdc42 mobility in the fission yeast Schizosaccharomyces pombe, we changed its membrane binding properties by replacing its prenylation site with 1, 2 or 3 repeats of the Rit1 C terminal membrane binding domain (ritC), yielding alleles with progressively lower unbinding and diffusion rates. Concordant modelling predictions and experimental observations show that lower Cdc42 mobility results in lower Cdc42 activation level and wider patches. Indeed, while Cdc42-1ritC cells are viable and polarized, Cdc42-2ritC polarize poorly and Cdc42-3ritC is inviable. The model further predicts that GAP depletion increases Cdc42 activity at the expense of loss of polarization. Experiments confirm this prediction, as deletion of Cdc42 GAPs restores viability to Cdc42-3ritC cells. Our combined experimental and modelling studies demonstrate how membrane flows are an integral part of Cdc42-driven pattern formation. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.21.550060v1?rss=1 Authors: Roberto, G. M., Boutet, A., Keil, S., Emery, G. Abstract: Collective cell migration occurs in various biological processes such as development, wound healing and metastasis. During Drosophila oogenesis, border cells (BC) form a cluster that migrates collectively inside the egg chamber. The Ste20-like kinase Misshapen (Msn) is a key regulator of BC migration coordinating the restriction of protrusion formation and contractile forces within the cluster. Here, we demonstrate that the kinase Tao acts as an upstream activator of Msn in BCs. Depletion of Tao significantly impedes BC migration and produces a phenotype similar to Msn loss-of-function. Furthermore, we show that the localization of Msn relies on its CNH domain, which interacts with the small GTPase Rap2l. Our findings indicate that Rap2l promotes the trafficking of Msn to the endolysosomal pathway. When Rap2l is depleted, the levels of Msn increase in the cytoplasm and at cell-cell junctions between BCs. Overall, our data suggest that Rap2l ensures that the levels of Msn are higher at the periphery of the cluster through the targeting of Msn to the degradative pathway. Together, we identified two distinct regulatory mechanisms that ensure the appropriate distribution and activation of Msn in BCs. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.21.549990v1?rss=1 Authors: Fujii, K., Kondo, T., Kimura, A. Abstract: The centrosome is a major microtubule-organizing center in animal cells. The intracellular positioning of the centrosomes is important for proper cellular function. One of the features of centrosome positioning is the spacing between centrosomes. The spacing activity is mediated by microtubules extending from the centrosomes; however, the underlying mechanisms are not fully understood. To characterize the spacing activity in the Caenorhabditis elegans embryo, a genetic setup was developed to produce enucleated embryos. The centrosome duplicated multiple times in the enucleated embryo, which enabled us to characterize the chromosome-independent spacing activity between sister and non-sister centrosome pairs. We knocked down genes in the enucleated embryo and found that the timely spacing was dependent on cytoplasmic dynein. Based on these results, we propose a stoichiometric model of cortical and cytoplasmic pulling forces for the spacing between centrosomes. We also found a dynein-independent but non-muscle myosin II-dependent movement of the centrosomes in a later cell cycle phase. The dynein-dependent spacing mechanisms for positioning the centrosomes revealed in this study is likely functioning in the cell with nucleus and chromosomes, including the processes of centrosome separation and spindle elongation. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.21.550040v1?rss=1 Authors: Reyes-Serratos, E., Santos, J. R. L., Puttagunta, L., Lewis, S., Watanabe, M., Gonshor, A., Buck, R., Befus, A. D., Marcet-Palacios, M. Abstract: Calcium binding protein, spermatid associated 1 (CABS1) is a protein most widely studied in spermatogenesis. However, mRNA for CABS1 has been found in numerous tissues, albeit with little information about the protein. Previously, we identified CABS1 mRNA and protein in human salivary glands and provided evidence that in humans CABS1 contains a heptapeptide near its carboxyl terminus that has anti-inflammatory activities. Moreover, levels of an immunoreactive form of CABS1 were elevated in psychological stress. To more fully characterize human CABS1 we developed additional polyclonal and monoclonal antibodies to different sections of the protein and used these antibodies to characterize CABS1 in an overexpression cell lysate, human salivary glands, saliva, serum and testes using western blot, immunohistochemistry and bioinformatics approaches exploiting the Gene Expression Omnibus (GEO) database. CABS1 appears to have multiple molecular weight forms, consistent with its recognition as a structurally disordered protein, a protein with structural plasticity. Interestingly, in human testes, its cellular distribution differs from that in rodents and pigs, and includes Leydig cells, primary spermatogonia, Sertoli cells and developing spermatocytes and spermatids, Geodata suggests that CABS1 is much more widely distributed than previously recognized, including in the urogenital, gastrointestinal and respiratory tracts, as well as in the nervous system, immune system and other tissues. Much remains to be learned about this intriguing protein. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.21.549976v1?rss=1 Authors: Horisawa, K., Miura, S., Araki, H., Miura, F., Ito, T., Suzuki, A. Abstract: Direct reprogramming is a technique for inducing the conversion of one type of somatic cell into another by the forced expression of defined transcription factors. Cell differentiation is generally determined by specific gene expression profiles based on distinct genome-wide epigenetic signatures. Although the CpG methylation of genomic DNA is an essential epigenetic factor that affects the transcriptional state of genes, little is known about how DNA methylation changes and what roles it plays in direct reprogramming. Here, we performed comparative genome-wide DNA methylation analyses of mouse embryonic fibroblasts (MEFs) and cells composing organoids formed by intestinal stem cells (ISCs) or induced ISCs (iISCs) that were directly induced from MEFs to investigate the impact of DNA methylation dynamics on direct reprogramming. We found that the methylation state of CpG was similar between cells forming ISC organoids and iISC organoids, while they differed widely from those in MEFs. Moreover, genomic regions that were differentially methylated between ISC organoid- and iISC organoid-forming cells did not significantly affect gene expression. These results demonstrate the accuracy and safety of iISC induction, as they show that the DNA methylation state transitions to a state close to that of ISCs during direct reprogramming from MEFs to iISCs. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.21.550091v1?rss=1 Authors: Bugga, P., Stoner, M., Manning, J., Mushala, B., Thapa, D., Scott, I. Abstract: GCN5L1, also known as BLOC1S1 and BLOS1, is a small intracellular protein involved in a number of key biological processes. Over the last decade, GCN5L1 has been implicated in the regulation of protein lysine acetylation, energy metabolism, endo-lysosomal function, and cellular immune pathways. An increasing number of published papers have used commercially-available reagents to interrogate GCN5L1 function. However, in many cases these reagents have not been rigorously validated, leading to potentially misleading results. In this report we tested several commercially-available antibodies for GCN5L1, and found that two-thirds of those available did not unambiguously detect the protein by western blot in cultured mouse cells or ex vivo liver tissue. These data suggest that previously published studies which used these unverified antibodies to measure GCN5L1 protein abundance, in the absence of other independent methods of corroboration, should be interpreted with appropriate caution. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.21.549845v1?rss=1 Authors: Jaulim, A., Cassidy, L. D., Young, A. R. J., Chan, A. S. L., Warren, A. Y., Taylor, A. E., Arlt, W., Lan, G., Blayney, M. L., Davidson, O., Barratt, C. L. R., Pacey, S., Narita, M. Abstract: Autophagy has been implicated in male fertility but its specific role in the post-testicular organs remains unclear. Here, we investigate this in mice expressing a doxycycline-inducible RNAi against Atg5 (Atg5i). Systemic autophagy inhibition in Atg5i mice resulted in the morphological and functional abrogation of the male accessory sex organs, leading to male subfertility. However, the testis was largely protected, likely due to the limited permeability of doxycycline through the blood-testis barrier. Interestingly, restoration of autophagy by doxycycline withdrawal in Atg5i mice led to substantial recovery of the phenotype in the accessory organs. This model offers a unique opportunity to dissect the pre- and post-testicular roles of autophagy, highlighting the non-autonomous impact of autophagy on male fertility. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.19.549752v1?rss=1 Authors: Dong, B., Mahapatra, S., Clark, M. G., Carlsen, M., Mohn, K. J., Ma, S., Brasseale, K., Crim, G., Zhang, C. Abstract: Controlling chemical processes in live cells is a challenging task. The spatial heterogeneity of biochemical reactions in cells is often overlooked by conventional means of incubating cells with desired chemicals. A comprehensive understanding of spatially diverse biochemical processes requires precise control over molecular activities at the subcellular level. Herein, we develop a closed-loop optoelectronic control system that allows the manipulation of biomolecular activities in live cells at high spatiotemporal precision. Chemical-selective fluorescence signals are utilized to command lasers that trigger specific chemical reactions or control the activation of photoswitchable inhibitors at desired targets. We demonstrate the capability to selectively produce reactive oxygen species (ROS) solely at targeted organelles using blue light. Notably, the induction of ROS in the endoplasmic reticulum leads to a more pronounced disruption of tubulin polymerization and a reduction in green fluorescent protein signals, in comparison to that in lipid droplets. Moreover, when combined with a photoswitchable inhibitor, we selectively inhibit tubulin polymerization within subcellular compartments. This technology enables spatiotemporal control over chemical processes and drug activities, exclusively at desired targets, while minimizing undesired effects on non-targeted locations. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.20.549847v1?rss=1 Authors: Aarts, M. T., Wagner, M., van der Wal, T., van Boxtel, A. L., van Amerongen, R. Abstract: Progesterone receptor (PR) signaling is required for mammary gland development and homeostasis. A major bottleneck in studying PR signaling is the lack of sensitive assays to measure and visualize PR pathway activity both quantitatively and spatially. Here, we develop new tools to study PR signaling in human breast epithelial cells. First, we generate optimized Progesterone Responsive Element (PRE)-luciferase constructs and demonstrate that these new reporters are a powerful tool to quantify PR signaling activity across a wide range of progesterone concentrations in two luminal breast cancer cell lines, MCF7 and T47D. We also describe a fluorescent lentiviral PRE-GFP reporter as a novel tool to visualize PR signaling at the single-cell level. Our reporter constructs are sensitive to physiological levels of progesterone. Second, we show that low background signaling, and high levels of PR expression are a prerequisite for robustly measuring PR signaling. Increasing PR expression by transient transfection, stable overexpression in MCF7 or clonal selection in T47D, drastically improves both the dynamic range of luciferase reporter assays, and the induction of endogenous PR target genes as measured by qRT-PCR. We find that the PR signaling response differs per cell line, target gene and hormone concentration used. Taken together, our tools allow a more rationally designed approach for measuring PR signaling in breast epithelial cells. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.20.548338v1?rss=1 Authors: Li, M., Wang, Y., Wei, X., Cai, W.-F., Zhu, M., Yao, L., Wang, Y., Liu, Y.-H., Wu, J., Xiong, J., Tian, X., Qu, Q., Xie, R., Li, X., Chen, S., Huang, X., Zhang, C., Xie, C., Wu, Y., Xu, Z., Zhang, B., Jiang, B., Yu, Y., Wang, Z.-C., Li, Q., Li, G., Lin, S.-Y., Yu, L., Piao, H.-l., Deng, X., Zhang, C.-S., Lin, S.-C. Abstract: The shift of carbon utilisation from glucose to other nutrients is a fundamental metabolic adaptation to cope with the decreased glucose oxidation during fasting or starvation. AMP-activated protein kinase (AMPK) plays crucial roles in manifesting physiological benefits accompanying glucose starvation or calorie restriction. However, the underlying mechanisms are unclear. Here, we show that low glucose-induced activation of AMPK plays a decisive role in the shift of carbon utilisation from glucose to glutamine. We demonstrate that endoplasmic reticulum (ER)-localised PDZD8, which we identify to be a new substrate of AMPK, is required for the glucose starvation-promoted glutaminolysis. AMPK phosphorylates PDZD8 at threonine 527 (T527), and promotes it to interact with and activate the mitochondrial glutaminase 1 (GLS1), a rate-limiting enzyme of glutaminolysis, and as a result the ER-mitochondria contact is strengthened. In vivo, PDZD8 enhances glutaminolysis, and triggers mitohormesis that is required for extension of lifespan and healthspan in Caenorhabditis elegans subjected to glucose starvation or caloric restriction. Muscle-specific re-introduction of wildtype PDZD8, but not the AMPK-unphosphorylable PDZD8-T527A mutant, to PDZD8-/- mice is able to rescue the increase of glutaminolysis, and the rejuvenating effects of caloric restriction in aged mice, including grip strength and running capacity. Together, these findings reveal an AMPK-PDZD8-GLS1 axis that promotes glutaminolysis and executes the anti-ageing effects of calorie restriction by promoting inter-organelle crosstalk between ER and mitochondria. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.19.549095v1?rss=1 Authors: Lyu, Z., Genereux, J. C. Abstract: Proximity labeling is a powerful approach for characterizing subcellular proteomes. We recently demonstrated that proximity labeling can be used to identify mistrafficking of secretory proteins, such as occurs during pre-emptive quality control (pre-QC) following endoplasmic reticulum (ER) stress. This assay depends on protein quantification by immunoblotting and densitometry, which is only semi-quantitative and suffers from poor sensitivity. Here, we integrate parallel reaction monitoring mass spectrometry to enable a more quantitative platform for ER import. PRM as opposed to densitometry improves quantification of transthyretin mistargeting while also achieving at least a ten-fold gain in sensitivity. The multiplexing of PRM also enabled us to evaluate a series of normalization approaches, revealing that normalization to auto-labeled APEX2 peroxidase is necessary to account for drug treatment-dependent changes in labeling efficiency. We apply this approach to systematically characterize the relationship between chemical ER stressors and ER pre-QC induction in HEK293T cells. Using dual-FLAG-tagged transthyretin (FLAGTTR) as a model secretory protein, we find that Brefeldin A treatment as well as ER calcium depletion cause pre-QC, while tunicamycin and dithiothreitol do not, indicating ER stress alone is not sufficient. This finding contrasts with the canonical model of pre-QC induction, and establishes the utility of our platform. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.20.549453v1?rss=1 Authors: Telerman, S. B., Hamilton, R. S., Shaw, B., Dimitrov, J. D., Steventon, B., Ferguson-Smith, A. C. Abstract: Perturbations in fundamental developmental pathways have a profound influence on tumorigenesis. Numb plays a pivotal role in vertebrate development, including neurogenesis and is a key negative regulator of Notch signaling. Perturbation of Numb expression affects brain morphology and cell fate. While extensive research has been conducted on canonical Notch ligands, regulation by vertebrate-specific non-canonical ligands is not understood. Here we identify Delta like non-canonical Notch ligand 2/EGFL9 (Dlk2) as a regulator of zebrafish neurogenesis with mutants exhibiting early increase and subsequent depletion of neural stem cells, decreased radial glial cells density, impaired neuronal cell distribution, and hypersensitivity to stimuli mimicking the embryonic murine Numb/Numblike null phenotype. Numb function is inactivated by aberrant phosphorylation, and we show that Dlk2 protein exhibits a high affinity direct interaction with Numb, with loss of Dlk2 in zebrafish telencephalon increasing Numb Ser276 phosphorylation with a concomitant increase in Notch signaling. Patients with tumors exhibiting reduced levels of Dlk2 have a poorer prognosis, while overexpression of Dlk2 in human cancer cell lines reduces cell proliferation. Our findings identify Dlk2 as a key partner of Numb, a gatekeeper of its activity, and an important player in a network of protein interactions regulating both neurogenesis and cancer with potential therapeutic implications. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.20.549860v1?rss=1 Authors: Link, F., Borges, A., Karo, O., Jungblut, M., Mueller, T., Meyer-Natus, E., Krueger, T., Sachs, S., Jones, N. G., Morphew, M., Sauer, M., Stigloher, C., McIntosh, J. R., Engstler, M. Abstract: Endocytosis is a common process observed in all eukaryotic cells, although its complexity varies among different organisms. In Trypanosoma brucei, the endocytic machinery is under special selective pressure because rapid membrane recycling is essential for immune escape. This unicellular parasite effectively removes host antibodies from its cell surface through hydrodynamic drag and fast endocytic internalization. The entire process of membrane recycling occurs exclusively through the flagellar pocket, an extracellular organelle situated at the posterior pole of the spindle-shaped cell. The high-speed dynamics of membrane flux in trypanosomes do not seem compatible with the conventional concept of distinct compartments for early, late and recycling endosomes. To investigate the underlying structural basis for the remarkably efficient membrane traffic in trypanosomes, we employed advanced techniques in light and electron microscopy to examine the three-dimensional architecture of the endosomal system. Our findings reveal that the endosomal system in trypanosomes exhibits a remarkably intricate structure. Instead of being compartmentalized, it constitutes a continuous membrane system, with specific functions of the endosome segregated into membrane subdomains enriched with classical markers for early, late, and recycling endosomes. These membrane subdomains can partly overlap or are interspersed with areas that are negative for endosomal markers. This continuous endosome allows fast membrane flux by facilitated diffusion that is not slowed by multiple fission and fusion events. Our study further suggests that in trypanosomes, the endosome has taken over the role of the trans-Golgi network as the master regulator of membrane trafficking. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.19.549693v1?rss=1 Authors: Zhu, S., Waeckel-Enee, E., Moser, A., Bessard, M.-A., Roger, K., Lipecka, J., Yilmaz, A., Bertocci, B., Diana, J., Saintpierre, B., Guerrera, I. C., Francesconi, S., Mauvais, F.-X., van Endert, P. Abstract: Appropriate tuning of protein homeostasis through mobilization of the unfolded protein response (UPR) is key to the capacity of pancreatic beta cells to cope with highly variable demand for insulin synthesis. An efficient UPR ensures a sufficient beta cell mass and secretory output but can also affect beta cell resilience to autoimmune aggression. The factors regulating protein homeostasis in the face of metabolic and immune challenges are insufficiently understood. We examined beta cell adaptation to stress in mice deficient for insulin-degrading enzyme (IDE), a ubiquitous protease with high affinity for insulin and genetic association with type 2 diabetes. IDE deficiency induced a low-level UPR in both C57BL/6 and autoimmune non-obese diabetic (NOD) mice, associated with rapamycin-sensitive beta cell proliferation strongly enhanced by proteotoxic stress. Moreover, in NOD mice, IDE deficiency protected from spontaneous diabetes and triggered an additional independent pathway, conditional on the presence of islet inflammation but inhibited by proteotoxic stress, highlighted by strong upregulation of regenerating islet-derived protein 2, a protein attenuating autoimmune inflammation. Our findings establish a key role of IDE in islet cell protein homeostasis, identify a link between low-level UPR and proliferation, and reveal an UPR-independent anti-inflammatory islet cell response uncovered in the absence of IDE of potential interest in autoimmune diabetes. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.19.549646v1?rss=1 Authors: Bergmann, T. C., Menssen, M., Schotte, C., Cox, R. J., Lee-Thedieck, C. Abstract: Fungal specialized metabolites often exhibit a wide range of potent bioactivities, with tropolone sesquiterpenoids (TS) representing a promising family with a variety of known bioactive properties. Here, we investigated bioactive properties of the novel TS compounds 4- hydroxyxenovulene B 4, 4-dehydroxy-norpycnidione 5 and the structurally-related compounds 4-hydroxy-norxenovulene B 6, and xenovulene B 3, for which no bioactive effects have been reported yet. We detected compound-specific changes in cell shapes and ramification patterns that were strongly correlated in samples treated with structurally-similar compounds and related to the tropolone moieties present on the molecule. The number of tropolone moieties per molecule had a significant influence on survival of FAIK3-5 cells, with an increased cytotoxic effect observed with the dual-substituted compounds compared to the monotropolones. Treatment with eupenifeldin 2, compound 4 and 5 demonstrated versatile influences on cellular behaviour and can reduce EPO content in hypomethylated FAIK3-5 cells. Monotropolone 5 showed significantly reduced proliferation with low cytotoxic effects and could serve as a potential compound for further (pre-)clinical drug testing for cancer treatment. Our results bring us one step closer to linking the molecular structures of bioactive TS compounds to their biological effects and may improve their potential for clinical applications. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.19.549701v1?rss=1 Authors: Ralliere, C., Jagot, S., Sabin, N., Gabillard, J.-C. Abstract: Essential for muscle fiber formation and hypertrophy, muscle stem cells, also called satellite cells, reside beneath the basal lamina of the muscle fiber. Satellite cells have been commonly localized by the expression of the Paired box 7 (Pax7) due to its specificity and the availability of antibodies in tetrapods. In fish, the identification of satellite cells remains difficult due to the lack of specific antibodies in most species. Based on the development of a highly sensitive in situ hybridization (RNAScope) for pax7, we showed that pax7+ cells were detected in the undifferentiated myogenic epithelium corresponding to the dermomyotome at day 14 post-fertilization. Then, from day 24, pax7+ cells gradually migrated into the deep myotome and were localized along the muscle fibers and reach their niche in satellite position of the fibres after hatching. Our results showed that 18 days after muscle injury, a large number of pax7+ cells accumulated at the wound site compared to the uninjured area. During the in vitro differentiation of satellite cells, the percentage of pax7+ cells decreased from 44% to 18% on day 7, and some differentiated cells still expressed pax7. Taken together, these results show the dynamic expression of pax7 genes and the follow-up of these muscle stem cells during the different situations of muscle fiber formation in trout. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.18.548845v1?rss=1 Authors: Penjweini, R., Pasut, A., Roarke, B., Alspaugh, G., Sackett, D. L., Knutson, J. R. Abstract: Molecular oxygen (O2) is one of the most functionally relevant metabolites. O2 is essential for mito-chondrial aerobic respiration. Changes in O2 affect muscle metabolism and play a critical role in the maintenance of skeletal muscle mass, with lack of sufficient O2 resulting in detrimental loss of muscle mass and function. How exactly O2 is used by muscle cells is less known, mainly due to the lack of tools to address O2 dynamics at the cellular level. Here we discuss a new imaging method for the real time quantification of intracellular O2 in muscle cells based on a genetically encoded O2-responsive sensor, Myoglobin-mCherry. We show that we can spatially resolve and quantify intracellular O2 con-centration in single muscle cells and that the spatiotemporal O2 gradient measured by the sensor is linked to, and reflects, functional metabolic changes occurring during the process of muscle differentia-tion. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.18.549609v1?rss=1 Authors: Choi, I., Jeong, J. Abstract: Tjp1 + is considered a crucial protein involved in the stepwise assembly of tight junctions (TJs) between compaction and blastocoel cavitation in early development. In this study, we investigated the specific role of Tjp1 + in TJ formation by employing an alternative splicing-specific knockdown of the Tjp1 + exon. To deplete Tjp1 + expression, we used siRNA targeting RNA-binding protein 47 (Rbm47), which induces the inclusion of the + exon in Tjp1 mRNA. The knockdown resulted in approximately 85% reduction in Rbm47 mRNA levels and 75% reduction in Tjp1 + mRNA levels in blastocysts. Surprisingly, despite this knockdown, blastocyst development and TJ permeability of trophectoderm were unaffected. Additionally, we observed an interaction between Tjp1 - and Ocln in Rbm47 knockdown blastocysts, suggesting a compensatory role of Tjp1 -. Overall, our findings indicate that Tjp1 + is not essential for the stepwise assembly of TJs and the completion of TJ biogenesis during blastocyst development in mice although a minimal amount of remaining Tjp1 + is sufficient for TJs assembly. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.18.549588v1?rss=1 Authors: Barman, B., Ramirez, M., Liu, Q., Weaver, A. Abstract: Extracellular vesicles (EVs) influence cell phenotypes and functions via protein, nucleic acid and lipid cargoes. EVs are heterogeneous, due to diverse biogenesis mechanisms that remain poorly understood. Our previous study revealed that the endoplasmic reticulum (ER) membrane contact site (MCS) linker protein VAP-A drives biogenesis of a subset of RNA-enriched EVs. Here, we examine the protein content of VAP-A-regulated EVs. Using label-free proteomics, we identified down- and up-regulated proteins in sEVs purified from VAP-A knockdown (KD) colon cancer cells. Gene set enrichment analysis (GSEA) of the data revealed protein classes that are differentially sorted to SEVs dependent on VAP-A. STRING protein-protein interaction network analysis of the RNA-binding protein (RBP) gene set identified several RNA functional machineries that are downregulated in VAP-A KD EVs, including ribosome, spliceosome, mRNA surveillance, and RNA transport proteins. We also observed downregulation of other functionally interacting protein networks, including cadherin-binding, unfolded protein binding, and ATP-dependent proteins. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.18.549529v1?rss=1 Authors: Rueda, A. D., Salvador-Martinez, I., Sospedra-Arrufat, I., Alcaina-Caro, A., Fernandez-Minan, A., Burgos-Ruiz, A. M., Cases, I., Mohedano, A., Tena, J. J., Heyn, H., Lopez-Rios, J., Nusspaumer, G. Abstract: The cellular complexity of the endochondral bone underlies its essential and pleiotropic roles during organismal life. While the adult bone has received significant attention, we still lack a deep understanding of the perinatal bone cellulome. Here, we have profiled the full composition of the murine endochondral bone at the single-cell level during the transition from fetal to newborn life and in comparison to the adult organ, with particular emphasis on the mesenchymal compartment. The perinatal bone contains different fibroblastic clusters with blastema-like characteristics in organizing and supporting skeletogenesis, angiogenesis, and hematopoiesis. Our data also points out to a dynamic inter- and intra-compartment interactions as well as a bone marrow milieu prone to anti-inflammation, which we hypothesize is necessary to ensure the proper program of lymphopoiesis and the establishment of central and peripheral tolerance in early life. Our study provides a integrative roadmap for the future design of genetic and cellular functional assays to validate cellular interactions and lineage relationships within the perinatal bone. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.18.549571v1?rss=1 Authors: Chang, L., Chen, Q., Wang, B., Liu, J., Zhang, M., Zhu, W., Jiang, J. Abstract: The evolution and development of vertebrate lungs have received extensive concerns for the significance in terrestrial adaptation. Amphibians possess the most primitive lungs among tetrapods, underscoring the evolutionary importance of lungs in bridging the transition from aquatic to terrestrial life. However, the intricate process of cell differentiation during amphibian lung development is still lacking. Using single cell RNA-seq, we identified 21 cell types in the developing lung of a land-dwelling frog (Microhyla fissipes). We elucidated that single type of alveolar epithelial cells (AECs) existed in amphibian and the diversity of AECs may correspond to the capacity for terrestrial adaptation in tetrapods. Based on pseudotime trajectories analysis, we revealed previously unrecognized developmental-specific transition cell states of epithelial and endothelial cells supporting the rapid morphogenesis of lung during metamorphic climax. We illustrated the cellular and molecular processes during lung functionalization. These findings uncover the cell differentiation trajectories and functionalization for air breathing of frog lungs and highlight the evolutionary peculiarity of the primitive lungs. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.19.549670v1?rss=1 Authors: Stratiievska, A., Filippova, O., Ozpolat, T., Byrne, D., Bailey, S. L., Mollica, M. Y., Harris, J., Esancy, K., Chen, J., Dhaka, A., Sniadecki, N. J., Lopez, J. A., Stolla, M. Abstract: Platelets are sensitive to temperature changes and akin to sensory neurons, are activated by a decrease in temperature. However, the molecular mechanism of this temperature-sensing ability is unknown. Yet, platelet activation by temperature could contribute to numerous clinical sequelae, most importantly to reduced quality of ex vivo-stored platelets for transfusion. In this interdisciplinary study, we present evidence for the expression of the temperature-sensitive ion channel transient receptor potential cation channel subfamily member 8 (TRPM8) in human platelets and precursor cells. We found the TRPM8 mRNA and protein in MEG-01 cells and platelets. Inhibition of TRPM8 prevented temperature-induced platelet activation and shape change. However, chemical agonists of TRPM8 did not seem to have an acute effect on platelets. When exposing platelets to below-normal body temperature, we detected a cytosolic calcium increase which was independent of TRPM8 but was completely dependent on the calcium release from the endoplasmic reticulum. Because of the high interindividual variability of TRPM8 expression, a population-based approach should be the focus of future studies. Our study suggests that the cold response of platelets is complex and TRPM8 appears to play a role in early temperature-induced activation of platelets, while other mechanisms likely contribute to later stages of temperature-mediated platelet response. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.17.549243v1?rss=1 Authors: Tran, H., Frank, D., Bergamasco, M., Mlodzianoski, M., Kueh, A., Tsui, E., Hall, C., Kastrappis, G., Voss, A. K., McLean, C., Faux, M. C., Rogers, K., Tran, B., Vincan, E., Komander, D., Dewson, G. Abstract: Trabid/ZRANB1 missense mutations have been identified in children diagnosed with a range of congenital disorders including reduced brain size, but how Trabid regulates neurodevelopment is not understood. We have characterised these patient mutations in cells and mice to identify a key role for Trabid in the regulation of neurite growth. One of the patient mutations flanked the catalytic cysteine of Trabid and its deubiquitylating (DUB) activity was abrogated. The second variant retained DUB activity, but failed to bind STRIPAK, a large multiprotein assembly implicated in cytoskeleton organisation and neural development. Trabid/ZRANB1 knock-in mice harbouring either of these patient mutations exhibited reduced neuronal and glial cell densities in the brain and a motor deficit consistent with fewer dopaminergic neurons and projections. Mechanistically, both DUB-impaired and STRIPAK-binding-deficient Trabid variants impeded the trafficking of adenomatous polyposis coli (APC) to microtubule plus-ends. Consequently, the formation of neuronal growth cones and the trajectory of neurite outgrowth from mutant midbrain progenitors were severely compromised. We propose that STRIPAK recruits Trabid to deubiquitylate APC, and that in cells with mutant Trabid, APC becomes hyperubiquitylated and mislocalised causing impaired organisation of the cytoskeleton that underlie the neuronal and developmental phenotypes. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.19.548188v1?rss=1 Authors: Chai, Y., Li, D., Gong, W., Ke, J., Tian, D., Chen, Z., Guo, A., Guo, Z., Li, W., Feng, W., Ou, G. Abstract: KIF1A, a microtubule-based motor protein responsible for axonal transport, is linked to a group of neurological disorders known as KIF1A-associated neurological disorder (KAND). Current therapeutic options for KAND are limited. Here, we introduced the clinically relevant KIF1A(R11Q) variant into the C. elegans homolog UNC-104, resulting in uncoordinated animal behaviors. Through genetic suppressor screens, we identified intragenic mutations in UNC-104's motor domain that rescued synaptic vesicle localization and coordinated movement. We showed that two suppressor mutations partially recovered motor activity in vitro by counteracting the structural defect caused by R11Q at KIF1A's nucleotide-binding pocket. We found that supplementation with fisetin, a plant flavonol, improved KIF1A(R11Q) worms' movement and morphology. Notably, our biochemical and single-molecule assays revealed that fisetin directly restored the ATPase activity and processive movement of human KIF1A(R11Q) without affecting wild-type KIF1A. These findings suggest fisetin as a potential intervention for enhancing KIF1A(R11Q) activity and alleviating associated defects in KAND. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.19.549635v1?rss=1 Authors: Marhuenda, E., Xanthis, I., Pandey, P., Azad, A., Richter, M., Pavlovic, D., Gehmlich, K., Faggian, G., Ehler, E., Levitt, J., Ameer-Beg, S., Iskratsch, T. Abstract: Mechanical properties are cues for many biological processes in health or disease. Likewise, in the heart it is becoming clearer that mechanical signals are critically involved in the disease progression. Cardiomyocytes sense the mechanical properties of their environment at costameres through integrins and associated proteins, including the mechanosensitive protein talin as an integral component. Our previous work indicated different modes of talin tension, depending on the extracellular matrix stiffness. Here, we wanted to study how this leads to downstream mechanotransduction changes, further influencing the cardiomyocyte phenotype. Combining immunoprecipitations and Fluorescence Recovery after Photobleaching (FRAP) experiments, we identify that the talin interacting proteins DLC1, RIAM and paxillin each preferentially bind to talin at specific extracellular matrix stiffness and this interaction is preserved even in absence of tension. This demonstrates a mechanical memory, which we confirm further in vivo in mouse hearts. The mechanical memory is regulated through adhesion related kinase pathways. Optogenetic experiments using the LOVTRAP systems confirm direct competition between the individual proteins, which again is altered through phosphorylation. DLC1 regulates RhoA activity in a stiffness dependent way and both loss and overexpression of DLC1 results in myofibrillar disarray. Together the study demonstrates a mechanism of imprinting mechanical information into the talin-interactome to finetune RhoA activity, with impacts on cardiac health and disease. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.18.549581v1?rss=1 Authors: Nguyen, V., Kravitz, J., Gao, C., Hochman, M. L., Meng, D., Chen, D., Wang, Y., Jegga, A. G., Nelson, S., Tan, W. Abstract: Port Wine Birthmark (PWB) is a congenital vascular malformation in the skin, occurring in 1-3 per 1,000 live births. We recently generated PWB-derived induced pluripotent stem cells (iPSCs) as clinically relevant disease models. The metabolites associated with the pathological phenotypes of PWB-derived iPSCs are unknown, which we aimed to explore in this study. Metabolites were separated by ultra-performance liquid chromatography and were screened with electrospray ionization mass spectrometry. Orthogonal partial least-squares discriminant analysis, multivariate and univariate analysis were used to identify differential metabolites (DMs). KEGG analysis was used for the enrichment of metabolic pathways. A total of 339 metabolites were identified. There were 22 DMs confirmed with 9 downregulated DMs including sphingosine and 13 upregulated DMs including glutathione in PWB iPSCs as compared to controls. Pathway enrichment analysis confirmed the upregulation of glutathione and downregulation of sphingolipid metabolism in PWB-derived iPSCs as compared to normal ones. We next examined the expression patterns of the key factors associated with glutathione metabolism in PWB lesions. We found that hypoxia-inducible factor 1 (HIF1), glutathione S-transferase Pi 1 (GSTP1), {gamma}-glutamyl transferase 7 (GGT7), and glutamate cysteine ligase modulatory subunit (GCLM) were upregulated in PWB vasculatures as compared to blood vessels in normal skins. Our data demonstrate that there are perturbations in sphingolipid and cellular redox homeostasis in the PWB vasculature, which may facilitate cell survival and pathological progression. Our data imply that upregulation of glutathione may contribute to laser-resistant phenotypes in the PWB vasculature. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.19.549730v1?rss=1 Authors: Peebles, K. E., LaFever, K. S., Page-McCaw, P. S., Colon, S., Wang, D., Stricker, A. M., Ferrell, N., Bhave, G., Page-McCaw, A. Abstract: Basement membranes are thin strong sheets of extracellular matrix. They provide mechanical and biochemical support to epithelia, muscles, nerves, and blood vessels, among other tissues. The mechanical properties of basement membranes are conferred in part by Collagen IV (Col4), an abundant protein of basement membrane that forms an extensive two-dimensional network through head-to-head and tail-to-tail interactions. After the Col4 network is assembled into a basement membrane, it is crosslinked by the matrix-resident enzyme Peroxidasin to form a large covalent polymer. Peroxidasin and Col4 crosslinking are highly conserved, indicating they are essential, but homozygous mutant mice have mild phenotypes. To explore the role of Peroxidasin, we analyzed mutants in Drosophila, including a newly generated catalytic null, and found that homozygotes were mostly lethal with 13% viable escapers. A Mendelian analysis of mouse mutants shows a similar pattern, with homozygotes displaying ~50% lethality and ~50% escapers. Despite the strong mutations, the homozygous escapers had low but detectable levels of Col4 crosslinking, indicating that inefficient alternative mechanisms exist and that are probably responsible for the viable escapers. Further, fly mutants have phenotypes consistent with a decrease in stiffness. Interestingly, we found that even after adult basement membranes are assembled and crosslinked, Peroxidasin is still required to maintain stiffness. These results suggest that Peroxidasin crosslinking may be more important than previously appreciated. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.18.549463v1?rss=1 Authors: de Jaime Soguero, A., Hattemer, J., Haas, A., Bufe, A., Di Marco, B., Bohly, N., Landry, J. J. M., Schoell, B., Rosa, V. S., Villacorta, L., Baskan, Y., Androulaki, S., Trapp, M., Benes, V., Das, B., Shahbazi, M., Jauch, A., Engel, U., Patrizi, A., Sotillo, R., Bageritz, J., Alfonso, J., Bastians, H., Acebron, S. P. Abstract: The development and homeostasis of organisms rely on the correct replication, maintenance and segregation of their genetic blueprints. How these intracellular processes are monitored across generations of different human cellular lineages, and why the spatio-temporal distribution of mosaicism varies during development remain unknown. Here, we identify several lineage specification signals that regulate chromosome segregation fidelity in both human and mouse pluripotent stem cells. Through epistatic analyses, we find that that WNT, BMP and FGF form a signalling rheostat upstream of ATM that monitors replication fork velocity, origin firing and DNA damage during S-phase in pluripotency, which in turn controls spindle polymerisation dynamics and faithful chromosome segregation in the following mitosis. Cell signalling control of chromosome segregation fidelity declines together with ATM activity after pluripotency exit and specification into the three human germ layers, or further differentiation into meso- and endoderm lineages, but re-emerges during neuronal lineage specification. In particular, we reveal that a tug-of-war between FGF and WNT signalling in neural progenitor cells results in DNA damage and chromosome missegregation during cortical neurogenesis, which could provide a rationale for the high levels of mosaicism in the human brain. Our results highlight a moonlighting role of morphogens, patterning signals and growth factors in genome maintenance during pluripotency and lineage specification, which could have important implications for our understanding on how mutations and aneuploidy arise during human development and disease. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.17.549432v1?rss=1 Authors: Mohammed, T. O., Lin, Y.-R., Weissenbruch, K., Ngo, K. X., Zhang, Y., Kodera, N., Bastmeyer, M., Miyanari, Y., Taoka, A., Franz, C. M. Abstract: S100A11 is a small Ca2+-activatable protein with an established role in different cellular processes involving actin cytoskeleton remodeling, such as cell migration, membrane protrusion formation, and plasma membrane repair. It also displays Ca2+-dependent F-actin binding activity and localizes to actin stress fibers (SFs), but its precise role in regulating these structures remains unclear. Analyzing endogenous S100A11 localization in HeLa and U2OS osteosarcoma cells confirmed SF association but in addition revealed steady localization to stable focal adhesions (FAs), typically at the end of dorsal stress fibers. In contrast, S100A11 levels at FAs increased sharply, but transiently, at the onset of peripheral FA disassembly. Elevating intracellular Ca2+ levels using the Ca2+ ionophore ionomycin reliably stimulated both S100A11 recruitment and subsequent FA disassembly. However, pre-incubation with the non-muscle myosin II (NM II) inhibitor blebbistatin, or with an inhibitor to the stretch-activatable Ca2+ channel Piezo1 effectively suppressed S100A11 recruitment, implicating S100A11 in an actomyosin contractility-driven FA disassembly mechanism involving Piezo1-dependent Ca2+ influx. Applying external mechanical forces on peripheral FAs via a micropipette likewise recruited S100A11 to FAs, even when NM II was inhibited by blebbistatin or in NM IIA knockout cells, corroborating the mechanosensitive recruitment mechanism of S100A11. However, extracellular Ca2+ and Piezo1 function was still indispensable, indicating that NM II-dependent contraction forces act upstream of Piezo1-mediated Ca2+ influx, in turn leading to S100A11 activation and FA recruitment. Moreover, S100A11 knockout cells feature enlarged FAs and display delayed FA disassembly during cell membrane retraction, consistent with impaired FA turnover in these cells. Our results thus demonstrate a novel mechano-sensitive function for S100A11 in promoting actomyosin contractility-driven FA disassembly. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.18.549509v1?rss=1 Authors: Cacho-Navas, C., Lopez-Pujante, C., Reglero-Real, N., Colas-Algora, N., Cuervo, A., Conesa, J. J., Barroso, S., Ciordia, S., Paradela, A., D'Agostino, G., Manzo, C., Feito, J., Andres, G., Correas, I., Carazo, J. M., Nourshargh, S., Huch, M., Millan, J. Abstract: Epithelial Intercellular Adhesion Molecule (ICAM)-1 is apically polarized, interacts with and guides leukocytes across epithelial barriers. Polarized hepatic epithelia organize their apical membrane domain into bile canaliculi and ducts, which are not accessible to circulating immune cells but that nevertheless confine most of ICAM-1. Here, by analyzing ICAM-1_KO human hepatic cells, liver organoids from ICAM-1_KO mice and rescue-of-function experiments, we show that ICAM-1 regulates epithelial apicobasal polarity in a leukocyte adhesion-independent manner. ICAM-1 signals to an actomyosin network at the base of canalicular microvilli, thereby controlling the dynamics and size of bile canalicular-like structures (BCs). We identified the scaffolding protein EBP50/NHERF1/SLC9A3R1, which connects membrane proteins with the underlying actin cytoskeleton, in the proximity interactome of ICAM-1. EBP50 and ICAM-1 form nano-scale domains that overlap in microvilli, from which ICAM-1 regulates EBP50 nano-organization. Indeed, EBP50 expression is required for ICAM-1-mediated control of BC morphogenesis and actomyosin. Our findings indicate that ICAM-1 regulates the dynamics of epithelial apical membrane domains beyond its role as a heterotypic cell-cell adhesion molecule and reveal potential therapeutic strategies for preserving epithelial architecture during inflammatory stress. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.18.549413v1?rss=1 Authors: Sun, Y., Guo, C., Chen, Z., Lin, J., Yang, L., Zhang, Y., Wu, C., Zhao, D., Jardin, B., Pu, W., Zhao, M., Dong, E., Hu, X., Zhang, S., Guo, Y. Abstract: Aims: Recombinant adeno-associated viruses (rAAVs) are federally approved gene delivery vectors for in vivo gene supplementation therapy. Loss-of-function truncating variants of LMNA, the coding gene for Lamin-A/C, are one of the primary causes of inherited dilate cardiomyopathy (DCM). Here we aim to study whether AAV-based LMNA supplementation could treat LMNA deficiency-triggered cardiac defects. Methods and Results: We compared whole-body, cardiomyocyte-specific and genetic-mosaic mouse models that carry Lmna truncating variants at the same genetic loci and uncovered primarily a non-cell autonomous impact of Lmna on cardiomyocyte maturation. Whole-body lamin-A supplementation by rAAVs moderately rescued the cardiac defects in Lmna germline mutants. By contrast, cardiomyocyte-specific lamin-A addback failed to restore the cardiomyocyte growth defects. A Cre-loxP-based AAV vector that expresses lamin-A throughout the body but excluding the heart was able to restore cardiomyocyte growth in Lmna germline mutants. Conclusions: Lmna regulates cardiomyocyte growth non-cell autonomously. Non-myocytes are the key cell targets for a successful gene therapy for LMNA-associated cardiac defects. Translational perspective: LMNA truncating mutations are among the major causes of inherited DCM. AAV gene supplementation therapy is emerging as a promising strategy to treat genetic cardiomyopathy, but whether this strategy is suitable for LMNA cardiomyopathy remained unclear. Our study counterintuitively showed that the cardiomyocytes are not necessarily the correct therapeutic cell targets for AAV-based treatment of LMNA cardiomyopathy. By contrast, careful elucidation of cell-autonomous versus non-cell-autonomous gene functions is essential for the proper design of a gene supplementation therapy for cardiomyopathy. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.16.549116v1?rss=1 Authors: Li, B., Rodrigo-Torres, D., Pelz, C., Innes, B. T., Canaday, P., Chai, S., Zandstra, P., Bader, G., Grompe, M. Abstract: In solid tissues homeostasis and regeneration after injury involve a complex interplay between many different cell types. The mammalian liver harbors numerous epithelial and non-epithelial cells and little is known about the global signaling networks that govern their interactions. To better understand the hepatic cell network, we isolated and purified 10 different cell populations from normal and regenerative mouse livers. Their transcriptomes were analyzed by bulk RNA-seq and a computational platform was used to analyze the cell-cell and ligand-receptor interactions among the 10 populations. Over 50,000 potential cell-cell interactions were found in both the ground state and after partial hepatectomy. Importantly, about half of these differed between the two states, indicating massive changes in the cell network during regeneration. Our study provides the first comprehensive database of potential cell-cell interactions in mammalian liver cell homeostasis and regeneration. With the help of this prediction model, we identified and validated two previously unknown signaling interactions involved in accelerating and delaying liver regeneration. Overall, we provide a novel platform for investigating autocrine/paracrine pathways in tissue regeneration, which can be adapted to other complex multicellular systems. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.14.549021v1?rss=1 Authors: Daheron, L., Becker, C., Aygar, S. Abstract: Copy number increases involving chromosome 20q with gain of the gene BCL2L1 are a prevalent form of genomic instability in hPSC. In addition to large aneuploidies, findings in this region often include microamplifications that are too small to detect by G-banded karyotyping. Gene editing procedures warrant especially close monitoring of 20q genomic stability because they involve p53-activating stressors that select for the survival of BCL2L1-aneuploid cells. Here we describe an optimized strategy for detecting BCL2L1 copy number increases in hPSC cultures using duplexed droplet digital PCR (ddPCR) with genomic DNA or cell lysate as the starting material. The procedure consists of droplet generation, thermocycling, droplet reading and data analysis. The expected result is a copy number estimate derived by comparing the number of droplets positive for BCL2L1 to the number positive for a reference template, PVRL2. This procedure generates same-day screening results for 1 to 96 samples, providing a convenient option for screening hPSC cultures that is easily integrated into a gene editing workflow. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.18.549521v1?rss=1 Authors: Wallace, N. S., Gadbery, J. E., Cohen, C. I., Kendall, A. K., Jackson, L. P. Abstract: Tepsin is an established accessory protein found in Adaptor Protein 4 (AP-4) coated vesicles, but the biological role of tepsin remains unknown. AP-4 vesicles originate at the trans-Golgi network (TGN) and target the delivery of ATG9A, a scramblase required for autophagosome biogenesis, to the cell periphery. Using in silico methods, we identified a putative LC3-Interacting Region (LIR) motif in tepsin. Biochemical experiments using purified recombinant proteins indicate tepsin directly binds LC3B, but not other members, of the mammalian ATG8 family. Calorimetry and structural modeling data indicate this interaction occurs with micromolar affinity using the established LC3B LIR docking site. Loss of tepsin in cultured cells dysregulates ATG9A export from the TGN as well as ATG9A distribution at the cell periphery. Tepsin depletion in a mRFP-GFP-LC3B HeLa reporter cell line using siRNA knockdown increases autophagosome volume and number, but does not appear to affect flux through the autophagic pathway. Re-introduction of wild-type tepsin partially rescues ATG9A cargo trafficking defects. In contrast, re-introducing tepsin with a mutated LIR motif or missing N-terminus does not fully rescue altered ATG9A subcellular distribution. Together, these data suggest roles for tepsin in cargo export from the TGN; delivery of ATG9A-positive vesicles at the cell periphery; and in overall maintenance of autophagosome structure. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.17.549391v1?rss=1 Authors: Rao, S., Strong, L. M., Ren, X., Skulsuppaisarn, M., Lazarou, M., Hurley, J. H., Hummer, G. Abstract: The covalent attachment of ubiquitin-like LC3 proteins prepares the autophagic membrane for cargo recruitment. We resolve key steps in LC3 lipidation by combining molecular dynamics simulations and experiments in vitro and in cellulo. We show how the E3-like ligase ATG12--ATG5-ATG16L1 in complex with the E2-like conjugase ATG3 docks LC3 onto the membrane in three steps by (1) the PI(3)P effector protein WIPI2, (2) helix 2 of ATG16L1, and (3) a membrane-interacting surface of ATG3. Phosphatidylethanolamine (PE) lipids concentrate in a region around the thioester bond between ATG3 and LC3, highlighting residues with a possible role in the catalytic transfer of LC3 to PE, including two conserved histidines. In a near-complete pathway from the initial membrane recruitment to the LC3 lipidation reaction, the three-step targeting of the ATG12--ATG5-ATG16L1 machinery establishes a high level of regulatory control. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.16.549237v1?rss=1 Authors: McCormick, L. E., Suarez, C., Herring, L. E., Cannon, K. S., Kovar, D. R., Brown, N. G., Gupton, S. L. Abstract: The actin cytoskeleton performs multiple cellular functions, and as such, actin polymerization must be tightly regulated. We previously demonstrated that reversible, non-degradative ubiquitination regulates the function of the actin polymerase VASP in developing neurons. However, the underlying mechanism of how ubiquitination impacts VASP activity was unknown. Here we show that mimicking multi-monoubiquitination of VASP at K240 and K286 negatively regulates VASP interactions with actin. Using in vitro biochemical assays, we demonstrate the reduced ability of multi-monoubiquitinated VASP to bind, bundle, and elongate actin filaments. However, multi-monoubiquitinated VASP maintained the ability to bind and protect barbed ends from capping protein. Lastly, we demonstrate the introduction of recombinant multi-monoubiquitinated VASP protein altered cell spreading morphology. Collectively, these results suggest a mechanism in which ubiquitination controls VASP-mediated actin dynamics. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.14.548550v1?rss=1 Authors: Jahn, D., Knapstein, P. R., Otto, E., Köhli, P., Sevecke, J., Graef, F., Graffmann, C., Fuchs, M., Jiang, S., Rickert, M., Erdmann, C., Appelt, J., Revend, L., Küttner, Q., Witte, J., Rahmani, A., Duda, G., Xie, W., Donat, A., Schinke, T., Ivanov, A., Ngokingha Tchouto, M., Beule, D., Frosch, K.-H., Baranowsky, A., Tsitsilonis, S., Keller, J. Abstract: Traumatic brain injury (TBI) is associated with a hyperadrenergic state and paradoxically causes systemic bone loss while accelerating fracture healing. Here, we identify the beta2-adrenergic receptor (Adrb2) as a central mediator of these skeletal manifestations. While the negative effects of TBI on the unfractured skeleton can be explained by the established impact of Adrb2 signaling on bone formation, Adrb2 promotes neovascularization of the fracture callus under conditions of high sympathetic tone, including TBI and advanced age. Mechanistically, norepinephrine stimulates the expression of Vegfa and Cgrp primarily in periosteal cells via Adrb2, both of which synergistically promote the formation of osteogenic type-H vessels in the fracture callus. Accordingly, the beneficial effect of TBI on bone repair is abolished in mice lacking Adrb2 or Cgrp, and aged Adrb2-deficient mice without TBI develop fracture nonunions despite high bone formation in uninjured bone. Pharmacologically, the Adrb2 antagonist propranolol impairs, and the agonist formoterol promotes fracture healing in aged mice by regulating callus neovascularization. Clinically, intravenous beta-adrenergic sympathomimetics are associated with improved callus formation in trauma patients with long bone fractures. Thus, Adrb2 is a novel target for promoting bone healing, and widely used beta-blockers may cause fracture nonunion under conditions of increased sympathetic tone. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.15.549151v1?rss=1 Authors: Anderson, A. G., Tansey, M. G. Abstract: Mutations in the progranulin (PGRN) encoding gene, GRN, cause familial frontotemporal dementia (FTD) and neuronal ceroid lipofuscinosis (NCL) and PGRN is also implicated in Parkinson's disease (PD). These mutations result in decreased PGRN expression. PGRN is highly expressed in peripheral immune cells and microglia and regulates cell growth, survival, repair, and inflammation. When PGRN is lost, the lysosome becomes dysfunctional, but the exact mechanism by which PGRN plays a role in lysosome function and how this contributes to inflammation and degeneration is not entirely understood. To better understand the role of PGRN in regulating lysosome function, this study examined how loss of GRN impacts total LAMP1 protein expression and cathepsin activities. Using mouse embryonic fibroblasts (MEFs), immunocytochemistry and immunoblotting assays were performed to analyze fluorescent signal from LAMP1 (lysosomal marker) and BMV109 (marker for pan-cathepsin activity). GRN-/- MEFs exhibit increased expression of pan-cathepsin activity relative to GRN+/+ MEFs, and significantly impacts expression of LAMP1. The significant increase in pan-cathepsin activity in the GRN-/- MEFs confirms that PGRN loss does alter cathepsin expression, which may be a result of compensatory mechanisms happening within the cell. Using NTAP PGRN added to GRN-/- MEFs, specific cathepsin activity is rescued. Further investigations should include assessing LAMP1 and BMV109 expression in microglia from GRN-/- mice, in the hopes of understanding the role of PGRN in lysosomal function in immune cells of the central nervous system and the diseases in which it is implicated. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.14.549031v1?rss=1 Authors: Seal, S., Carreras-Puigvert, J., Carpenter, A. E., Spjuth, O., Bender, A. Abstract: Cell Painting assays generate morphological profiles that are versatile descriptors of biological systems and have been used to predict in vitro and in vivo drug effects. However, Cell Painting features are based on image statistics, and are, therefore, often not readily biologically interpretable. In this study, we introduce an approach that maps specific Cell Painting features into the BioMorph space using readouts from comprehensive Cell Health assays. We validated that the resulting BioMorph space effectively connected compounds not only with the morphological features associated with their bioactivity but with deeper insights into phenotypic characteristics and cellular processes associated with the given bioactivity. The BioMorph space revealed the mechanism of action for individual compounds, including dual-acting compounds such as emetine, an inhibitor of both protein synthesis and DNA replication. In summary, BioMorph space offers a more biologically relevant way to interpret cell morphological features from the Cell Painting assays and to generate hypotheses for experimental validation. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.15.549172v1?rss=1 Authors: Zheng, J.-X., Du, T.-Y., Shao, G.-C., Ma, Z.-H., Jiang, Z.-D., Hu, W., Suo, F., He, W., Dong, M.-Q., Du, L.-L. Abstract: Killer meiotic drivers (KMDs) skew allele transmission in their favor by killing meiotic progeny not inheriting the driver allele. Despite their widespread presence in eukaryotes, the molecular mechanisms behind their selfish behavior are poorly understood. Here we investigate how the poison and antidote products of a fission yeast wtf-family KMD gene can act antagonistically. Both the poison and the antidote are multi-transmembrane proteins, differing only in their N-terminal cytosolic tails. We find that the antidote employs N-terminal PY motifs to bind Rsp5/NEDD4 family ubiquitin ligases, which ubiquitinate the antidote. Mutating PY motifs or attaching a deubiquitinating enzyme transforms the antidote into a toxic protein. Ubiquitination promotes the transport of the antidote from the trans-Golgi network to the endosome, thereby neutralizing its toxicity and that of the bound poison. We propose that post-translational modification-mediated protein localization and/or activity changes may be a common mechanism governing the antagonistic duality of single-gene KMDs. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.15.548973v1?rss=1 Authors: Upton, R., Calatayud, N. E., Clulow, S., Brett, D., Burton, A. L., Colyvas, K., Mahony, M., Clulow, J. Abstract: There are strong potential benefits of incorporating assisted reproductive technologies (ARTs) into conservation programs for the management of threatened amphibians as the global amphibian decline continues. As sperm cryopreservation and other ARTs advance in common species, focus on non-lethal sperm collection methods for threatened amphibians is imperative. We aimed to realise this goal by testing various doses of exogenous hormones for non-lethal induction of spermiation in a threatened frog (Litoria aurea) and develop cold storage and cryopreservation protocols following the recovery of urinic sperm. Our major findings include: (1) that sperm release could be induced in high concentrations with 20 IU/g bodyweight of human chorionic gonadotrophin (hCG); (2) high levels ( greater than 50%) of live, motile sperm could be recovered post-cryopreservation by treating the sperm with 15% v/v DMSO and 1% w/v sucrose pre-freeze; and (3) urinic sperm stored at 5{degrees}C retained motility over a 14-day period. Our findings demonstrate that it is possible to obtain and store large quantities of quality sperm from a threatened amphibian via non-lethal means, representing an important step forward for the use of ARTs in conservation programs for rare and threatened species. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.15.549141v1?rss=1 Authors: Polino, A. J., Ng, X. W., Rooks, R., Piston, D. W. Abstract: Just under the plasma membrane of most animal cells lies a dense meshwork of actin filaments called the cortical cytoskeleton. In insulin-secreting pancreatic beta cells, a longstanding model posits that the cortical actin layer primarily acts to restrict access of insulin granules to the plasma membrane. Here we test this model and find that stimulating beta cells with pro-secretory stimuli (glucose and/or KCl) has little impact on the cortical actin layer. Chemical perturbations of actin polymerization, by either disrupting or enhancing filamentation, dramatically enhances glucose-stimulated insulin secretion. We find that this enhancement does not correlate with the state of the cortical actin layer, suggesting filament disruptors act on insulin secretion independently of the cortical cytoskeleton. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.16.549168v1?rss=1 Authors: Schrad, J. R., Fu, G., Hable, W. E., Tayar, A. M., Oliveira, K., Nicastro, D. Abstract: Cilia and flagella play a crucial role in the development and function of most eukaryotic organisms. The structural core of the flagellum, the axoneme, is conserved in most eukaryotes and careful regulation of dynein motors within the axoneme is required for proper ciliary beating. The sperm flagellum from the American eel (Anguilla rostrata) has been shown to lack many of the canonical axonemal proteins, including the radial spokes, the central pair complex, and possibly even the outer row of dynein arms, presenting a "minimal" flagellar system. Here, we present cryo-electron tomography analysis of the eel sperm flagellum. We identified two states for the eel sperm flagellum within our tomograms, narrow and wide, and found that the flagellum started narrow near the sperm head and widened distally. Subtomogram averages revealed that the eel sperm flagellum has retained remnants of the missing regulatory complexes, including a short radial spoke 3 complex, basal components of radial spokes 1 and 2, and an outer dynein arm docking complex. We also describe unique structural features of the A. rostrata sperm flagellum, such as a unique pattern of holes at the inner junction and an accessory complex located at the "outer" junction. Finally, we discuss the consequences of losing key regulatory factors for the eel sperm flagellum and hypothesize several evolutionary factors that may have led to their loss. Together, our results shed light onto the structure and function of the eel sperm axoneme and provide insight into the minimum requirements for proper ciliary beating. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.16.549168v1?rss=1 Authors: Schrad, J. R., Fu, G., Hable, W. E., Tayar, A. M., Oliveira, K., Nicastro, D. Abstract: Cilia and flagella play a crucial role in the development and function of most eukaryotic organisms. The structural core of the flagellum, the axoneme, is conserved in most eukaryotes and careful regulation of dynein motors within the axoneme is required for proper ciliary beating. The sperm flagellum from the American eel (Anguilla rostrata) has been shown to lack many of the canonical axonemal proteins, including the radial spokes, the central pair complex, and possibly even the outer row of dynein arms, presenting a "minimal" flagellar system. Here, we present cryo-electron tomography analysis of the eel sperm flagellum. We identified two states for the eel sperm flagellum within our tomograms, narrow and wide, and found that the flagellum started narrow near the sperm head and widened distally. Subtomogram averages revealed that the eel sperm flagellum has retained remnants of the missing regulatory complexes, including a short radial spoke 3 complex, basal components of radial spokes 1 and 2, and an outer dynein arm docking complex. We also describe unique structural features of the A. rostrata sperm flagellum, such as a unique pattern of holes at the inner junction and an accessory complex located at the "outer" junction. Finally, we discuss the consequences of losing key regulatory factors for the eel sperm flagellum and hypothesize several evolutionary factors that may have led to their loss. Together, our results shed light onto the structure and function of the eel sperm axoneme and provide insight into the minimum requirements for proper ciliary beating. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.16.549168v1?rss=1 Authors: Schrad, J. R., Fu, G., Hable, W. E., Tayar, A. M., Oliveira, K., Nicastro, D. Abstract: Cilia and flagella play a crucial role in the development and function of most eukaryotic organisms. The structural core of the flagellum, the axoneme, is conserved in most eukaryotes and careful regulation of dynein motors within the axoneme is required for proper ciliary beating. The sperm flagellum from the American eel (Anguilla rostrata) has been shown to lack many of the canonical axonemal proteins, including the radial spokes, the central pair complex, and possibly even the outer row of dynein arms, presenting a 'minimal' flagellar system. Here, we present cryo-electron tomography analysis of the eel sperm flagellum. We identified two states for the eel sperm flagellum within our tomograms, narrow and wide, and found that the flagellum started narrow near the sperm head and widened distally. Subtomogram averages revealed that the eel sperm flagellum has retained remnants of the missing regulatory complexes, including a short radial spoke 3 complex, basal components of radial spokes 1 and 2, and an outer dynein arm docking complex. We also describe unique structural features of the A. rostrata sperm flagellum, such as a unique pattern of holes at the inner junction and an accessory complex located at the 'outer' junction. Finally, we discuss the consequences of losing key regulatory factors for the eel sperm flagellum and hypothesize several evolutionary factors that may have led to their loss. Together, our results shed light onto the structure and function of the eel sperm axoneme and provide insight into the minimum requirements for proper ciliary beating. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.15.549173v1?rss=1 Authors: Kumar, V., Bermea, K. C., Kumar, D., Singh, A., Verma, A., Kaileh, M., Sen, R., Lakatta, E. G., Adamo, L. Abstract: Mice with cardiac-specific overexpression of adenylyl cyclase (AC) type 8 (TGAC8) are under a constant state of severe myocardial stress and have been shown to have a remarkable ability to adapt to this stress. However, they eventually develop accelerated cardiac aging and cardiac fibrosis, and experience reduced longevity. Here we show that young (3-month-old) TGAC8 animals are characterized by a broad and extensive inflammatory state, that precedes the development of cardiac fibrosis. We demonstrate that activation of ACVIII in the cardiomyocytes results in cell-autonomous RelA-mediated NF-{kappa}B signaling. This is associated with non-cell-autonomous activation of proinflammatory and age-associated signaling in myocardial endothelial cells, increases in serum levels of inflammatory cytokines, changes in myocardial immune cells, and changes in the size or composition of lymphoid organs. Finally, we provide evidence suggesting that ACVIII-driven RelA activation in cardiomyocytes might be mediated by calcium-Protein Kinase A (PKA) signaling. Our findings highlight a novel mechanistic connection between cardiomyocyte stress, myocardial para-inflammation, systemic inflammation, and aging, and therefore point to novel potential therapeutic targets to reduce age-associated myocardial deterioration. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.14.548998v1?rss=1 Authors: De Matteis, M. A., Zappa, F., Intartaglia, D., Guarino, A. M., De Cegli, R., Wilson, C., Salierno, F. G., Polishchuk, E., Sorrentino, N. C., Conte, I. Abstract: The skeletal dysplasia spondyloepiphyseal dysplasia tarda (SEDT) is caused by mutations in the TRAPPC2 gene, which encodes Sedlin, a component of the trafficking protein particle (TRAPP) complex that we have shown previously to be required for the export of type II collagen (Col2) from the endoplasmic reticulum. No vertebrate model for SEDT has been generated thus far. To address this gap, we generated a Sedlin knockout animal by mutating the orthologous TRAPPC2 gene (olSedl) of Oryzias latipes (medaka) fish. OlSedl deficiency leads to embryonic defects, short size, diminished skeletal ossification, and altered Col2 production and secretion, resembling human defects observed in SEDT patients. Moreover, SEDT knock-out animals display photoreceptor degeneration and gut morphogenesis defects, suggesting a key role for Sedlin in the development of these organs. Thus, by studying Sedlin function in vivo, we provide evidence for a mechanistic link between TRAPPC2-mediated membrane trafficking, Col2 export, and developmental disorders. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.13.548759v1?rss=1 Authors: Serebrenik, Y. V., Mani, D., Maujean, T., Burslem, G. M., Shalem, O. Abstract: The field of induced proximity therapeutics is in its ascendancy but is limited by a lack of scalable tools to systematically explore effector-target protein pairs in an unbiased manner. Here, we combined Scalable POoled Targeting with a LIgandable Tag at Endogenous Sites (SPOTLITES) for the high-throughput tagging of endogenous proteins, with generic small molecule-based protein recruitment to screen for novel proximity-based effectors. We apply this methodology in two orthogonal screens for targeted protein degradation: the first using fluorescence to monitor target protein levels directly, and the second using a cellular growth phenotype that depends on the degradation of an essential protein. Our screens revealed a multitude of potential new effector proteins for degradation and converged on members of the CTLH complex which we demonstrate potently induce degradation. Altogether, we introduce a platform for pooled induction of endogenous protein-protein interactions that can be used to expand our toolset of effector proteins for targeted protein degradation and other forms of induced proximity. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.14.549027v1?rss=1 Authors: Yasue, S., Ozeki, M., Nozawa, A., Endo, S., Ohnishi, H. Abstract: Recently, a low-level somatic mutation in NRAS gene (c.182 A greater than G, Q61R) was identified in the specimens of patients with kaposiform lymphangiomatosis (KLA). However, it is unknown how these low-frequency mutated cells can affect the characterization and surrounding environment of their lesions. To understand the pathogenesis and association of these gene abnormalities, we established NRASQ61R mutated lymphatic endothelial cells (LECs) transfected with lentivirus vector and undertook morphological and functional characterization, protein expression profiling, and metabolome analysis. NRASQ61R human dermal LECs showed poor tube formation and high cell proliferation and migration ability with increasing ratios of mutated cells. Analysis of signaling pathways showed inactivation of the PIK3/AKT/mTOR pathway and hyperactivation of the RAS/MAPK/ERK pathway, which was improved by MEK inhibitor treatment. This study may show the theoretical circumstances in vitro induced by NRASQ61R-mutated cells in the affected lesions of KLA patients. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.13.548953v1?rss=1 Authors: Kim, S., Kang, N., Ra, J. S., Park, S. H., Myung, K., Lee, K.-y. Abstract: Replication stresses are the major source of break-induced replication (BIR). Here, we show that in alternative lengthening of telomeres (ALT) cells, replication stress-induced polyubiquitinated PCNA (polyUb-PCNA) triggers BIR at telomeres and the common fragile site (CFS). Consistently, depleting RAD18, a PCNA ubiquitinating enzyme, reduces the occurrence of ALT-associated PML bodies (APBs) and mitotic DNA synthesis at telomeres and CFS, both of which are mediated by BIR. In contrast, inhibiting USP1, an Ub-PCNA deubiquitinating enzyme, results in an increase in the above phenotypes in a RAD18- and UBC13 (the PCNA polyubiquitinating enzyme)-dependent manner. Furthermore, deficiency of ATAD5, which facilitates USP1 activity and unloads PCNAs, augments recombination-associated phenotypes. Mechanistically, telomeric polyUb-PCNA accumulates SLX4, a nuclease scaffold, at telomeres through its ubiquitin-binding domain and increases telomere damage. Consistently, APB increase induced by Ub-PCNA depends on SLX4 and SLX4-associated nucleases. Taken together, our results identified the polyUb-PCNA-SLX4 axis as a trigger for directing BIR. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.14.549060v1?rss=1 Authors: Stanczyk, P. J., Tatekoshi, Y., Shapiro, J. S., Nayudu, K., Chen, Y., Zeilber, Z., Schipma, M., De Jesus, A., Mahmoodzadeh, A., Akrami, A., Chang, H.-C., Ardehali, H. Abstract: BACKGROUND: Proper nuclear organization is critical for cardiomyocyte (CM) function, as global structural remodeling of nuclear morphology and chromatin structure underpins the development and progression of cardiovascular disease. Previous reports have implicated a role for DNA damage in cardiac hypertrophy, however, the mechanism for this process is not well delineated. AMPK family of proteins regulate metabolism and DNA damage response (DDR). Here, we examine whether a member of this family, SNF1-related kinase (SNRK), which plays a role in cardiac metabolism, is also involved in hypertrophic remodeling through changes in DDR and structural properties of the nucleus. METHODS: We subjected cardiac specific (cs)-Snrk-/- mice to trans-aortic banding (TAC) to assess the effect on cardiac function and DDR. In parallel, we modulated SNRK in vitro and assessed its effects on DDR and nuclear parameters. We also used phospho-proteomics to identify novel proteins that are phosphorylated by SNRK. Finally, co-immunoprecipitation (co-IP) was used to verify Destrin (DSTN) as the binding partner of SNRK that modulates its effects on the nucleus and DDR. RESULTS: cs-Snrk-/- mice display worse cardiac function and cardiac hypertrophy in response to TAC, and an increase in DDR marker pH2AX in their hearts. Additionally, in vitro Snrk knockdown results in increased DNA damage and chromatin compaction, along with alterations in nuclear flatness and 3D volume. Phospho-proteomic studies identified a novel SNRK target, DSTN, a member of F-actin depolymerizing factor (ADF) proteins that directly binds to and depolymerize F-actin. SNRK binds to DSTN, and DSTN downregulation reverses excess DNA damage and changes in nuclear parameters, in addition to cellular hypertrophy, with SNRK knockdown. We also demonstrate that SNRK knockdown promotes excessive actin depolymerization, measured by the increased ratio of globular (G-) actin to F-actin. Finally, Jasplakinolide, a pharmacological stabilizer of F-actin, rescues the increased DNA damage and aberrant nuclear morphology in SNRK downregulated cells. CONCLUSIONS: These results indicate that SNRK is a key player in cardiac hypertrophy and DNA damage through its interaction with DSTN. This interaction fine-tunes actin polymerization to reduce DDR and maintain proper CM nuclear shape and morphology. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.13.548611v1?rss=1 Authors: Sansbury, S. E., Serebrenik, Y. V., Lapidot, T., Burslem, G. M., Shalem, O. Abstract: System-level understanding of proteome organization and function requires methods for direct visualization and manipulation of proteins at scale. We developed an approach enabled by high-throughput gene tagging for the generation and analysis of complex cell pools with endogenously tagged proteins. Proteins are tagged with HaloTag to enable visualization or direct perturbation. Fluorescent labeling followed by in situ sequencing and deep learning-based image analysis identifies the localization pattern of each tag, providing a birds-eye-view of cellular organization. Next, we use a hydrophobic HaloTag ligand to unfold tagged proteins, inducing spatially restricted proteotoxic stress that is read out by single cell RNA sequencing. By integrating optical and perturbation data, we map compartment-specific responses to protein misfolding, revealing inter-compartment organization and direct cross-talk, and assigning proteostasis functions to uncharacterized genes. Altogether, we present a powerful and efficient method for large-scale studies of proteome dynamics, function, and homeostasis. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.13.548819v1?rss=1 Authors: Warner, H. M., Chen, T., Mahajan, S., ter Beest, M., Linders, P., Franciosa, G., Bianchi, F., van den Bogaart, G. Abstract: The mounting of an adaptive immune response is critical for removing pathogens from the body and generating immunological memory. Central to this process are myeloid cells, which sense pathogens through a variety of cell surface receptors, engulf and destroy pathogens and become activated. Activation is essential for the release of cytokines as well as the cell-surface presentation of pathogen-derived-antigens. Activation-induced cytokine release by myeloid cells requires a complex series of molecular events to facilitate cytokine expression. However, although the transcriptional machinery regulating cytokine expression is well defined, it is becoming increasingly clear that trafficking machinery has to be re-programmed through post-translational modifications to dynamically regulate cytokine secretory events. We demonstrate through quantitative total internal-resonance fluorescence (TIRF) microscopy that short-term stimulation with the pathogenic stimulus lipopolysaccharide (LPS) is sufficient to up-regulate IL-6 secretion rates in human blood monocyte-derived dendritic cells and that this secretion is asymmetric and thus polarised. Using bioinformatics analysis of our phosphoproteomic data, we demonstrate that LPS stimulation of monocyte-derived dendritic cells rapidly reprograms SNARE-associated membrane trafficking machinery, through phosphorylation/dephosphorylation events. Finally, we link this enhanced rate of secretion to the phosphorylation of the SNARE protein VAMP3 at serine 44 (48 in mice), by showing that this phosphorylation drives the release of VAMP3 by its chaperone WDFY2 and the complexing of VAMP3 with STX4 at the plasma membrane. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.13.548868v1?rss=1 Authors: de Caestecker, C., Macara, I. Abstract: Apical sorting of epithelial membrane proteins is an essential process but remains incompletely understood. Apical cytoplasmic domains are significantly smaller than those of basolateral proteins; however, the reason for this attribute is unknown. We asked if a diffusion barrier at the trans-Golgi network might impede apical sorting of proteins with large cytoplasmic tails. We used Crumbs3 and Ace2 as example apical transmembrane proteins with short cytoplasmic tails. FKBP was attached to the C-termini for inducible dimerization to FRB-tagged proteins. A streptavidin-binding peptide on the extracellular domain traps the proteins in the endoplasmic reticulum (ER). Biotin addition triggers release to the Golgi, then departure in vesicles to the apical cortex. Increasing cytoplasmic bulk by dimerization to FRB-SNAPtag moieties significantly delayed departure. Crb3 binds through its cytoplasmic tail to the Pals1 protein, and although Crb3 and Pals1 are associated at the ER and Golgi, Pals1 disassociates before Crb3 departure. A non-dissociable mutant Pals1 impedes Crb3 exit. We conclude that small cytoplasmic domains facilitate apical sorting. Reducing cytoplasmic domain size by timely Pals1 release is essential for normal kinetics of Crb3 sorting. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.12.548696v1?rss=1 Authors: Karnewar, S., Karnewar, V., Shankman, L. S., Owens, G. K. Abstract: The use of senolytic agents to remove senescent cells from atherosclerotic lesions is controversial. A common limitation of previous studies is the failure to rigorously define the effects of senolytic agent ABT-263 (Navitoclax) on smooth muscle cells (SMC) despite studies claiming that they are the major source of senescent cells. Moreover, there are no studies of the effect of ABT-263 on endothelial cells (EC), which along with SMC comprise 90% of -SMA+ myofibroblast-like cells in the protective fibrous cap. Here we tested the hypothesis that treatment of advanced atherosclerotic mice with the ABT-263 will reduce lesion size and increase plaque stability. SMC (Myh11-CreERT2-eYFP) and EC (Cdh5-CreERT2-eYFP) lineage tracing Apoe-/- mice were fed a WD for 18 weeks, followed by ABT-263 100mg/kg/bw for six weeks or 50mg/kg/bw for nine weeks. ABT-263 treatment did not change lesion size or lumen area of the brachiocephalic artery (BCA). However, ABT-263 treatment reduced SMC by 90% and increased EC-contributions to lesions via EC-to-mesenchymal transition (EndoMT) by 60%. ABT-263 treatment also reduced -SMA+ fibrous cap thickness by 60% and increased mortality by greater than 50%. Contrary to expectations, treatment of WD-fed Apoe-/- mice with the senolytic agent ABT-263 resulted in multiple detrimental changes including reduced indices of stability, and increased mortality. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.12.548716v1?rss=1 Authors: Zhan, H., Zhang, H., Kafeiti, N., Lee, S., Masarik, K., Zheng, H. Abstract: Patients with JAK2V617F-positive myeloproliferative neoplasms (MPNs) and clonal hematopoiesis of indeterminate potential (CHIP) are at a significantly higher risk of cardiovascular diseases (CVDs). Endothelial cells (ECs) carrying the JAK2V617F mutation can be detected in many MPN patients. Here, we investigated the impact of endothelial JAK2V617F mutation on CVD development using both transgenic murine models and human induced pluripotent stem cell lines. Our findings revealed that JAK2V617F mutant ECs promote CVDs by impairing endothelial function and undergoing endothelial-to-mesenchymal transition (EndMT). Importantly, we found that inhibiting the endothelial thrombopoietin receptor MPL suppressed JAK2V617F-induced EndMT and prevented cardiovascular dysfunction caused by mutant ECs. These findings propose that targeting the endothelial MPL receptor could be a promising therapeutic approach to manage CVD complications in patients with JAK2V617F-positive MPNs and CHIP. Further investigations into the impact of other CHIP-associated mutations on endothelial dysfunction are needed to improve risk stratification for individuals with CHIP. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.13.548906v1?rss=1 Authors: Hu, M., Bonas-Guarch, S., Kim, I., Moran, I., Bonnefond, A., Froguel, P., Rutter, G. A. Abstract: Variants at the SLC30A8 locus are associated with type 2 diabetes (T2D) risk. The lead variant, rs13266634, encodes an amino acid change, Arg325Trp (R325W), at the C-terminus of the secretory granule-enriched zinc transporter, ZnT8. Although this protein-coding variant was previously thought to be the sole driver of altered disease risk, recent studies have provided evidence for lowered expression of SLC30A8 mRNA in protective allele carriers. In the present study, combined allele-specific expression (cASE) analysis in human islets revealed that multiple variants affect the expression SLC30A8. Chromatin accessibility and epigenomic analyses imply the existence at the SLC30A8 locus of an islet-selective super-enhancer cluster hosting multiple diabetes-associated variants. The variant region is spatially associated with both the SLC30A8 promoter and with the regulatory regions of the neighbouring RAD21, RAD21-AS1, UTP23 and other genes. Deletion of variant-bearing regions from human-derived EndoC-BH3 cells using CRISPR-Cas9 lowered the expression of SLC30A8 and several neighbouring genes, suggesting their co-regulation by this enhancer cluster. Whilst deletion of SLC30A8 had no effect on beta cell survival under the conditions examined, loss of RAD21 or UTP23 markedly reduced cell viability. Thus, the protective effects of variants that lower SLC30A8 activity may be modulated by the altered expression of nearby genes. Direct evidence for this possibility was not, however, obtained by cASE or eQTL analysis of human islet samples. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.12.548679v1?rss=1 Authors: Duot, M., Viel, R., Viet, J., Le Goff-Gaillard, C., Paillard, L., Lachke, S., Gautier-Courteille, C., Reboutier, D. Abstract: The ocular lens, along with the cornea, focuses light on the retina to generate sharp images. Opacification of the lens, or cataract, is the leading cause of blindness worldwide. Presently, the best approach for cataract treatment is to surgically remove the diseased lens and replace it with an artificial implant. Although effective, this is costly and can have post-surgical complications. Toward identifying alternate treatments, it is imperative to develop organoid models relevant for lens studies and anti-cataract drug screening. Here, we demonstrate that by culturing mouse lens epithelial cells under defined 3-dimensional (3D) culture conditions, it is possible to generate organoids that display optical properties and recapitulate many aspects of lens organization at the tissue, cellular and transcriptomic levels. These 3D cultured lens organoids can be rapidly produced in large amounts. High-throughput RNA-sequencing (RNA-seq) on specific organoid regions isolated by laser capture microdissection (LCM) and immunofluorescence assays demonstrate that these lens organoids display spatiotemporal expression of key lens genes, e.g., Jag1, Pax6, Prox1, Hsf4 and Cryab. Further, these lens organoids are amenable to induction of opacities. Finally, knockdown of a cataract-linked RNA-binding protein encoding gene, Celf1, induces opacities in these organoids, indicating their use in rapidly screening for genes functionally relevant to lens biology and cataract. In sum, this lens organoid model represents a compelling new tool to advance the understanding of lens biology and pathology, and can find future use in the rapid screening of compounds aimed at preventing and/or treating cataract. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.12.548547v1?rss=1 Authors: Prislusky, M. I. A., Lam, J. G., Ruiz Contreras, V., Ng, M., Chamberlain, M., Fields, M., Zhang, X., Amer, A., Seveau, S. M. Abstract: Mammalian cells are frequently exposed to mechanical and biochemical stresses resulting in plasma membrane injuries. Repair mechanisms rapidly reseal the plasma membrane to restore homeostasis and prevent cell death. In the present work, a silencing RNA (siRNA) screen was performed to uncover the plasma membrane repair mechanisms of cells injured by the bacterial pore-forming toxin listeriolysin O (LLO). The screen identified a novel role for the septin cytoskeleton in mediating plasma membrane repair. Upon cell injury, the septin cytoskeleton partially dissociates from actin stress fibers and remodels with cortical F-actin and myosin-II to form loop (and ring)-like domains that protrude from the cell surface. These domains strictly colocalize with the calcium-dependent phospholipid-binding protein, annexin A2 (ANXA2). Importantly, formation of the SEPT/F-actin/ANXA2 domains are dependent on SEPT7 expression and is functionally correlated with the plasma membrane repair efficiency. Our studies open new research avenues by identifying a novel role for the septin cytoskeleton in remodeling the plasma membrane for its repair. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.12.548706v1?rss=1 Authors: Wu, Y., Ding, C., Weinreb, A., Manning, L., Swaim, G., Yogev, S., Colon-Ramos, D., Hammarlund, M. Abstract: Mitochondria transport is crucial for mitochondria distribution in axons and is mediated by kinesin-1-based anterograde and dynein-based retrograde motor complexes. While Miro and Milton/TRAK were identified as key adaptors between mitochondria and kinesin-1, recent studies suggest the presence of additional mechanisms. In C. elegans, ric-7 is the only single gene described so far, other than kinesin-1, that is absolutely required for axonal mitochondria localization. Using CRISPR engineering in C. elegans, we find that Miro is important but is not essential for anterograde traffic, whereas it is required for retrograde traffic. Both the endogenous RIC-7 and kinesin-1 act at the leading end to transport mitochondria anterogradely. RIC-7 recruitment to mitochondria requires its N-terminal domain and partially relies on MIRO-1, whereas RIC-7 accumulation at the leading end depends on its disordered region, kinesin-1 and metaxin2. We conclude that polarized transport complexes containing kinesin-1 and RIC-7 form at the leading edge of mitochondria, and that these complexes are required for anterograde axonal transport. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.12.548688v1?rss=1 Authors: Wang, S., Cheng, H., Li, M., Wu, H., Zhang, S., Gao, D., Huang, Y., Guo, K. Abstract: Although increasing studies has demonstrated that cell competition widely involved in the growth and homeostasis of multicellular organisms is closely linked to tumorigenesis and development, the mechanistic contributions to the association between tumor cell competition-driven heterogeneity and drug resistance remains ill-defined. In our study, lenvitinib-resistant hepatocellular carcinoma (HCC) cells display obviously competitive growth dominance against sensitive cells through reprogramming energy metabolism. Mechanistically, when BCL2 interacting protein3 (BNIP3) overexpression activates mitophagy activity in lenvatinib-resistant HCC cells, energy imbalance signal caused by reduced mitochondrial oxidative phosphorylation levels provokes the phosphorylation of AMP-activated protein kinase (AMPK) sensor; subsequently, enabled AMPK specifically targets enolase 2 (ENO2) to enhance glycolysis and eventually promots the competitive capacity and dominant growth. Of note, BNIP3 deficiency shows certain inhibition of cell competition outcome. Our findings emphasize a vital role for BNIP3-AMPK-ENO2 signaling in maintaining the competitive outcome of lenvitinib-resistant HCC cells via regulating energy metabolism; meanwhile this work recognaizes BNIP3 as a promising target to overcome HCC drug resistance. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.12.548636v1?rss=1 Authors: Lu, C., Chen, K., Qiu, H., Chen, X., Chen, G., Qi, X., Jiang, H. Abstract: Electron microscopy (EM) revolutionized the way to visualize cellular ultrastructure. Volume EM (vEM) has further broadened its three-dimensional nanoscale imaging capacity. However, intrinsic trade-offs between imaging speed and quality of EM restrict the attainable imaging area and volume. Isotropic imaging with vEM for large biological volumes remains unachievable. Here we developed EMDiffuse, a suite of algorithms designed to enhance EM and vEM capabilities, leveraging the cutting-edge image generation diffusion model. EMDiffuse demonstrates outstanding denoising and super-resolution performance, generates realistic predictions without unwarranted smoothness, improves prediction resolution by ~30%, and exhibits excellent transferability by taking only one pair of images to fine-tune. EMDiffuse also pioneers the isotropic vEM reconstruction task, generating isotropic volume similar to that obtained using advanced FIB-SEM even in the absence of isotropic training data. We demonstrated the robustness of EMDiffuse by generating isotropic volumes from six public datasets obtained from different vEM techniques and instruments. The generated isotropic volume enables accurate organelle reconstruction, making 3D nanoscale ultrastructure analysis faster and more accessible and extending such capability to larger volumes. More importantly, EMDiffuse features self-assessment functionalities and guarantees reliable predictions for all tasks. We envision EMDiffuse to pave the way for more in-depth investigations into the intricate subcellular nanoscale structures within large areas and volumes of biological systems. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.12.548764v1?rss=1 Authors: Schwartz, L. S., Young, K. A., Stearns, T. M., Boyer, N., Mujica, K. D., Trowbridge, J. J. Abstract: Age-associated clonal hematopoiesis (CH) occurs due to somatic mutations accrued in hematopoietic stem cells (HSCs) that confer a selective advantage in the context of aging. The mechanisms by which CH-mutant HSCs gain this advantage with aging are not comprehensively understood. Using unbiased transcriptomic approaches, we identify Oncostatin M (OSM) signaling as a candidate contributor to aging-driven Dnmt3a-mutant CH. We find that Dnmt3a-mutant HSCs from young mice do not functionally respond to acute OSM stimulation with respect to proliferation, apoptosis, hematopoietic engraftment, or myeloid differentiation. However, young Dnmt3a-mutant HSCs transcriptionally upregulate an inflammatory cytokine network in response to acute OSM stimulation including genes encoding IL-6, IL-1{beta} and TNF. In addition, OSM-stimulated Dnmt3a-mutant HSCs upregulate the anti-inflammatory genes Socs3, Atf3 and Nr4a1, creating a negative feedback loop limiting sustained activation of the inflammatory network. In the context of an aged bone marrow (BM) microenvironment with chronically elevated levels of OSM, Dnmt3a-mutant HSCs upregulate pro-inflammatory genes but do not upregulate Socs3, Atf3 and Nr4a1. Together, our work suggests that chronic inflammation with aging exhausts the regulatory mechanisms in young CH-mutant HSCs that resolve inflammatory states, and that OSM is a master regulator of an inflammatory network that contributes to age-associated CH. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.11.548530v1?rss=1 Authors: Sharma, D. K., Soni, I., Misra, H. S., Rajpurohit, Y. S. Abstract: Deinococcus radiodurans exhibits remarkable survival under extreme conditions, including ionizing radiation, desiccation, and various DNA-damaging agents. It employs unique repair mechanisms, such as single-strand annealing (SSA) and extended synthesis-dependent strand annealing (ESDSA), to efficiently restore damaged DNA fragments. In this study, we investigate the regulatory role of the NT-specific protein DprA in DNA repair pathways following acute gamma radiation exposure. Our findings demonstrate that the absence of DprA leads to rapid repair of gamma radiation-induced DNA double-strand breaks (DSBs), with diminished involvement of the ESDSA pathway. Furthermore, our data suggest that the SSA pathway becomes the primary mechanism for DNA DSB repair in the absence of DprA. Overall, our results highlight the regulatory function of DprA in modulating the choice between SSA and ESDSA pathways for DNA repair in the radiation-resistant bacterium D. radiodurans. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.11.548599v1?rss=1 Authors: Chavan, A., Skrutl, L., Uliana, F., Pfister, M., Burke, D. F., Braendle, F., Beltrao, P., Jagannathan, M. Abstract: The pericentromeric heterochromatin of eukaryotic chromosomes primarily consists of abundant non-coding repeats known as satellite DNA, which promote accurate chromosome segregation and genome stability. During interphase, sequence-specific satellite DNA-binding proteins cluster repeats from multiple chromosomes into foci known as chromocenters, which function to encapsulate the entire genome in a single nucleus. Despite the pivotal role of satellite DNA-binding proteins and chromocenters in cellular function, the proteins associated with these repetitive sequences remains incompletely characterized. Here, we use quantitative mass spectrometry to characterize the chromocenter-associated proteome in Drosophila embryos, ovaries and testes using two satellite DNA-binding proteins, D1 and Prod, as baits. We identify nearly 500 interactions, including known heterochromatin-associated proteins as well as proteins previously unlinked to satellite DNA or chromocenters. Among these interactions, we find that multiple components of the transposon-silencing piRNA pathway are associated with chromocenters. Strikingly, we reveal that proper satellite DNA clustering plays a role in transgenerational transposon repression, such that mothers with disrupted chromocenters give rise to progeny that exhibit transposon de-repression, germ cell loss and gonadal atrophy. Overall, our study highlights a novel link between satellite DNA repeats and transposon repression and lays the foundation for a more comprehensive understanding of satellite DNA function across tissues. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.12.548725v1?rss=1 Authors: Song, A., Phandthong, R., Talbot, P. Abstract: Both spill over and spill back of SARS-CoV-2 virus have been reported on mink farms in Europe and the United States. Zoonosis is a public health concern as dangerous mutated forms of the virus could be introduced into the human population through spillback. The purpose of our study was to determine the SARS-CoV-2 entry mechanism using mink lung epithelial cell line (Mv1Lu) and to block entry with drug inhibitors. Mv1Lu cells were susceptible to SARS-CoV-2 viral pseudoparticle infection, validating them as a suitable disease model for COVID-19. Inhibitors of TMPRSS2 and of endocytosis, two pathways of viral entry, were tested to identify those that blocked infection. Dyngo4a, a small molecule endocytosis inhibitor, significantly reduced infection, while TMPRSS2 inhibitors had minimal impact, supporting the conclusion that the entry of the SARS-CoV-2 virus into Mv1Lu cells occurs primarily through endocytosis. The small molecule inhibitors that were effective in this study could potentially be used therapeutically to prevent SARS-CoV-2 infection in mink populations. This study will facilitate the development of therapeutics to prevent zoonotic transmission of SARS-CoV-2 variants to other animals, including humans. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.10.548452v1?rss=1 Authors: Schmied, C., Ebner, M., Samso, P. F., Haucke, V., Lehmann, M. Abstract: Eukaryotic cells are highly compartmentalized by a variety of organelles that carry out specific cellular processes. The position of these organelles within the cell is elaborately regulated and vital for their function. For instance, the position of lysosomes relative to the nucleus controls their degradative capacity and is altered in pathophysiological conditions. The molecular components orchestrating the precise localization of organelles remain incompletely understood. A confounding factor in these studies is the fact that organelle positioning is surprisingly non-trivial to address. E.g., perturbations that affect the localization of organelles often lead to secondary phenotypes such as changes in cell or organelle size. These phenotypes could potentially mask effects or lead to the identification of false positive hits. To uncover and test potential molecular components at scale, accurate and easy to use analysis tools are required that allow robust measurements of organelle positioning. Here, we present an analysis workflow for the faithful, robust, and quantitative analysis of organelle positioning phenotypes. Our workflow consists of an easy to use Fiji plugin and an R Shiny App. These tools enable users without background in image or data analysis to (1) segment single cells and nuclei and to detect organelles, (2) to measure cell size and the distance between detected organelles and the nucleus, (3) to measure intensities in the organelle channel plus one additional channel, and (4) to plot the results in informative graphs. Using simulated data and immunofluorescent images of cells in which the function of known factors for lysosome positioning has been perturbed, we show that the workflow is robust against common problems for the accurate assessment of organelle positioning such as changes of cell shape and size, organelle size and background. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.10.548429v1?rss=1 Authors: Warner, H., Franciosa, G., van der Borg, G., Faas, F., Koenig, C., de Boer, R., Classens, R., Maassen, S., Baranov, M., Mahajan, S., Dabral, D. D., Coenen, B., Bianchi, F., van Hilten, N., Risselada, H. J., Roos, W. H., Olsen, J., Querol Cano, L., van den Bogaart, G. V. Abstract: To mount an adaptive immune response, dendritic cells must process antigens, migrate to lymph nodes and form synapses with T cells. Critical to 3D migration and mechanosensing is the nucleus, which is the size-limiting barrier for navigation through gaps in the extracellular matrix. Here, we show that inflammatory activation of dendritic cells leads to the nucleus becoming spherically deformed, and enables dendritic cells to overcome the typical 2 to 3 micron pore limit for 3D migration. We show that the nuclear shape-change is partially attained through reduced cell adhesion, whereas improved migration through extracellular matrix is achieved through reprogramming of the actin cytoskeleton. Specifically we show that phosphorylation of cofilin-1 at serine 41 drives the assembly of a CofilinActoMyosin (CAM)ring proximal to the nucleus and enhances migration through 3D collagen gels. In summary, these data describe novel signaling events through which dendritic cells simultaneously deform their nucleus and enhance their migratory capacity; molecular events that may be recapitulated in other contexts such as wound healing and cancer. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.10.548473v1?rss=1 Authors: Singh, S. B., Rajput, S. S., Sharma, A., Ananthanarayanan, V., Nandi, A., Patil, S. P. V., majumdar, a., Subramanyam, D. Abstract: Protein aggregation is a common underlying feature of neurodegenerative disorders. Cells expressing neurodegeneration associated mutant proteins show altered uptake of ligands, suggestive of impaired endocytosis, in a manner as yet unknown. Using live cell imaging, we show that clathrin mediated endocytosis (CME) is affected due to altered actin cytoskeletal organization in the presence of Huntingtin aggregates. Additionally, we find that cells containing Huntingtin aggregates are stiffer and less viscous than their wild type counterparts due to altered actin conformation, and not merely due to the physical presence of aggregate(s). We further demonstrate that CME and cellular viscosity can be rescued by overexpressing Hip1, Arp2/3 or transient LatrunculinA treatment. Examination of other pathogenic aggregates revealed that only a subset of these display defective CME, along with altered actin organization and increased stiffness. Together, our results point to an intimate connection between functional CME, actin organization and cellular stiffness in the context of neurodegeneration. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.11.548601v1?rss=1 Authors: Campos, Y., Rodriguez-Enriquez, R., Palacios, G., Van de Vlekkert, D., Qiu, X., Weesner, J., Gomero, E., Demmers, J., Bertorini, T., Opferman, J. T., Grosveld, G. T., d'Azzo, A. Abstract: High energy-demanding tissues, such as skeletal muscle, require mitochondrial proteostasis to function properly. Two quality-control mechanisms, the ubiquitin proteasome system (UPS) and the release of mitochondria-derived vesicles, safeguard mitochondrial proteostasis. However, whether these processes interact is unknown. Here we show that the E3 ligase CRL5Ozz, a member of the UPS, and its substrate Alix control the mitochondrial concentration of Slc25A4, a solute carrier that is essential for ATP production. The mitochondria in Ozz-/- or Alix-/- skeletal muscle share overt morphologic alterations (they are supernumerary, swollen, and dysmorphic) and have abnormal metabolomic profiles. We found that CRL5Ozz ubiquitinates Slc25A4 and promotes its proteasomal degradation, while Alix facilitates SLC25A4 loading into exosomes destined for lysosomal destruction. The loss of Ozz or Alix offsets steady-state levels of Slc25A4, which disturbs mitochondrial metabolism and alters muscle fiber composition. These findings reveal hitherto unknown regulatory functions of Ozz and Alix in mitochondrial proteostasis Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.10.548332v1?rss=1 Authors: Herruzo, E., Sanchez-Diaz, E., Gonzalez-Arranz, S., Santos, B., Carballo, J. A., San-Segundo, P. A. Abstract: The meiotic recombination checkpoint reinforces the order of events during meiotic prophase I, ensuring the accurate distribution of chromosomes to the gametes. The AAA+ ATPase Pch2 remodels the Hop1 axial protein enabling adequate levels of Hop1-T318 phosphorylation to support the ensuing checkpoint response. While these events are focalized at chromosome axes, the checkpoint activating function of Pch2 relies on its cytoplasmic population. In contrast, forced nuclear accumulation of Pch2 leads to checkpoint inactivation. Here, we reveal the mechanism by which Pch2 travels from the cell nucleus to the cytoplasm to maintain Pch2 cellular homeostasis. Leptomycin B treatment provokes the nuclear accumulation of Pch2, indicating that its nucleocytoplasmic transport is mediated by the Crm1 exportin recognizing proteins containing Nuclear Export Signals (NESs). Consistently, leptomycin B leads to checkpoint inactivation and impaired Hop1 axial localization. Pch2 nucleocytoplasmic traffic is independent of its association with Zip1 and Orc1. We also identify a conserved functional NES in the non-catalytic N-terminal domain of Pch2 that is required for its nucleocytoplasmic traffic and proper checkpoint activity. In sum, we unveil another layer of control of Pch2 function during meiosis involving the nuclear export via the exportin pathway that is crucial to maintain the critical balance of Pch2 distribution among different cellular compartments. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.10.548405v1?rss=1 Authors: Schwartz, A. Z., Abdu, Y., Nance, J. Abstract: Rapid and conditional protein depletion is the gold standard genetic tool for deciphering the molecular basis of developmental processes. Previously, we showed that by conditionally expressing the E3 ligase substrate adaptor ZIF-1 in Caenorhabditis elegans somatic cells, proteins tagged with the first CCCH Zn finger (ZF1) domain from the germline regulator PIE-1 degrade rapidly, resulting in loss-of-function phenotypes. The described role of ZIF-1 is to clear PIE-1 and several other CCCH Zn finger proteins from early somatic cells, helping to enrich them in germline precursor cells. Here, we show that proteins tagged with the PIE-1 ZF1 domain are subsequently cleared from primordial germ cells in embryos and from undifferentiated germ cells in larvae and adults by ZIF-1. We harness germline ZIF-1 activity to degrade a ZF1-tagged heterologous protein from PGCs and show that its depletion produces phenotypes equivalent to those of a null mutation. Our findings reveal that ZIF-1 switches roles from degrading CCCH Zn finger proteins in somatic cells to clearing them from undifferentiated germ cells, and that ZIF-1 activity can be harnessed as a new genetic tool to study the early germ line. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.10.548399v1?rss=1 Authors: Suda, Y., Tachikawa, H., Suda, T., Kurokawa, K., Nakano, A., Irie, K. Abstract: Gametogenesis in budding yeast involves large-scale rearrangement of membrane traffic to allow de novo formation of a membrane, called the prospore membrane (PSM). However, the mechanism underlying this event is not fully elucidated. Here, we show that the number of endoplasmic reticulum exit sites (ERES) per cell fluctuates and switches from decreasing to increasing upon the onset of PSM formation. Reduction in ERES number is accompanied by a transient stall in membrane traffic, resulting in loss of the preexisting Golgi apparatus from the cell, as well as local ERES regeneration, leading to Golgi reassembly in nascent spores. We have revealed that protein phosphatase-1 (PP-1) and its development-specific subunit, Gip1, promote ERES regeneration through Sec16 foci formation. Furthermore, a mutant with impaired ERES formation showed defects in PSM growth and spore formation. Thus, ERES regeneration in nascent spores facilitates the segregation of membrane traffic organelles, leading to PSM growth. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.10.548302v1?rss=1 Authors: Richardson, H. E., Portela, M., Mukherjee, S., Paul, S., La Marca, J. E., Parsons, L. M., Veraksa, A. Abstract: The tumour suppressor, Lethal (2) giant larvae (Lgl), is an evolutionarily conserved protein that was discovered in the vinegar fly, Drosophila, where its depletion results in tissue overgrowth and loss of cell polarity and tissue architecture. Our previous studies have revealed a new role for Lgl in linking cell polarity and tissue growth through regulation of the Notch (proliferation and differentiation) and the Hippo (negative tissue growth control) signalling pathways. Moreover, Lgl regulates vesicle acidification, via the Vacuolar ATPase (V-ATPase), and we showed that Lgl inhibits V-ATPase activity through Vap33 (a Vamp (v-SNARE)-associated protein, involved in endo-lysosomal trafficking) to regulate the Notch pathway. However, how Lgl acts to regulate the Hippo pathway was unclear. In this current study, we show that V-ATPase activity inhibits the Hippo pathway, whereas Vap33 acts to activate Hippo signalling. Using an in vivo affinity-purification approach we found that Vap33 binds to the actin cytoskeletal regulators RtGEF (Pix, a Rho-type guanine nucleotide exchange factor) and Git (G protein-coupled receptor kinase interacting ArfGAP), which also bind to the Hpo protein kinase, and are involved in the activation of the Hippo pathway. Vap33 genetically interacts with RtGEF and Git in Hippo pathway regulation. Additionally, we show that the ADP ribosylation factor Arf79F (Arf1), which is a Hpo interactor, is involved in the inhibition of the Hippo pathway. Altogether our data suggests that Lgl acts via Vap33 to activate the Hippo pathway by a dual mechanism, 1) through interaction with RtGEF/Git/Arf79F, and 2) through interaction and inhibition of the V-ATPase, thereby controlling epithelial tissue growth. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.10.548366v1?rss=1 Authors: Hayman, D. J., Lin, H., Prior, A., Charlesworth, G., Johnson de Sousa Brito, F. M., Hao, Y., Patel, K., Soul, J., Clark, I. M., Pirog, K. A., Barter, M. J., van 't Hof, R. J., Young, D. A. Abstract: microRNAs (miRNAs) are non-coding RNAs which modulate the expression of other RNA molecules. One miRNA can target many transcripts, allowing each miRNA to play key roles in many biological pathways. miR-324 is a miRNA previously implicated in bone and cartilage maintenance, defects of which result in common age-related diseases, such as osteoporosis or osteoarthritis (OA). In global miR-324-null mice cartilage damage was increased in both surgically and ageing-induced OA, despite minimal changes to the cartilage transcriptome, with few predicted miR-324 targets dysregulated. However, micro-computed tomography and histology demonstrated that global miR-324-null the mice had an increase in bone mineral density, trabecular thickness and cortical thickness, with many parameters increasing with age. The bone marrow of miR-324-null mice also had reduced lipid content while and in vivo TRAP staining revealed a decrease in osteoclasts, with histomorphometry demonstrating an increased rate of bone formation in miR-324-null mice. Ex vivo assays revealed that the high bone mass phenotype of the miR-324-null mice resulted from increased osteoblast activity and decreased osteoclastogenesis. RNA-seq and qRT-PCR followed by miR-324 target prediction and validation in osteoblasts, osteoclasts and bone marrow macrophages identified the osteoclast fusion regulator Pin1 as a miR-324 target in the osteoclast lineage and the master osteogenic regulator Runx2 as a target of miR-324-5p in osteoblasts, the in vitro overexpression of which recapitulated the increased osteogenesis and decreased adipogenesis phenotype observed in vivo. These data point to important roles of miR-324 in skeletal biology with altered bone homeostasis in miR-324-null mice potentially causal for the increased cartilage damage observed during OA and ageing. Elucidation of pathways regulated by miR-324 offer promise for the treatment of bone diseases such as osteoporosis. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.09.548259v1?rss=1 Authors: McCafferty, C. L., Papoulas, O., Lee, C., Bui, K. H. L., Taylor, D. W., Marcotte, E. M., Wallingford, J. B. Abstract: Motile cilia are ancient, evolutionarily conserved organelles whose dysfunction underlies motile ciliopathies, a broad class of human diseases. Motile cilia contain myriad different proteins that assemble into an array of distinct machines, so understanding the interactions and functional hierarchies among them presents an important challenge. Here, we defined the protein interactome of motile axonemes using cross-linking mass spectrometry (XL/MS) in Tetrahymena thermophila. From over 27,000 XLs, we identified 9,208 unique amino acid interactions among 1,368 distinct proteins, providing both macromolecular and atomic-scale insights into diverse ciliary machines, including the Intraflagellar Transport system, axonemal dynein arms, radial spokes, the 96 nm ruler, and microtubule inner proteins, among others. Guided by this dataset, we used vertebrate multiciliated cells to reveal novel functional interactions among several poorly-defined human ciliopathy proteins. The dataset therefore provides a powerful resource for studying the basic biology of an ancient organelle and the molecular etiology of human genetic disease. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.09.545884v1?rss=1 Authors: Teranishi, K., Wagatsuma, K., Toda, K., Nomaru, H., Yanagihashi, Y., Ochiai, H., Akai, S., Mochizuki, E., Onda, Y., Nakagawa, K., Sugimoto, K., Takahashi, S., Yamaguchi, H., Ota, S. Abstract: Automation and quality control (QC) are critical in manufacturing safe and effective cell and gene therapy products. However, current QC methods, reliant on molecular staining, pose difficulty in in-line testing and can increase manufacturing costs. Here we demonstrate the potential of using label-free ghost cytometry (LF-GC), a machine learning-driven, multidimensional, high-content, and high-throughput flow cytometry approach, in various stages of the cell therapy manufacturing processes. LF-GC accurately quantified T cells in human peripheral blood mononuclear cells (PBMCs) and discriminated between live and dead cells, non-apoptotic live cells and early apoptotic/dead cells, T cells and non-T cells, activated T cells and quiescent T cells, and particulate impurities in PBMCs. The data support that LF-GC is a non-destructive label-free cell analytical method that can be used to monitor cell numbers, assess viability, identify specific cell subsets or phenotypic states, and remove impurities during cell therapy manufacturing. Thus, LF-GC holds the potential to enable full automation in the manufacturing of cell therapy products with reduced cost and increased efficiency. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.09.548244v1?rss=1 Authors: Raz, E., Westerich, K. J., Tarbashevich, K., Schick, J., Gupta, A., Zhu, M., Hull, K., Romo, D., Zeuschner, D., Goudarzi, M., Gross-Thebing, T. Abstract: Germ granules, condensates of phase-separated RNA and protein, are organelles essential for germline development in different organisms The patterning of the granules and its relevance for germ cell fate are not fully understood. Combining three-dimensional in vivo structural and functional analyses, we study the dynamic spatial organization of molecules within zebrafish germ granules. We find that localization of RNA molecules to the periphery of the granules, where ribosomes are localized depends on translational activity at this location. In addition, we find that the vertebrate-specific Dead end (Dnd1) protein is essential for nanos3 RNA localization at the condensates periphery. Accordingly, in the absence of Dnd1, or when translation is inhibited, nanos3 RNA translocates into the granule interior, away from the ribosomes, a process that is correlated with loss of germ cell fate. These findings highlight the relevance of sub-granule compartmentalization for posttranscriptional control, and its importance for preserving germ cell totipotency. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.06.547985v1?rss=1 Authors: Migliori, B., Bose, N., Paull, D. Abstract: High-content imaging (HCI) is a popular technique that leverages high throughput datasets to uncover phenotypes of cell populations in vitro. When the differences between populations (such as a healthy and disease state) are completely unknown, it is crucial to build very large HCI screens to account for individual (donor) variation, as well as having enough replicates to create a reliable model. One approach to highlight phenotypic differences is to reduce images into a set of features using unbiased methods, such as embeddings or autoencoders. These methods are powerful at preserving the predictive power contained in each image while removing most of the unimportant image features and noise (e.g., background). However, they do not provide interpretable information about the features driving the decision process of the AI algorithm used. While tools have been developed to address this issue, such as CellProfiler, scaling this tool to large sample batches containing hundreds of thousands of images poses computational challenges. Additionally, the resulting feature vector, computationally expensive to have generated, is very large in size (containing over 3000 features) with many redundant features, making it challenging to perform further analysis and identify the truly relevant features. Ultimately, there is an increased risk of overfitting due to the presence of too many non-meaningful features that can ultimately skew downstream predictions. To address this issue, we have developed ScaleFExSM, a Python pipeline that extracts multiple generic fixed features at the single cell level that can be deployed across large high-content imaging datasets with low computational requirements. This pipeline efficiently and reliably computes features related to shape, size, intensity, texture, granularity as well as correlations between channels. Additionally, it allows the measurement of additional features specifically related to mitochondria and RNA only, as they represent important channels with characteristics worth to be measured on their own. The measured features can be used to not only separate populations of cells using AI tools, but also highlight the specific interpretable features that differ between populations. We applied ScaleFExSM to identify the phenotypic shifts that multiple cell lines undergo when exposed to different compounds. We used a combination of recursive feature elimination, logistic regression, correlation analysis and dimensionality reduction representations to narrow down to the most meaningful features that described the drug shifts. Furthermore, we used the best scoring features to extract images of cells for each class closest to the average to visually highlight the phenotypic shifts caused by the drugs. Using this approach, we were able to identify features linked to the drug shifts in line with literature, and we could visually validate their involvement in the morphological changes of the cells. ScaleFExSM can be used as a powerful tool to understand the underlying phenotypes of complex diseases and subtle drug shifts at the single cell level, bringing us a step closer to identifying disease-modifying compounds for the major diseases of our time. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.07.548108v1?rss=1 Authors: Jerabkova-Roda, K., Mousson, A., Peralta, M., Karali, R., Justiniano, H., Lisii, L.-M., Carl, P., Asokan, N., Busnelli, I., Larnicol, A., Lefebvre, O., Lachuer, H., Pichot, A., Stemmelen, T., Molitor, A., Hirschler, A., Delalande, F., Sick, E., Carapito, R., Carapito, C., Hyenne, V., Schauer, K., Ronde, P., GOETZ, J. G. Abstract: Emerging evidences suggest that both function and position of organelles are pivotal for tumor cell dissemination. Among them, lysosomes stand out as they integrate metabolic sensing with gene regulation and secretion of proteases. Yet, how the function of lysosomes is linked to their position and how this controls metastatic progression remains elusive. Here, we analyzed lysosome subcellular distribution in micropatterned patient-derived melanoma cells and found that lysosome spreading scales with their aggressiveness. Peripheral lysosomes promote invadopodia-based matrix degradation and invasion of melanoma cells, which is directly linked to their lysosomal and cell transcriptional programs. When controlling lysosomal positioning using chemo-genetical heterodimerization in melanoma cells, we demonstrated that perinuclear clustering impairs lysosomal secretion, matrix degradation and invasion. Impairing lysosomal spreading in a zebrafish metastasis model significantly reduces invasive outgrowth. Our study provides a mechanistic demonstration that lysosomal positioning controls cell invasion, illustrating the importance of organelle adaptation in carcinogenesis. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.07.548175v1?rss=1 Authors: Wilson, Z. N., Balasubramaniam, S. S., Wopat, M., Hughes, A. Abstract: The outer mitochondrial membrane (OMM) creates a boundary that imports most of the mitochondrial proteome while removing extraneous or damaged proteins. How the OMM senses aberrant proteins and remodels to maintain OMM integrity remains unresolved. Previously, we identified a piecemeal autophagic mechanism called the mitochondrial-derived compartment (MDC) that removes a subset of the mitochondrial proteome. Here we show that MDCs specifically sequester proteins localized only at the OMM, providing an explanation for how select mitochondrial proteins are removed by MDCs. Remarkably, selective sorting into MDCs also occurs within the OMM, as subunits of the translocase of the outer membrane (TOM) complex are excluded from MDCs unless assembly of the TOM complex is impaired. Considering that overloading the OMM with mitochondrial membrane proteins or mistargeted tail-anchored membrane proteins induces MDCs to form and sequester these proteins, we propose that one functional role of MDCs is to create an OMM-enriched trap that segregates and sequesters excess proteins from the mitochondrial surface. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.06.547894v1?rss=1 Authors: Rajshekar, S., Adame-Arana, O., Bajpai, G., Lin, K., Colmenares, S., Safran, S., Karpen, G. H. Abstract: Nucleoli are surrounded by Peri-Centromeric Heterochromatin (PCH), reflecting a close spatial association between the two largest biomolecular condensates in eukaryotic nuclei. We have investigated how this highly conserved organization is established de novo during early Drosophila development and whether these distinct condensates influence each other's 3D organization. High-resolution live imaging revealed a highly dynamic process in which the PCH progressively surrounds nucleoli through a series of stage-specific intermediates. To assess interplay between the condensates, nucleolus assembly was eliminated by deleting the ribosomal RNA genes (rDNA), resulting in increased PCH compaction and subsequent reorganization to a hollow shell. In addition, in embryos lacking rDNA, some nucleolar proteins were abnormally redistributed into new bodies or 'neocondensates,' including enrichment in the PCH hollow core. These observations, combined with computational modeling, led to the hypothesis that nucleolar-PCH associations are mediated by a hierarchy of affinities between PCH, nucleoli, and 'amphiphilic' protein(s) that interact with both nucleolar and PCH components. We identified the nucleolar protein Pitchoune as a candidate for such an amphiphilic protein because it also contains a PCH-interaction motif and fills the PCH hollow core in embryos lacking rDNA. Together, these results unveil a dynamic program for establishing nucleolar-PCH associations during animal development, demonstrate that nucleoli are required for normal PCH organization, and identify Pitchoune as a likely molecular link for stabilizing PCH-nucleolar associations. Finally, we propose that disrupting affinity hierarchies could cause cellular disease phenotypes by liberating components that form 'neocondensates' or other abnormal structures through self-association and secondary affinities. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.06.548044v1?rss=1 Authors: Zhang, Z., Xu, Z., McGuire, H., Essam, C., Nicholson, A., Hamilton, T. J., Li, J., Eshraghian, J. K., Yong, K.-T., Vigolo, D., Kavehei, O. Abstract: Flow cytometry is a widespread and high-throughput technology that can measure the features of cells and can be combined with fluorescence analysis for additional phenotypical characterisations but only provide low-dimensional output and spatial resolution. Imaging flow cytometry is another technology that offers rich spatial information, allowing more profound insight into single-cell analysis. However, offering such high-resolution, full-frame feedback can compromise speed and has become a significant trade-off challenge to tackle during development. In addition, the current dynamic range offered by conventional photosensors can only capture limited fluorescence signals, exacerbating the difficulties in elevating performance speed. Neuromorphic photo-sensing architecture focuses on the events of interest via individual-firing pixels to reduce data redundancy and provide low latency in data processing. With the inherent high dynamic range, this architecture has the potential to drastically elevate the performance in throughput by incorporating motion-activated spatial resolution. Herein, we presented an early demonstration of neuromorphic cytometry with the implementation of object counting and size estimation to measure 8~$\mu$m and 15~$\mu$m polystyrene-based microparticles and human monocytic cell line (THP-1). In this work, our platform has achieved highly consistent outputs with a widely adopted flow cytometer (CytoFLEX) in detecting the total number and size of the microparticles. Although the current platform cannot deliver multiparametric measurements on cells, future endeavours will include further functionalities and increase the measurement parameters (granularity, cell condition, fluorescence analysis) to enrich cell interpretation. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.06.547935v1?rss=1 Authors: Kalampouka, I., Mould, R. R., Botchway, S. W., Mackenzie, A., Nunn, A. V., Thomas, E. L., Bell, J. D. Abstract: The application of near infrared (NIR)-light to living systems has been suggested as a potential method to enhance tissue repair, decrease inflammation, and possibly mitigate cancer therapy-associated side effects. In this study, we examined the effect of exposing three cell lines: breast cancer (MCF7), non-cancer breast cells (MCF10A), and lung fibroblasts (IMR-90), to 734 nm NIR-light for 20 minutes per day for six days, and measuring changes in cellular senescence. Positive senescent populations were induced using doxorubicin. Flow cytometry was used to assess relative levels of senescence together with mitochondria-related variables. Exposure to NIR-light significantly increased the level of senescence in MCF7 cells (13.5%; P less than 0.01), with no observable effects on MCF10A or IMR-90 cell lines. NIR-induced senescence was associated with significant changes in mitochondria homeostasis, including raised ROS level (36.0%; P less than 0.05) and mitochondrial membrane potential (14.9%; P less than 0.05), with no changes in mitochondrial Ca2+. These results suggest that NIR-light exposure can significantly arrest the proliferation of breast cancer cells via inducing senescence, while leaving non-cancerous cell lines unaffected. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.06.547926v1?rss=1 Authors: Pauwels, J., Van de Steene, T., Van de Velde, J., Eyckerman, S., Gevaert, K. Abstract: Extracellular vesicles (EVs), membrane-delimited nanovesicles that are secreted by cells into the extracellular environment, are gaining substantial interest due to their involvement in cellular homeostasis and their contribution to disease pathology. The latter in particular has led to an exponential increase in interest in EVs as they are considered to be circulating packages containing potential biomarkers and are also a possible biological means to deliver drugs in a cell-specific manner. However, several challenges hamper straightforward analysis of EVs as they are generally low abundant and reside in complex biological matrices. These matrices typically contain protein concentrations that vastly exceed those of the EV proteome and contain particles in the same size and density range (e.g. protein aggregates and apolipoprotein particles). Therefore, extensive EV isolation and purification protocols are imperative and many have been developed, including (density) ultracentrifugation, size-exclusion and precipitation methods. Here, we describe an approach based on 300 kDa MWCO filtration, which allows processing of multiple samples in parallel within a reasonable timeframe and at moderate cost. We demonstrate that our strategy is capable of quantitatively retaining EV particles on filters, whilst allowing extensive washing with relatively high percentages of the mild detergent TWEEN-20. In addition, we provide evidence that the retained EVs can be recuperated from the filter for qualitative studies or can be directly lysed on the filter for the recovery of the EV protein cargo for proteome analysis. Applying this strategy on MCF7 conditioned medium using different percentages of serum, we observed dramatic changes in the EV proteome. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.06.547951v1?rss=1 Authors: Cloots, E., Guilbert, P., Provost, M., Neidhardt, L., Van de Velde, E., Fayazpour, F., De Sutter, D., Savvides, S. N., Eyckerman, S., Janssens, S. Abstract: As secretory cells specialized in the production of mucins, intestinal goblet cells are challenged by the need for efficient protein folding. Goblet cells express Inositol-Requiring Enzyme 1{beta} (IRE1{beta}), a unique unfolded protein response (UPR) sensor that is part of an adaptive mechanism that regulates the demands of mucin production and secretion. However, how IRE1{beta} activity is tuned to mucus folding load remains unknown. We identified the disulfide isomerase and mucin chaperone AGR2 as a goblet cell specific protein that crucially regulates IRE1{beta}-, but not IRE1-mediated signaling. AGR2 binding to IRE1{beta} disrupts IRE1{beta} dimerization, thereby blocking its downstream endonuclease activity. Depletion of endogenous AGR2 from goblet cells induces spontaneous IRE1{beta} activation, suggesting that alterations in AGR2 availability in the endoplasmic reticulum sets the threshold for IRE1{beta} activation. We found that AGR2 mutants lacking their catalytic cysteine or displaying the disease-associated mutation H117Y were no longer able to dampen IRE1{beta} activity. Collectively, these results demonstrate that AGR2 is a central chaperone regulating the goblet cell UPR by acting as a rheostat of IRE1{beta} endonuclease activity. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.06.547997v1?rss=1 Authors: Suzuki, S., West, M., Zhang, Y., Fan, J. S., Roberts, R. T., Odorizzi, G., Emr, S. D. Abstract: Endosomes are specialized organelles that function in the secretory and endocytic protein sorting pathways. Endocytosed cell surface receptors and transporters destined for lysosomal degradation are sorted into intralumenal vesicles (ILVs) at endosomes by Endosomal Sorting Complex Required for Transport (ESCRT) proteins. The endosomes (multivesicular bodies, MVBs) then fuse with the lysosome. During endosomal maturation, the number of ILVs increases, but the size of endosomes does not decrease despite consumption of the limiting membrane during ILV formation. Vesicle-mediated trafficking is thought to provide lipids to support MVB biogenesis. However, we have uncovered an unexpected contribution of a large bridge-like lipid transfer protein, Vps13, in this process. Here, we reveal that Vps13-mediated lipid transfer at ER-endosome contact sites is required for the ESCRT pathway. We propose that Vps13 may play a critical role in supplying lipids to the endosome, ensuring continuous ESCRT-mediated sorting during MVB formation. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.06.547837v1?rss=1 Authors: Bagley, D. C., Morham, S. G., Kaplan, J., Ward, D. M. Abstract: Mon1a has been shown to function in the endolysosomal pathway and the secretory pathway, interact with dynein and affecting ER to Golgi traffic. Here we show that Mon1a is also required for maintenance of the Golgi apparatus. We identified the F-BAR protein FCHO2 as a Mon1a-interacting protein by both yeast two-hybrid analysis and co-immunoprecipitation. siRNA-dependent reductions in Mon1a or FCHO2 resulted in Golgi fragmentation. Membrane trafficking through the secretory apparatus in FCHO2-depleted cells was unaltered, however, reduction of FCHO2 affected the uniform distribution of Golgi enzymes necessary for carbohydrate modification. Fluorescence recovery after photobleaching analysis showed that the Golgi ministacks in Mon1a- or FCHO2-silenced cells did not exchange resident membrane proteins. The effect of FCHO2 silencing on Golgi structure was partially cell cycle-dependent and required mitosis-dependent Golgi fragmentation, whereas the effect of Mon1a-silencing on Golgi disruption was not cell cycle-dependent. mCherry-FCHO2 transiently colocalized on Golgi structures independent of Mon1a. These findings suggest that Mon1a has functions throughout the secretory pathway including interacting with dynein at the ER-Golgi interface in vesicle formation and then interacting with FCHO2 at the Golgi to generate lateral links between ministacks, thus creating Golgi ribbons. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.07.548115v1?rss=1 Authors: Listian, S. A., Kol, M., Ufelmann, E., Eising, S., Froehlich, F., Walter, S., Holthuis, J. C. M., Barisch, C. Abstract: Dictyostelium discoideum is a professional phagocyte frequently used as experimental model to study cellular processes underlying the recognition, engulfment and infection course of microbial pathogens. Sphingolipids are abundant components of the plasma membrane that bind cholesterol, control vital membrane properties, participate in signal transmission and serve as adhesion molecules in recognition processes relevant to immunity and infection. While the pathway of sphingolipid biosynthesis has been well characterized in plants, animals and fungi, the identity of sphingolipids produced in D. discoideum, an organism at the crossroads between uni- and multicellular life, is not known. Combining lipidomics with a bioinformatics-based cloning strategy for key sphingolipid biosynthetic enzymes, we show here that D. discoideum produces phosphoinositol-containing sphingolipids with predominantly phytoceramide backbones. Cell-free expression of candidate inositol-phosphorylceramide (IPC) synthases from D. discoideum in defined lipid environments enabled identification of an enzyme that selectively catalyses the transfer of phosphoinositol from phosphatidylinositol onto ceramide. The corresponding IPC synthase, DdIPCS1, is non-homologous to but shares multiple sequence motifs with yeast IPC and human sphingomyelin synthases and localizes to the Golgi apparatus as well as the contractile vacuole of D. discoideum. Collectively, these findings open up important opportunities for exploring a role of sphingolipids in phagocytosis and infection across major evolutionary boundaries. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.07.548169v1?rss=1 Authors: Wilson, Z. N., West, M., English, A. M., Odorizzi, G., Hughes, A. Abstract: Preserving the health of the mitochondrial network is critical to cell viability and longevity. To do so, mitochondria employ several membrane remodeling mechanisms, including the formation of mitochondrial-derived vesicles (MDVs) and compartments (MDCs) to selectively remove portions of the organelle. In contrast to well-characterized MDVs, the distinguishing features of MDC formation and composition remain unclear. Here we used electron tomography to observe that MDCs form as large, multilamellar domains that generate concentric spherical compartments emerging from mitochondrial tubules at ER-mitochondria contact sites. Time-lapse fluorescence microscopy of MDC biogenesis revealed that mitochondrial membrane extensions repeatedly elongate, coalesce, and invaginate to form these compartments that encase multiple layers of membrane. As such, MDCs strongly sequester portions of the outer mitochondrial membrane, securing membrane cargo into a protected domain, while also enclosing cytosolic material within the MDC lumen. Collectively, our results provide a model for MDC formation and describe key features that distinguish MDCs from other previously identified mitochondrial structures and cargo-sorting domains. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.07.548172v1?rss=1 Authors: Darden, C., Donkor, J. E., Korolkova, O., Khan Barozai, M. Y., Chaudhuri, M. Abstract: Nuclear-encoded mitochondrial proteins are correctly translocated to their proper sub-mitochondrial destination using location specific mitochondrial targeting signals (MTSs) and via multi-protein import machineries (translocases) in the outer and inner mitochondrial membranes (TOM and TIMs, respectively). However, MTSs of multi-pass Tims are less defined. Here we report the characterization of the MTSs of Trypanosoma brucei Tim17 (TbTim17), an essential component of the most divergent TIM complex. TbTim17 possesses a characteristic secondary structure including four predicted transmembrane (TM) domains in the center with hydrophilic N- and C- termini. After examining mitochondrial localization of various deletion and site-directed mutants of TbTim17 in T. brucei using subcellular fractionation and confocal microscopy we located at least two internal signals, 1) within TM1 (31-50 AAs) and 2) TM4 + Loop 3 (120-136 AAs). Both signals are required for proper targeting and integration of TbTim17 in the membrane. Furthermore, a positively charged residue (K122) is critical for mitochondrial localization of TbTim17. This is the first report of characterizing the internal mitochondrial targeting signals (ITS) for a multipass inner membrane protein in a divergent eukaryote, like T. brucei. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.06.547980v1?rss=1 Authors: Barger, S. R., Penfield, L., Bahmanyar, S. Abstract: Barrier-to-autointegration factor (BAF) is a DNA binding protein that crosslinks chromatin to assemble the nuclear envelope (NE) after mitosis. BAF also binds the Lap2b-Emerin-Man1 (LEM) domain family of NE proteins to repair interphase ruptures. The NE adaptors to ESCRTs, LEMD2-CHMP7, seal NE holes surrounding mitotic spindle microtubules (MTs), but whether NE hole closure in mitosis involves BAF-LEM binding is not known. Here, we analyze NE sealing after meiosis II in C. elegans oocytes to show that BAF-LEM binding and LEM-2-CHMP-7 have distinct roles in hole closure around spindle MTs. LEM-2/EMR-1 (emerin) function redundantly with BAF-1 to seal the NE. Compromising BAF-LEM binding revealed an additional role for EMR-1 in maintenance of the NE permeability barrier and an essential role for LEM-2-CHMP-7 in preventing NE assembly failure. The WH domain of LEM-2 recruits the majority of CHMP-7 to the NE in C. elegans and a LEM-2 -independent pool of CHMP-7, which is mostly enriched in the nucleoplasm, also contributes to NE stability. Thus, NE hole closure surrounding spindle MTs requires redundant mechanisms that safeguard against failure in NE assembly to support embryogenesis. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.06.548042v1?rss=1 Authors: Fu, H., Li, J., Zhang, C., Du, P., Gao, G., Ge, Q., Guan, X., Cui, D. Abstract: Senile plaque blue autofluorescence in the Alzheimers disease (AD) was discovered around 40 years ago, however, its impact on AD pathology is not fully examined. We analyzed senile plaques with immunohistochemistry and fluorescence imaging on AD brain pathological sections and also the A{beta} aggregation process in vitro in test tubes. In DAPI or Hoechst staining experiments, the data showed that the nuclear blue fluorescence could only be correctly assigned after subtracting the blue autofluorescence background. The plaque cores have very strong blue autofluorescence which is roughly 2.09 times of average DAPI nuclear staining and roughly 1.78 times of average Hoechst nuclear staining. The composite flower-like structures formed by Cathepsin D lysosomal staining wrapping dense core blue fluorescence should not be considered as central-nucleated neurons filled with defective lysosomes since there was no nuclear staining in the plaque core when the blue autofluorescence was subtracted. Furthermore, the dense cores were shown to be completely lack of nuclear signals by PI staining. The A{beta} aggregation assay indicated that both A{beta} self-oligomers and A{beta}/Hemoglobin (Hb) heterocomplexes had significant blue autofluorescence. However, the blue autofluorescence intensity was not always proportional to the intensity of A{beta} immunostaining. The majority of aggregates in the A{beta}/Hb incubations were sensitive to Proteinase K (PK) digestion while the rest were PK resistant. The blue autofluorescence of A{beta} aggregates not only labels senile plaques but also illustrates red blood cell aggregation, hemolysis, CAA, vascular amyloid plaques, vascular adhesion and microaneurysm. In summary, we conclude that A{beta}-aggregation-generated blue autofluorescence is an excellent amyloid pathology marker in the senile plaques, blood and vascular pathologies in the Alzheimers disease. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.06.548042v1?rss=1 Authors: Fu, H., Li, J., Zhang, C., Du, P., Gao, G., Ge, Q., Guan, X., Cui, D. Abstract: Senile plaque blue autofluorescence in the Alzheimer's disease (AD) was discovered around 40 years ago, however, its impact on AD pathology is not fully examined. We analyzed senile plaques with immunohistochemistry and fluorescence imaging on AD brain pathological sections and also the A{beta} aggregation process in vitro in test tubes. In DAPI or Hoechst staining experiments, the data showed that the nuclear blue fluorescence could only be correctly assigned after subtracting the blue autofluorescence background. The plaque cores have very strong blue autofluorescence which is roughly 2.09 times of average DAPI nuclear staining and roughly 1.78 times of average Hoechst nuclear staining. The composite flower-like structures formed by Cathepsin D lysosomal staining wrapping dense core blue fluorescence should not be considered as central-nucleated neurons filled with defective lysosomes since there was no nuclear staining in the plaque core when the blue autofluorescence was subtracted. Furthermorethe dense cores were shown to be completely lack of nuclear signals by PI staining. The A{beta} aggregation assay indicated that both A{beta} self-oligomers and A{beta}/Hemoglobin (Hb) heterocomplexes had significant blue autofluorescence. However, the blue autofluorescence intensity was not always proportional to the intensity of A{beta} immunostaining. The majority of aggregates in the A{beta}/Hb incubations were sensitive to Proteinase K (PK) digestion while the rest were PK resistant. The blue autofluorescence of A{beta} aggregates not only labels senile plaques but also illustrates red blood cell aggregation, hemolysis, CAA, vascular amyloid plaques, vascular adhesion and microaneurysm. In summary, we conclude that A{beta}-aggregation-generated blue autofluorescence is an excellent amyloid pathology marker in the senile plaques, blood and vascular pathologies in the Alzheimer's disease. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.06.548042v1?rss=1 Authors: Fu, H., Li, J., Zhang, C., Du, P., Gao, G., Ge, Q., Guan, X., Cui, D. Abstract: Senile plaque blue autofluorescence in the Alzheimers disease (AD) was discovered around 40 years ago, however, its impact on AD pathology is not fully examined. We analyzed senile plaques with immunohistochemistry and fluorescence imaging on AD brain pathological sections and also the A{beta} aggregation process in vitro in test tubes. In DAPI or Hoechst staining experiments, the data showed that the nuclear blue fluorescence could only be correctly assigned after subtracting the blue autofluorescence background. The plaque cores have very strong blue autofluorescence which is roughly 2.09 times of average DAPI nuclear staining and roughly 1.78 times of average Hoechst nuclear staining. The composite flower-like structures formed by Cathepsin D lysosomal staining wrapping dense core blue fluorescence should not be considered as central-nucleated neurons filled with defective lysosomes since there was no nuclear staining in the plaque core when the blue autofluorescence was subtracted. Furthermore, the dense cores were shown to be completely lack of nuclear signals by PI staining. The A{beta} aggregation assay indicated that both A{beta} self-oligomers and A{beta}/Hemoglobin (Hb) heterocomplexes had significant blue autofluorescence. However, the blue autofluorescence intensity was not always proportional to the intensity of A{beta} immunostaining. The majority of aggregates in the A{beta}/Hb incubations were sensitive to Proteinase K (PK) digestion while the rest were PK resistant. The blue autofluorescence of A{beta} aggregates not only labels senile plaques but also illustrates red blood cell aggregation, hemolysis, CAA, vascular amyloid plaques, vascular adhesion and microaneurysm. In summary, we conclude that A{beta}-aggregation-generated blue autofluorescence is an excellent amyloid pathology marker in the senile plaques, blood and vascular pathologies in the Alzheimers disease. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.06.548042v1?rss=1 Authors: Fu, H., Li, J., Zhang, C., Du, P., Gao, G., Ge, Q., Guan, X., Cui, D. Abstract: Senile plaque blue autofluorescence in the Alzheimer's disease (AD) was discovered around 40 years ago, however, its impact on AD pathology is not fully examined. We analyzed senile plaques with immunohistochemistry and fluorescence imaging on AD brain pathological sections and also the A{beta} aggregation process in vitro in test tubes. In DAPI or Hoechst staining experiments, the data showed that the nuclear blue fluorescence could only be correctly assigned after subtracting the blue autofluorescence background. The plaque cores have very strong blue autofluorescence which is roughly 2.09 times of average DAPI nuclear staining and roughly 1.78 times of average Hoechst nuclear staining. The composite flower-like structures formed by Cathepsin D lysosomal staining wrapping dense core blue fluorescence should not be considered as central-nucleated neurons filled with defective lysosomes since there was no nuclear staining in the plaque core when the blue autofluorescence was subtracted. Furthermorethe dense cores were shown to be completely lack of nuclear signals by PI staining. The A{beta} aggregation assay indicated that both A{beta} self-oligomers and A{beta}/Hemoglobin (Hb) heterocomplexes had significant blue autofluorescence. However, the blue autofluorescence intensity was not always proportional to the intensity of A{beta} immunostaining. The majority of aggregates in the A{beta}/Hb incubations were sensitive to Proteinase K (PK) digestion while the rest were PK resistant. The blue autofluorescence of A{beta} aggregates not only labels senile plaques but also illustrates red blood cell aggregation, hemolysis, CAA, vascular amyloid plaques, vascular adhesion and microaneurysm. In summary, we conclude that A{beta}-aggregation-generated blue autofluorescence is an excellent amyloid pathology marker in the senile plaques, blood and vascular pathologies in the Alzheimer's disease. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.05.547885v1?rss=1 Authors: Zhang, H., Wang, J., Jin, L., Zhu, Y., Guo, Y., Zhang, M., Zhang, Y., Wang, Z., Su, Y., Wu, Y., Ji, B., Toomre, D., Liu, X., Xu, Y. Abstract: Structured illumination microscopy (SIM) is a versatile super-resolution technique known for its compatibility with a wide range of probes and fast implementation. While 3D SIM is capable of achieving a spatial resolution of ~120 nm laterally and ~300 nm axially, attempting to further enhance the resolution through methods such as nonlinear SIM or 4-beam SIM introduces complexities in optical configurations, increased phototoxicity, and reduced temporal resolution. Here, we have developed a novel method that combines SIM with augmented super-resolution radial fluctuations (aSRRF) utilizing a single image through image augmentation. By applying aSRRF reconstruction to SIM images, we can enhance the SIM resolution to ~50 nm isotopically, without requiring any modifications to the optical system or sample acquisition process. Additionaly, we have incorporated the aSRRF approach into an ImageJ plugin and demonstrated its versatility across various fluorescence microscopy images, showcasing a remarkable two-fold resolution increase. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.04.547690v1?rss=1 Authors: Hensel, I. V., Elias, S., Steinhauer, M., Stoll, B., Benfatto, S., Merkt, W., Krienke, S., Lorenz, H.-M., Haas, J., Wildeman, B., Resnik-Docampo, M. Abstract: Human intestinal epithelial cells are the interface between potentially harmful luminal content and basally residing immune cells. Their role is not only nutrient absorption but also the formation of a tight monolayer that constantly secrets mucus creating a multi-layered protective barrier. Alterations in this barrier can lead to increased gut permeability which is frequently seen in individuals with chronic extraintestinal autoimmune diseases, such as Systemic Lupus Erythematosus (SLE). Despite recent advances in identifying alterations in gut microbiota composition in SLE patients, not much attention has been given to the epithelial barrier itself. To date, it remains largely unexplored which role and function intestinal epithelial cells have in SLE pathology. Here, we present a unique near-physiologic in vitro model specifically designed to examine the effects of SLE on the epithelial cells. We utilize human colon organoids that are stimulated with serum obtained from SLE patients. Combining bulk and scRNA transcriptomic analysis with functional assays revealed that SLE serum stimulation induced a unique expression profile marked by a type I interferon gene signature. Additionally, organoids exhibited decreased mitochondrial fitness, alterations in mucus composition and imbalanced cellular composition. Similarly, transcriptomic analysis of SLE human colon biopsies revealed a downregulation of epithelial secretory markers. Our work uncovers a crucial connection between SLE and intestinal homeostasis that might be promoted in vivo through the blood, offering insights into the causal connection of barrier dysfunction and autoimmune diseases. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.03.547506v1?rss=1 Authors: Kehlenbach, R. H., James, C., Möller, U., König, S., Urlaub, H. Abstract: ELYS is a nucleoporin that localizes to the nuclear side of the nuclear envelope in interphase cells. In mitosis, it serves as an assembly platform that interacts with chromatin and then with nucleoporin subcomplexes to initiate the formation of novel nuclear pore complexes. Here we describe the interaction of ELYS with the membrane protein VAPB. In mitosis, ELYS becomes phosphorylated at many sites, including a predicted FFAT (two phenylalanines in an acidic tract) motif, which is shown to mediate interaction with the MSP (major sperm protein)-domain of VAPB. Phosphorylation-dependent binding of VAPB to ELYS is demonstrated by peptide binding assays and co-immunoprecipitation experiments. In anaphase, the two proteins co-localize to the non-core region of the newly forming nuclear envelope. Depletion of VAPB resulted in prolonged mitosis and slow progression from meta- to anaphase and also to chromosome segregation defects. Together, our results suggest an active role of VAPB in recruiting membrane fragments to chromatin and in the biogenesis of a novel nuclear envelope during mitosis. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.05.547782v1?rss=1 Authors: Luu, O., Barua, D., Winklbauer, R. Abstract: Convergent extension of the chordamesoderm is the best-examined gastrulation movement in Xenopus. Here we study general features of cell-cell contacts in this tissue by combining depletion of adhesion factors C-cadherin, Syndecan-4, fibronectin, and hyaluronic acid, the analysis of respective contact width spectra and contact angles, and La3+ staining of the pericellular matrix. We provide evidence that like in other gastrula tissues, cell-cell adhesion in the chordamesoderm is largely mediated by different types of pericellular matrix. Specific glycocalyx structures previously identified in Xenopus gastrula tissues are absent in chordamesoderm but other contact types like 10-20 nm wide La3+ stained structures are present instead. Knockdown of any of the adhesion factors reduces the abundance of cell contacts but not the average relative adhesiveness of the remaining ones: a decrease of adhesiveness at low contact widths is compensated by an increase of contact widths and an increase of adhesiveness proportional to width. From the adhesiveness-width relationship, we derive a model of chordamesoderm cell adhesion that involves the interdigitation of distinct pericellular matrix units. Quantitative description of pericellular matrix deployment suggests that reduced contact abundance upon adhesion factor depletion is due to some contact types becoming non-adhesive and others being lost. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.05.547674v1?rss=1 Authors: Demirbas, B., Filina, O., Louisse, T., Goos, Y., Sanchez-Romero, M. A., Olmedo, M., van Zon, J. Abstract: FOXO transcription factors are highly conserved effectors of insulin and insulin-like growth factor signaling, that are crucial for mounting responses to a broad range of stresses. Key signaling step is the stress-induced translocation of FOXO proteins to the nucleus, where they induce expression of stress response genes. Insulin signaling and FOXO proteins often control responses that impact the entire organism, such as growth or starvation-induced developmental arrest, but how body-wide coordination is achieved is poorly understood. Here, we leverage the small size of the nematode C. elegans, to quantify translocation dynamics of DAF-16, the sole C. elegans FOXO transcription factor, with single-cell resolution, yet in a body-wide manner. Surprisingly, when we exposed individual animals to constant levels of stress that cause larval developmental arrest, DAF-16/FOXO translocated between the nucleus and cytoplasm in stochastic pulses. Even though the occurrence of translocation pulses was random, they nevertheless exhibited striking synchronization between cells throughout the body. DAF-16/FOXO pulse dynamics were strongly linked to body-wide growth, with isolated translocation pulses causing transient reduction of growth and full growth arrest observed only when pulses were of sufficiently high frequency or duration. Finally, we observed translocation pulses of FOXO3A in mammalian cells under nutrient stress. The link between DAF-16/FOXO pulses and growth provides a rationale for their synchrony, as uniform proportions are only maintained when growth and, hence, pulse dynamics are tightly coordinated between all cells. Long-range synchronization of FOXO translocation dynamics might therefore be integral also to growth control in more complex animals. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.04.547746v1?rss=1 Authors: Ly, C. H., Chung, J. D., Nguyen, J. H., Tian, L., Schroeder, J., Knaupp, A. S., Su, S., Trieu, J., Salmi, T. M., Zalcenstein, D., Jabbari, J. S., Boughton, B. A., Cox, A. G., Naik, S. H., Polo, J. M., Ritchie, M. E., Lynch, G. S., Ryall, J. G. Abstract: Skeletal muscle contains a resident population of somatic stem cells capable of both self-renewal and differentiation. The signals that regulate this important decision have yet to be fully elucidated. Here we use metabolomics and mass spectrometry imaging (MSI) to identity a state of localized hyperglycaemia following skeletal muscle injury. We show that committed muscle progenitor cells exhibit an enrichment of glycolytic and TCA cycle genes and that extracellular monosaccharide availability regulates intracellular citrate levels and global histone acetylation. Muscle stem cells exposed to a reduced (or altered) monosaccharide environment demonstrate reduced global histone acetylation and transcription of myogenic determination factors (including myod1). Importantly, reduced monosaccharide availability was linked directly to increased rates of asymmetric division and muscle stem cell self-renewal in regenerating skeletal muscle. Our results reveal an important role for the extracellular metabolic environment in the decision to undergo self-renewal or myogenic commitment during skeletal muscle regeneration. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.04.547647v1?rss=1 Authors: Amran, A., Pigatto, L., Farley, J., Godini, R., Pocock, R., Gopal, S. Abstract: The extracellular matrix (matrisome) provides chemical and mechanical cues to control the structure and function of cells and tissues. Yet, comprehensive understanding of how matrisome factors individually and collectively control cell and tissue behavior in vivo is lacking. Here, we systematically investigate the function of 443 conserved matrisome-coding genes in controlling germ cell behavior within a complex tissue - the Caenorhabditis elegans germline. Using high-content imaging, 3D reconstruction and cell behavior analysis of greater than 3500 germlines and greater than 7 million germ cells, we identify specific matrisome factors that regulate germline structure, protein distribution, germ cell cycle and fate, apoptosis, and oocyte health. These findings reveal matrisome networks acting autonomously and non-autonomously to coordinate germ cell behavior, providing new avenues to study and manipulate cell fates. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.04.547644v1?rss=1 Authors: Otcenaskova, T., Stopkova, R., Stopka, P. Abstract: Speciation and sperm competition have been shown to be the major driving forces for sperm morphology variation, swimming velocity and metabolism. We used light microscopy to measure sperm traits and nLC-MS/MS to detect proteomic variation in three species of rodents: promiscuous Apodemus flavicollis, less promiscuous Microtus arvalis, and the least promiscuous Mus musculus musculus. We show that the length of sperm apical hook containing the acrosome is the most variable trait and that this variation is reflected by proteomes on interspecific and intraspecific levels. Thus, we provide potential markers of selection such as Ldhc (in Mus and Apodemus) for long acrosomes which is a gene coding L-lactate dehydrogenase that is involved in sperm motility and Spaca1, which is important in sperm-oocyte fusion), and e.g. Mup17 for short acrosomes, a gene coding a Major urinary protein that likely chelates lipophilic compounds after spermiogenesis. In short, longer acrosomes are characteristic of proteins involved in fertilisation and gluconeogenesis, while shorter acrosomes contain more cytoskeletal proteins important for spermiogenesis. For the first time, we demonstrate that there is an innate and evolvable variability in sperm morphology and corresponding proteomes within species that can be driven by sperm competition to species-specific reproductive optima. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.03.547611v1?rss=1 Authors: Forsgren, E., Cloarec, O., Jonsson, P., Trygg, J. Abstract: Cell Painting is an established community-based, microscopy-assay platform that provides high-throughput, high-content data for biological readouts. In November 2022, the JUMP-Cell Painting Consortium released the largest annotated, publicly available dataset, comprising more than 2 billion cell images. This dataset is designed for predicting the activity and toxicity of 100k drug compounds, with the aim to make cell images as computable as genomes and transcriptomes. In this paper, we have developed a data analytics workflow that is both scalable and computationally efficient, while providing significant, biologically relevant insights for biologists estimating and comparing the effects of different drug treatments. The two main objectives proposed include: 1) a simple, yet sophisticated, scalable data analytics metric that utilizes negative controls for comparing morphological cell profiles. We call this metric the equivalence score (Eq. score). 2) A workflow to identify and amplify subtle morphological image profile changes caused by drug treatments, compared to the negative controls. In summary, we provide a data analytics workflow to assist biologists in interpreting high-dimensional image features, not necessarily limited to morphological ones. This enhances the efficiency of drug candidate screening, thereby streamlining the drug development process. By increasing our understanding of using complex image-based data, we can decrease the cost and time to develop new, life-saving treatments. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.04.547731v1?rss=1 Authors: Tennakoon, M., Thotamune, W., Payton, J. L., Karunarathne, A. Abstract: Prenylation is a universal and irreversible post-translational modification that supports membrane interactions of proteins involved in various cellular processes, including migration, proliferation, and survival. Thus, dysregulation of prenylation contributes to multiple disorders, including cancers, vascular diseases, and neurodegenerative diseases. During prenylation, prenyltransferase enzymes tether metabolically produced isoprenoid lipids to proteins via a thioether linkage. Pharmacological inhibition of the lipid synthesis pathway by statins has long been a therapeutic approach to control hyperlipidemia. Building on our previous finding that statins inhibit membrane association of G protein {gamma} (G{gamma}) in a subtype-dependent manner, we investigated the molecular reasoning for this differential. We examined the prenylation efficacy of carboxy terminus (Ct) mutated G{gamma} in cells exposed to Fluvastatin and prenyl transferase inhibitors and monitored the subcellular localization of fluorescently tagged G{gamma} subunits and their mutants using live-cell confocal imaging. Reversible optogenetic unmasking-masking of Ct residues was used to probe their contribution to the prenylation process and membrane interactions of the prenylated proteins. Our findings suggest that specific Ct residues regulate membrane interactions of the G{gamma} polypeptide statin sensitivity and prenylation efficacy. Our results also show that a few hydrophobic and charged residues at the Ct are crucial determinants of a protein's prenylation ability, especially under suboptimal conditions. Given the cell and tissue-specific expression of different G{gamma} subtypes, our findings explain how and why statins differentially perturb heterotrimeric G protein signaling in specific cells and tissues. Our results may provide molecular reasoning for repurposing statins as Ras oncogene inhibitors and the failure of using prenyltransferase inhibitors in cancer treatment. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.02.546740v1?rss=1 Authors: Yoshinaga, D., Feng, R., Prondzynski, M., Shani, K., Tharani, Y., Milosh, J., Walker, D., Carreon, K., Boss, B. M., Parker, K. K., Pu, W. T., Bezzerides, V. J. Abstract: BACKGROUND: N-terminal-acetyltransferases catalyze N-terminal acetylation (Nt-acetylation), an evolutionarily conserved co-translational modification. Nt-acetylation regulates diverse signaling pathways, yet little is known about its effects in the heart. To gain insights, we studied NAA10-related syndrome, in which mutations in NAA10, which catalyzes Nt-acetylation, causes severe QT prolongation, hypotonia, and neurodevelopmental delay. METHODS: We identified a missense variant in NAA10 (c.10C greater than A; p.R4S) that segregated with severe QT prolongation, arrhythmia, cardiomyopathy, and sudden death in a large kindred. We developed patient-derived and genome-edited human induced pluripotent stem cell (iPSC) models and deeply phenotyped iPSC-derived cardiomyocytes (iPSC-CMs) to dissect the mechanisms underlying NAA10-mediated cardiomyocyte dysfunction. RESULTS: The NAA10-R4S mutation reduced enzymatic activity, decreased expression levels of NAA10/NAA15 proteins, and destabilized the NatA complex. In iPSC-CM models of NAA10 dysfunction, dysregulation of the late sodium and slow rectifying potassium currents caused severe repolarization abnormalities, consistent with clinical QT prolongation and increased risk for arrhythmogenesis. Engineered heart tissues generated from mutant NAA10 cell lines had significantly decreased contractile force and sarcomeric disorganization, consistent with the cardiomyopathic phenotype in the identified family members. Diastolic calcium levels were increased with corresponding alterations in calcium handling pathways. We identified small molecule and genetic therapies that reversed the effects of NAA10 dysregulation of iPSC-CMs. CONCLUSIONS: Our study defines novel roles of Nt-acetylation in cardiac ion channel regulation and delineates mechanisms underlying QT prolongation, arrhythmia, and cardiomyopathy caused by NAA10 dysfunction. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.03.547452v1?rss=1 Authors: Punzon-Jimenez, P., Machado-Lopez, A., Perez-Moraga, R., Llera-Oyola, J., Grases, D., Galvez-Viedma, M., AlSibai, M., Satorres, E., Badenes, R., Ferrer, C., Porta-Pardo, E., Roson, B., Simon, C., Mas, A. Abstract: The myometrial dysfunction associated with aging can prompt complications during pregnancy and labor, causing a 7.8-fold increase in maternal mortality in women over 40. Using single-cell/single-nucleus RNA sequencing and spatial transcriptomics, we constructed a cellular atlas of the aging myometrium from 186,120 cells across twenty peri- and post-menopausal women. We identified 23 myometrial cell subpopulations, including novel contractile capillary, venous capillary, immune-modulated fibroblasts, and nervous system regulatory fibroblasts. Myometrial aging leads to fewer contractile capillary cells, a reduced level of ion channel expression in smooth muscle cells, and impaired gene expression in endothelial, smooth muscle, fibroblast, perivascular, and immune cells. We observed altered myometrial cell-to-cell communication as an aging hallmark associated with the loss of 25/229 signaling pathways, including those related to angiogenesis, tissue repair, contractility, immunity, and nervous system regulation. These insights may contribute to a better understanding of the complications faced by older women during pregnancy and labor. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.03.547539v1?rss=1 Authors: Zhang, C., Saurat, N., Cornacchia, D., Chung, S. Y., Skider, T., Minotti, A., Studer, L., Betel, D. Abstract: The differentiation of human pluripotent stem cells (hPSCs) provides access to most cell types and tissues. However, hPSC-derived lineages capture a fetal-stage of development and methods to accelerate progression to an aged identity are limited. Understanding the factors driving cellular age and rejuvenation is also essential for efforts aimed at extending human life and health span. A prerequisite for such studies is the development of methods to score cellular age and simple readouts to assess the relative impact of various age modifying strategies. Here we established a transcriptional score (RNAge) in young versus old primary fibroblasts, frontal cortex and substantia nigra tissue. We validated the score in independent RNA-seq datasets and demonstrated a strong cell and tissue specificity. In fibroblasts we observed a reset of RNAge during iPSC reprogramming while direct reprogramming of aged fibroblasts to induced neurons (iN) resulted in the maintenance of both a neuronal and a fibroblast aging signature. Increased RNAge in hPSC-derived neurons was confirmed for several age-inducing strategies such as SATB1 loss, progerin expression or chemical induction of senescence (SLO). Using RNAge as a probe set, we next performed an in-silico screen using the L1000 dataset. We identified and validated several novel age-inducing and rejuvenating compounds, and we observed that RNAage captures age-related changes associated with distinct cellular hallmarks of age. Our study presents a simple tool to score age manipulations and identifies compounds that greatly expand the toolset of age-modifying strategies in hPSC derived lineages. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.03.547521v1?rss=1 Authors: Fellows, A. D., Bruntraeger, M., Burgold, T., Bassett, A. R., Carter, A. P. Abstract: The microtubule motor dynein drives retrograde transport from the axon tip back to the cell body and is essential for neuronal survival. Its function requires its cofactor dynactin and regulators LIS1 and NDEL1. However, it is unclear if all dynein components can travel along the axon, how far they move and whether they do so together. Here, we use neuron-inducible (NGN2-OPTi-OX) human-stem-cell lines to endogenously tag dynein components and track them under a near single molecule regime. In the retrograde direction dynein and dynactin can move the entire greater than 500 um length of the axon in one go. Furthermore, LIS1 and NDEL1 also undergo long-distance movement, despite being mainly implicated with initiation of transport. Intriguingly, in the anterograde direction dynein/LIS1 move faster than dynactin/NDEL1 implying they can travel on different cargos. Therefore, neurons ensure efficient transport by keeping dynein/dynactin on cargos over long distances, but keeping them separate until their function is required. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.03.547485v1?rss=1 Authors: Allshire, R., Median-Pritchard, B., Spanos, C., Rappsilber, J., Jeyaprakash, A. A., London, N. Abstract: CENP-A chromatin specifies mammalian centromere identity, and its chaperone HJURP replenishes CENP-A when recruited by the Mis18 complex (Mis18C) via M18BP/KNL2 to CENP-C at kinetochores during interphase. However, the Mis18C recruitment mechanism remains unresolved in species lacking M18BP1, such as fission yeast. Fission yeast centromeres cluster at G2 spindle pole bodies (SPBs) when CENP-ACnp1 is replenished and where Mis18C also localizes. We show that SPBs play an unexpected role in concentrating Mis18C near centromeres through the recruitment of Mis18 by direct binding to the major SPB LInker of Nucleoskeleton and Cytoskeleton (LINC) complex component Sad1. Mis18 recruitment by Sad1 is important for CENP-ACnp1 chromatin establishment and acts in parallel with a CENP-C-mediated Mis18C recruitment pathway to maintain centromeric CENP-ACnp1, but is independent of Sad1-mediated centromere clustering. SPBs therefore provide a non-chromosomal scaffold for both Mis18C recruitment and centromere clustering during G2. This centromere-independent Mis18-SPB recruitment provides a mechanism that governs de novo CENP-ACnp1 chromatin assembly by the proximity of appropriate sequences to SPBs and highlights how nuclear spatial organization influences centromere identity. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.03.547458v1?rss=1 Authors: Kweyamba, P., Hofer, L. M., Kibondo, U. A., Mwanga, R. Y., Sayi, R. M., Matwewe, F., Austin, J. W., Stutz, S., Moore, S. J., Müller, P., Tambwe, M. M. Abstract: Pyrethroid resistance in the key malaria vectors threatens the success of pyrethroid-treated nets. To overcome pyrethroid resistance, Interceptor(R) G2 (IG2), a first-in-class dual insecticidal net that combines alpha-cypermethrin with chlorfenapyr was developed. Chlorfenapyr is a pro-insecticide, requiring bio-activation by oxidative metabolism within the insects mitochondria, constituting a mode of action preventing cross-resistance to pyrethroids. Recent epidemiological trials conducted in Benin and Tanzania confirm IG2s public health value in areas with pyrethroid-resistant Anopheles mosquitoes. As chlorfenapyr might also interfere with the metabolic mechanism of the Plasmodium parasite, we hypothesised that chlorfenapyr may provide additional transmission-reducing effects even if a mosquito survives a sub-lethal dose. Therefore, we tested the effect of chlorfenapyr netting to reduce Plasmodium falciparum transmission using a modified WHO tunnel test with a dose yielding sub-lethal effects. Pyrethroid-resistant Anopheles gambiae s.s. with established mixed-function oxidases and Vgsc-L995F knockdown resistance alleles were exposed to untreated netting and netting treated with 200 mg/m3 chlorfenapyr for 8 hours overnight and then fed on gametocytemic blood meals from naturally infected individuals. Prevalence and intensity of oocysts and sporozoites were determined on day 8 and day 16 after feeding. Both prevalence and intensity of P. falciparum infection in the surviving mosquitoes were substantially reduced in the chlorfenapyr-exposed mosquitoes compared to untreated nets. The odds ratios in the prevalence of oocysts and sporozoites were 0.33 (95% confidence interval; 95% CI: 0.23- 0.46) and 0.43 (95% CI: 0.25-0.73), respectively, while only the incidence rate ratio for oocysts was 0.30 (95% CI: 0.22-0.41). We demonstrated that sub-lethal exposure of pyrethroid-resistant mosquitoes to chlorfenapyr substantially reduces the proportion of infected mosquitoes and the intensity of the P. falciparum infection. This will likely also contribute to the reduction of malaria in communities beyond the direct killing of mosquitoes. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.03.547512v1?rss=1 Authors: Muranaka, Y., Shigetomi, R., Iwasaki, Y., Hamamoto, A., Nakayama, K., Takatsu, H., Shin, H.-W. Abstract: Phosphatidylinositol is a precursor of various phosphoinositides, which play crucial roles in intracellular signaling and membrane dynamics and have impact on diverse aspects of cell physiology. Phosphoinositide synthesis and turnover occur in the cytoplasmic leaflet of the organellar and plasma membranes. P4-ATPases (lipid flippases) are responsible for translocating membrane lipids from the exoplasmic (luminal) to the cytoplasmic leaflet, thereby regulating membrane asymmetry. However, the mechanism underlying phosphatidylinositol translocation across cellular membranes remains elusive. Here, we discovered that the phosphatidylcholine flippases ATP8B1, ATP8B2, and ATP10A can also translocate phosphatidylinositol at the plasma membrane. To explore the function of these phosphatidylinositol flippases, we used cells depleted of CDC50A, a protein necessary for P4-ATPase function. Upon activation of the Gq-coupled receptor, depletion of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] was accelerated in CDC50A knockout cells compared with control cells, suggesting a decrease in PtdIns4,5P2 levels within the plasma membrane of the knockout cells. These findings highlight the pivotal role of P4-ATPases in maintaining phosphoinositide homeostasis and suggest a mechanism for enrichment of phosphatidylinositol in the cytoplasmic leaflet of the plasma membrane. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.03.547412v1?rss=1 Authors: Rigal, S., Casas, B., Kanebratt, K. P., Wennberg Huldt, C., Magnusson, L. U., Mullers, E., Kalrsson, F., Clausen, M., Hansson, S. F., Jansson Lofmark, R., Ammala, C., Marx, U., Gennemark, P., Cedersund, G., Andersson, T. B., Vilen, L. K. Abstract: Current research on metabolic disorders such as type 2 diabetes relies on animal models because multi-organ diseases cannot be well studied with the standard in vitro assays. Here, we connect models of key metabolism organs, pancreas and liver, on a microfluidic chip to enable diabetes research in a human-based preclinical system. Aided by mechanistic mathematical modelling, we developed a two-organ microphysiological system (MPS) that replicates clinically-relevant phenotypes of diabetic dysregulation both in the liver and pancreas compartments. Exposure to hyperglycemia and high cortisone created a diseased pancreas-liver MPS which displayed beta-cell dysfunction, steatosis, elevated ketone-body secretion, increased glycogen storage, and upregulated gluconeogenic machinery. In turn, normoglycemia and physiological cortisone concentration maintained glucose tolerance and stable liver and beta-cell functions. This method was evaluated for repeatability in two laboratories and was effective in multiple pancreatic islet donors. The model also provides a platform to identify new therapeutic targets as demonstrated with a liver-secreted IL-1R2 protein that induced islet proliferation. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.02.547450v1?rss=1 Authors: Kang, M., Ko, U. H., Oh, E.-J., Kim, H. M., Chung, H. Y., Shin, J. H. Abstract: A scar is considered a natural consequence of the wound-healing process. However, the mechanism by which scars form remains unclear. Here, we suggest a new mechanism of wound healing and scar formation that involves the mechanosensitive regulation of HOX genes. RNA-sequencing of fibroblasts from different types of scars revealed differential HOX gene expression. Computational simulations predicted injury-induced tension loss in the skin, and in vitro experiments revealed a negative correlation between tension and fibroblast proliferation. Remarkably, exogenous tensile stress in fibroblasts has been shown to alter HOX gene expression levels in different scar types. Overall, we propose a model for normal wound healing and scar formation and show that successful wound healing requires tensional homeostasis in the skin tissue, which is regulated by tension-sensitive HOX genes. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.03.547488v1?rss=1 Authors: Romenskaja, D., Jonavice, U., Pivoriunas, A. Abstract: Autophagy dysfunction has been closely related with pathogenesis of many neurodegenerative diseases and therefore represents a potential therapeutic target. Extracellular vesicles (EVs) may act as a potent anti-inflammatory agents and also modulators of autophagy in target cells. However, the molecular mechanisms by which EVs modulate autophagy flux in human microglia remain largely unexplored. In the present study we investigated the effects of EVs derived from human oral mucosa stem cells on the autophagy in human microglia. We demonstrate that EVs promoted autophagy and autophagic flux in human microglia and that this process was dependent on the integrity of lipid rafts. LPS also activated autophagy, but combined treatment with EVs and LPS suppressed autophagy response indicating interference between these signalling pathways. Blockage of Toll-like receptor 4 (TLR4) with anti-TLR4 antibody suppressed EV-induced autophagy. Furthermore, blockage of EV- asscoiated HSP70 chaperone which is one of the endogenous ligands of the TLR4 also suppressed EV- induced lipid raft formation and autophagy. Pre-treatment of microglia with selective inhibitor of v{beta}3/v{beta}5 integrins cilengitide inhibited EV-induced autophagy. Finally, blockage of purinergic P2X4 receptor (P2X4R) with selective inhibitor 5-BDBD also suppressed of EV-induced autophagy. In conclusion, we demonstrate that EVs activate autophagy in human microglia through interaction with HSP70/TLR4, V{beta}3/V{beta}5, and P2X4R signalling pathways and that these effects depend on the integrity of lipid rafts. Our findings could be used for development of new therapeutic strategies targeting disease-associated microglia. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.02.547380v1?rss=1 Authors: Lewis, T. R., Makia, M. S., Castillo, C. M., Hao, Y., Al-Ubaidi, M. R., Skiba, N. P., Conley, S. M., Arshavsky, V. Y., Naash, M. I. Abstract: Visual signal transduction takes place within a stack of flattened membranous "discs" enclosed within the light-sensitive photoreceptor outer segment. The highly curved rims of these discs, formed in the process of disc enclosure, are fortified by large hetero-oligomeric complexes of two homologous tetraspanin proteins, PRPH2 (a.k.a. peripherin-2 or rds) and ROM1. While mutations in PRPH2 affect the formation of disc rims, the role of ROM1 remains poorly understood. In this study, we found that the knockout of ROM1 causes a compensatory increase in the disc content of PRPH2. Despite this increase, discs of ROM1 knockout mice displayed a delay in disc enclosure associated with a large diameter and lack of incisures in mature discs. Strikingly, further increasing the level of PRPH2 rescued these morphological defects. We next showed that disc rims are still formed in a knockin mouse in which the tetraspanin body of PRPH2 was replaced with that of ROM1. Together, these results demonstrate that, despite its contribution to the formation of disc rims, ROM1 can be replaced by an excess of PRPH2 for timely enclosure of newly forming discs and establishing normal outer segment structure. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.02.547408v1?rss=1 Authors: Nguyen, V., Gao, C., Hochman, M., Kravitz, J., Chen, E., Friedman, H., Wenceslau, C., Chen, D., Wang, Y., Nelson, J. S., Jegga, A. G., Tan, W. Abstract: Background: Port Wine Birthmark (PWB) is a congenital vascular malformation resulting from developmentally defective endothelial cells (ECs). Developing clinically relevant disease models is an unmet need for PWB studies. Objective: This study aims to generate PWB-derived induced pluripotent stem cells (iPSCs) and those-iPSC-derived ECs that preserve disease-related phenotypes. Method: PWB iPSCs were generated by reprogramming lesional dermal fibroblasts and were differentiated into ECs. Bulk RNA-seq and ATAC-seq were performed to identify enriched pathways. The functional phenotypes of iPSC-derived ECs were characterized using capillary-like structure (CLS) formation in vitro and Geltrex plug-in assay in vivo. Result: Human PWB and normal iPSC lines were generated through reprogramming of dermal fibroblasts by introducing the Yamanaka factors (Oct3/4, Sox2, Klf4, c-Myc) into them; The iPSCs were differentiated into ECs. These iPSCs and their-derived ECs were validated by expression of series of stem cell and EC biomarkers, respectively. PWB EC showed impaired CLS in vitro with larger perimeters and thicker branches comparing with control iPSC-derived ECs. In plug-in assay, perfused human vasculature formed by PWB iPSC-derived ECs showed bigger perimeters and greater densities than those formed by control iPSC-derived ECs in SCID mice. The transcriptome analysis showed that the impaired pathways of stem cell differentiation, Hippo, Wnt, and focal adhersion persisted through PWB iPSCs to ECs during differentiation. Interactive networks showed that the Hippo and Wnt pathway-related differentially expressed genes (DEGs) significantly function in vasculature development, tube morphology, endothelium development, and EC differentiation. Members of zinc-finger (ZNF) gene family were among the top changed DEGs in both PWB iPSCs and ECs. The ZNF DEGs confer significant functions in transcriptional regulation, chromatin remodeling, protein ubiquitination, and retinol acid pathway. In addition, NF-kappa B, TNF, MAPK, and cholesterol metabolism pathways were upregulated in PWB ECs as readouts of impaired differentiation. Conclusion: PWB iPSC-derived ECs can be served as novel and clinically relevant disease models by retaining pathological phenotypes. Our data suggests the impaired Hippo and Wnt pathways underlie the development of differentiation-defective ECs in PWB lesions. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.02.547436v1?rss=1 Authors: Ozato, K., Wu, T., Hou, H., Dey, A., Bachu, M., Chen, X., Wisniewski, J., Kudoh, F., Chen, C., Chauhan, S., Xiao, H., Pan, R. Abstract: BRD4 binds to acetylated histones to regulate transcription and drive cancer cell proliferation. However, the role of BRD4 in normal cell growth remains to be elucidated. Here we investigated the question by using mouse embryonic fibroblasts with conditional Brd4 knockout (KO). We found that Brd4KO cells grow more slowly than wild type cells: they do not complete replication, fail to achieve mitosis, and exhibit extensive DNA damage throughout all cell cycle stages. BRD4 was required for expression of more than 450 cell cycle genes including genes encoding core histones and centromere/kinetochore proteins that are critical for genome replication and chromosomal segregation. Moreover, we show that many genes controlling R-loop formation and DNA damage response (DDR) require BRD4 for expression. Finally, BRD4 constitutively occupied genes controlling R-loop, DDR and cell cycle progression. We suggest that BRD4 epigenetically marks those genes and serves as a master regulator of normal cell growth. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.02.547209v1?rss=1 Authors: Kamp, M., Surmacki, J., Segarra Mondejar, M., Young, T., Chrabaszcz, K., Joud, F., Zecchini, V., Speed, A., Frezza, C., Bohndiek, S. Abstract: Aberrantly accumulated metabolites such as fumarate elicit intra- and inter-cellular prooncogenic cascades, yet current methods to measure them require sample perturbation or disruption and lack spatio-temporal resolution, limiting our ability to fully characterize their function and distribution in cells and within a tissue. Raman spectroscopy (RS) is a powerful bio-analytical tool that directly characterizes the chemical composition of a sample based solely on the optical fingerprint of vibrational modes. Here, we show for the first time that RS can directly detect fumarate in living cells in vivo and animal tissues ex vivo. Using the observed linear relationship between Raman scattered intensity and fumarate concentration, we demonstrate that RS can distinguish between Fumarate hydratase (Fh1)-deficient and Fh1-proficient cells based on their fumarate concentration. Moreover, RS reveals the spatial compartmentalization of fumarate within cellular organelles: consistent with disruptive methods, in Fh1-deficient cells we observe the highest fumarate concentration (37 +/- 19 mM) in the mitochondria, where the TCA cycle operates, followed by the cytoplasm (24 +/- 13 mM) and then the nucleus (9 +/- 6 mM). Finally, we apply RS to tissues from an inducible mouse model of FH loss in the kidney, demonstrating that RS can accurately classify FH status in these tissues. These results suggest that RS could be adopted as a valuable tool for small molecule metabolic imaging, enabling in situ dynamic evaluation of fumarate compartmentalization. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.02.547417v1?rss=1 Authors: Kato-Inui, T., Ono, T., Miyaoka, Y. Abstract: As a versatile genome editing tool, the CRISPR-Cas9 system induces DNA double-strand breaks at targeted sites to activate mainly two DNA repair pathways: HDR which allows precise editing via recombination with a homologous template DNA, and NHEJ which connects two ends of the broken DNA, which is often accompanied by random insertions and deletions. Therefore, how to enhance HDR while suppressing NHEJ is a key to successful applications that require precise genome editing. Histones are small proteins with a lot of basic amino acids that generate electrostatic affinity to DNA. Since H2A.X is involved in DNA repair processes, we fused H2A.X to Cas9 and found that this fusion protein could improve the HDR/NHEJ ratio. As various post-translational modifications of H2A.X play roles in the regulation of DNA repair, we also fused H2A.X mimicry variants to replicate these post-translational modifications including phosphorylation, methylation, and acetylation. However, none of them were effective to improve the HDR/NHEJ ratio. We further fused other histone variants to Cas9 and found that H2A.1 exhibited the improved HDR/NHEJ ratio better than H2A.X. Thus, the fusion of histone variants to Cas9 is a promising option to enhance precise genome editing. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.01.547333v1?rss=1 Authors: Fong, H., Mendel, M., Jascur, J., Najmi, L., Kim, K., Lew, G., Garimalla, S., Schock, S., Hu, J., Villegas, A., Conway, A., Fontenot, J., Zompi, S. Abstract: iPSCs can serve as a renewable source of a consistent edited cell product, overcoming limitations of primary cells. While feeder-free generation of clinical grade iPSC-derived CD8 T cells has been achieved, differentiation of iPSC-derived CD4sp and regulatory T cells requires mouse stromal cells in an artificial thymic organoid. Here we report a serum- and feeder-free differentiation process suitable for large-scale production. Using an optimized concentration of PMA/Ionomycin, we generated iPSC-CD4sp T cells at high efficiency and converted them to Tregs using TGF{beta} and ATRA. Using zinc finger nucleases, we demonstrated high non-viral, targeted integration of an HLA-A2 CAR in iPSCs. iPSC-Tregs +/- HLA-A2 CAR phenotypically, transcriptionally and functionally resemble primary Tregs and suppress T cell proliferation in vitro. Our work is the first to demonstrate an iPSC-based platform amenable to manufacturing CD4 T cells to complement iPSC-CD8 oncology products and functional iPSC-Tregs to deliver Treg cell therapies at scale. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.06.30.547267v1?rss=1 Authors: Zamal, M. Y., Venkataramana, C., Subramanyam, R. Abstract: In the phototrophic alphaproteobacteria, photosynthesis is performed by pigment-protein complexes, including the light-harvesting complexes known as LH1 and LH2. The photosystem also encompasses carotenoids to assist in well-functioning of photosynthesis. Most photosynthetic bacteria are exposed to various abiotic stresses, and here, the Rhodobacter (R.) alkalitolerans were extracted from the alkaline pond. We report the comparative study of photosynthetic apparatus of R. alkalitolerans in various light intensities in relation to this bacterium's high pH tolerance ability. We found that as the light intensity increased, the stability of photosystem complexes decreased in normal pH (npH pH 6.8{+/-}0.05) conditions, whereas in high pH (hpH pH 8.6{+/-}0.05) acclimation was observed. The content of bacteriochlorophyll a, absorbance spectra, and circular dichroism data shows that the integrity of photosystem complexes is less affected in hpH compared to npH conditions. Sucrose density and LP-BN of photosystem complexes also shows that LH2 is more affected in npH than hpH, whereas RC-LH1 monomer or dimer has shown interplay between monomer and dimer in hpH although the dimer and monomer both increased in npH. Additionally, the phosphatidylcholine (PC) levels have increased in hpH conditions. Moreover, qPCR data showed that the subunit -c of ATPase levels was overexpressed in hpH. Consequently, the P515 measurement shows that more ATP production is required in hpH, which dissipates the protons from the chromatophore lumen. This could be the reason the photosystem protein complex destabilized due to more lumen acidification. To maintain homeostasis in hpH, the antiporter NhaD expressed more than in the npH condition. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.01.547305v1?rss=1 Authors: Liou, R. H.-C., Chen, S.-W., Cheng, H.-C., Wu, P.-C., Chang, Y.-F., Wang, A.-G., Fann, M.-J., Wong, Y.-H. Abstract: Retinal ganglion cells (RGCs) are essential for vision perception. In glaucoma and other optic neuropathies, RGCs and their optic axons undergo degenerative change and cell death; this can result in irreversible vision loss. Here we developed a rapid protocol for directly inducing RGC differentiation from human induced pluripotent stem cells (iPSCs) by the overexpression of ATOH7, BRN3B and SOX4. The hiPSC-derived RGC-like cells (iRGCs) show robust expression of various RGC-specific markers by whole transcriptome profiling. A functional assessment was also carried out and this demonstrated that these iRGCs display stimulus-induced neuronal activity, as well as spontaneous neuronal activity. Ethambutol (EMB), an effective first-line anti-tuberculosis agent, is known to cause serious visual impairment and irreversible vision loss due to the RGC degeneration in a significant number of treated patients. Using our iRGCs, EMB was found to induce significant dose-dependent and time-dependent increases in cell death and neurite degeneration. Western blot analysis revealed that the expression levels of p62 and LC3-II were upregulated, and further investigations revealed that EMB caused a blockade of lysosome-autophagosome fusion; this indicates that impairment of autophagic flux is one of the adverse effects of that EMB has on iRGCs. In addition, EMB was found to elevate intracellular reactive oxygen species (ROS) levels increasing apoptotic cell death. This could be partially rescued by the co-treatment with the ROS scavenger NAC. Taken together, our findings suggest that this iRGC model, which achieves both high yield and high purity, is suitable for investigating optic neuropathies, as well as being useful when searching for potential drugs for therapeutic treatment and/or disease prevention. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.06.30.547240v1?rss=1 Authors: Vasudevan, A., Ratnakaran, N., Murthy, K., Ahlawat, S., Koushika, S. P. Abstract: Asymmetric transport of cargo across axonal branches is a field of active research. Mechanisms contributing to preferential cargo transport along specific branches in vivo in wild type neurons are poorly understood. We find that anterograde synaptic vesicles preferentially enter the synaptic branch or pause at the branch point in C. elegans PLM neurons. The anterograde motor UNC-104/KIF1A regulates this vesicle behaviour at the branch point. Reduced levels of functional UNC-104 cause vesicles to predominantly pause at the branch point and lose their preference for turning into the synaptic branch. SAM-4/Myrlysin, which aids in recruitment/activation of UNC-104 on synaptic vesicles, regulates vesicle behaviour at the branch point similar to UNC-104. Increasing the levels of UNC-104 increases the preference of vesicles to go straight towards the asynaptic end. This suggests that the neuron optimises UNC-104 levels on the cargo surface to maximise the fraction of vesicles entering the branch and minimise the fraction going to the asynaptic end. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.06.30.547235v1?rss=1 Authors: Kang, S. S., Lee, E. J., Kim, K., Otgonnamjil, D., Shin, S. H. Abstract: The TRPV4 cation channel, is expressed in a broad range of tissues where it participates in generation of Ca2+ signal and/or depolarization of membrane potential. Here, we identified post synaptic density protein 95 (PSD95) as an interacting protein of this epithelial Ca2+ channel using confocal microscopy analysis and immunological assay. Using co-immunoprecipitation assays, we demonstrated that PSD95 was part of the TRPV4 protein complex. PSD95 protein was specifically associated with the C-terminal tail of TRPV4 to form a complex. A TRPV4 tail deletion mutant ({Delta}DAPL871: 4d) exhibited a diminished capacity to bind PSD95. Confocal microscopy analysis suggested that apical localization of TRPV4 required PSD95-TRPV4 interaction. Our data clearly suggest that formation of a complex between TRPV4 and PSD95 can regulate TRPV4 membrane localization. Both TRPV4 Ca2+ channel and its autophagy activity of 4d were reduced by more than 80% compared to those of the TRPV4 wild type. Our observation suggests that PSD95-TRPV4 complex plays crucial roles in routing TRPV4 to the apical plasma membrane and maintaining its authentic Ca2+ channel and biological function. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.01.547341v1?rss=1 Authors: Haran, V., Lenka, N. Abstract: Wnt, an evolutionarily conserved morphogen, is vital for various cell fates specification during early development. However, a concrete mechanistic understanding of the precise and fine-tuned regulation of Wnt underlying these processes is yet to be uncovered. Using the murine embryonic stem cells (ESCs) model, we have identified USP3, a histone deubiquitinase (DUB), displaying bimodal action, serving both as a downstream Wnt target and a regulator of canonical Wnt signalling. Using both loss- and gain-of-function approaches, we could identify USP3 as essential for mesoderm specification, exerting a differential influence during further differentiation. While cardiogenic mesoderm was negatively regulated and so also cardiomyogenesis, USP3 positively regulated hemangioblasts differentiation. Interestingly, however, these induced hemangioblasts promoted the haematopoietic program at the expense of endothelial differentiation. The mechanistic underpinning revealed USP3 localizing to chromatin and differentially modulating these fate choices by precise and contextual deposition of H2AUb/H2BUb in the promoters of mesoderm genes. Collectively our study underscored the Wnt-USP3 link underlying differential mesodermal fate modulation. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.01.547314v1?rss=1 Authors: Weng, Y., Yang, X., Zhang, Q., Chen, Y., Xu, Y., Zhu, C., Xie, Q., Wang, Y., Yang, H., Liu, M., Lu, W., Song, G. Abstract: The adenosine subfamily G protein-coupled receptors A2AR and A2BR were identified as promising candidates for cancer immunotherapy within recent years. One of the A2AR/A2BR dual antagonist, AB928, has progressed to phase II clinic trial for the treatment of rectal cancer. However, the precise mechanism underlying its dual-antagonistic properties remains elusive. Herein, we report crystal structures of A2AR in complex with AB928 and a selective A2AR antagonist, 2-118. The structures reveal a common binding mode on A2AR, wherein the ligands establish extensive interactions with residues from both the orthosteric pocket and the secondary pocket. Conversely, the cAMP assay together with molecular dynamics simulations conducted on both A2AR and A2BR indicate that the ligands adopt distinct binding modes on A2BR. Detailed analysis of their chemical structures suggests that AB928 can readily adapt to the A2BR pocket, while 2-118 cannot due to its intrinsic differences. This disparity potentially accounts for their divergent inhibitory efficacies between A2BR and A2AR. The findings from this study can serve as valuable structural templates for future development of selective or dual inhibitors targeting A2AR/A2BR in the context of cancer therapy. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.06.30.547235v1?rss=1 Authors: Kang, S. S., Lee, E. J., Kim, K., Otgonnamjil, D., Shin, S. H. Abstract: The TRPV4 cation channel, is expressed in a broad range of tissues where it participates in generation of Ca2+ signal and/or depolarization of membrane potential. Here, we identified post synaptic density protein 95 (PSD95) as an interacting protein of this epithelial Ca2+ channel using confocal microscopy analysis and immunological assay. Using co-immunoprecipitation assays, we demonstrated that PSD95 was part of the TRPV4 protein complex. PSD95 protein was specifically associated with the C-terminal tail of TRPV4 to form a complex. A TRPV4 tail deletion mutant ({Delta}DAPL871: 4d) exhibited a diminished capacity to bind PSD95. Confocal microscopy analysis suggested that apical localization of TRPV4 required PSD95-TRPV4 interaction. Our data clearly suggest that formation of a complex between TRPV4 and PSD95 can regulate TRPV4 membrane localization. Both TRPV4 Ca2+ channel and its autophagy activity of 4d were reduced by more than 80% compared to those of the TRPV4 wild type. Our observation suggests that PSD95-TRPV4 complex plays crucial roles in routing TRPV4 to the apical plasma membrane and maintaining its authentic Ca2+ channel and biological function. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.06.30.547235v1?rss=1 Authors: Kang, S. S., Lee, E. J., Kim, K., Otgonnamjil, D., Shin, S. H. Abstract: The TRPV4 cation channel, is expressed in a broad range of tissues where it participates in generation of Ca2+ signal and/or depolarization of membrane potential. Here, we identified post synaptic density protein 95 (PSD95) as an interacting protein of this epithelial Ca2+ channel using confocal microscopy analysis and immunological assay. Using co-immunoprecipitation assays, we demonstrated that PSD95 was part of the TRPV4 protein complex. PSD95 protein was specifically associated with the C-terminal tail of TRPV4 to form a complex. A TRPV4 tail deletion mutant ({Delta}DAPL871: 4d) exhibited a diminished capacity to bind PSD95. Confocal microscopy analysis suggested that apical localization of TRPV4 required PSD95-TRPV4 interaction. Our data clearly suggest that formation of a complex between TRPV4 and PSD95 can regulate TRPV4 membrane localization. Both TRPV4 Ca2+ channel and its autophagy activity of 4d were reduced by more than 80% compared to those of the TRPV4 wild type. Our observation suggests that PSD95-TRPV4 complex plays crucial roles in routing TRPV4 to the apical plasma membrane and maintaining its authentic Ca2+ channel and biological function. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.06.29.547098v1?rss=1 Authors: O'Brien, J. G., Willis, A. B., Long, A. M., Kwon, J., Lee, G., Li, F., Page, P. G., Vo, A. H., Hadhazy, M., Crosbie, R. H., Demonbreun, A. R., McNally, E. M. Abstract: Genetic background shifts the severity of muscular dystrophy. In mice, the DBA/2J strain confers a more severe muscular dystrophy phenotype, whereas the Murphys Roth Large (MRL) strain has super-healing properties that reduce fibrosis. A comparative analysis of the Sgcg null model of Limb Girdle Muscular Dystrophy in the DBA/2J versus MRL strain showed the MRL background was associated with greater myofiber regeneration and reduced structural degradation of muscle. Transcriptomic profiling of dystrophic muscle in the DBA/2J and MRL strains indicated strain-dependent expression of the extracellular matrix (ECM) and TGF-b signaling genes. To investigate the MRL ECM, cellular components were removed from dystrophic muscle sections to generate decellularized myoscaffolds. Decellularized myoscaffolds from dystrophic mice in the protective MRL strain had significantly less deposition of collagen and matrix-bound TGF-b1 and TGF-b3 throughout the matrix, and dystrophic myoscaffolds from the MRL background were enriched in myokines. C2C12 myoblasts were seeded onto decellularized matrices from Sgcg-/- MRL and Sgcg-/- DBA/2J matrices. Acellular myoscaffolds from the dystrophic MRL background induced myoblast differentiation and growth compared to dystrophic myoscaffolds from the DBA/2J matrices. These studies establish that the MRL background also generates its effect through a highly regenerative ECM, which is active even in muscular dystrophy. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.06.29.547074v1?rss=1 Authors: Langseth, C. M., Kukanja, P., Rodriguez-Kirby, L. A. R., Agirre, E., Raman, A., Yokota, C., Avenel, C., Tiklova, K., Guerreiro-Cacais, A. O., Olsson, T., Hilscher, M. M., Nilsson, M., Castelo-Branco, G. Abstract: Multiple sclerosis (MS) is a neurological disease characterised by multifocal lesions and smouldering pathology. While single-cell analyses have provided insights into neuropathology, cellular processes underlying MS remain poorly understood. We modelled the cellular dynamics of MS by examining temporal and regional rates of disease progression in the experimental autoimmune encephalomyelitis (EAE) mouse model. By performing single-cell spatial expression profiling using In situ sequencing, we annotated disease neighbourhoods during lesion evolution and found centrifugal propagation of active lesions. We demonstrated that disease-associated (DA) glia are dynamic and induced independently of lesions, preceding their formation. Single-cell spatial analysis of human archival MS spinal cord confirmed differential distribution of DA-glia, enabled deconvolution of active and inactive lesions into sub-compartments, and identification of new lesion areas. By establishing a spatial resource of mouse and human MS neuropathology at a single-cell resolution, our study unveils the intricate cellular dynamics underlying MS disease evolution. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.06.29.547114v1?rss=1 Authors: Kaya, H. E. K., Ward, M. A., Vangala, J. R., Byers, H. A., Diaz, A., Kaushik, S., Cuervo, A. M., Radhakrishnan, S. K. Abstract: Cells exposed to proteotoxic stress invoke adaptive responses aimed at restoring proteostasis. Our previous studies have established a firm role for the transcription factor Nuclear factor erythroid derived 2-related factor 1 (Nrf1, also called NFE2L1) in responding to proteotoxic stress elicited by inhibition of cellular proteasome. Following proteasome inhibition, Nrf1 mediates new proteasome synthesis, thus enabling the cells to mitigate the proteotoxic stress. Here we report that under similar circumstances, multiple components of the autophagy lysosomal pathway (ALP) are transcriptionally upregulated in an Nrf1-dependent fashion, thus providing the cells with an additional route to cope with proteasome insufficiency. In response to proteasome inhibitors, Nrf1-deficient cells displayed profound defects in invoking autophagy and clearance of aggresomes. This phenomenon was also recapitulated in NGLY1 knockout cells (a model for NGLY1 disease) where Nrf1 is known to be non-functional. Overall, our results significantly expand the role of Nrf1 in shaping the cellular response to proteotoxic stress. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.06.29.547092v1?rss=1 Authors: Leeds, B. K., Kostello, K. F., Liu, Y. Y., Biggins, S., Asbury, C. L. Abstract: During mitosis, kinetochore-attached microtubules form bundles (k-fibers) in which many filaments grow and shorten in near-perfect unison to align and segregate each chromosome. However, individual microtubules grow at intrinsically variable rates, which must be tightly regulated for a k-fiber to behave as a single unit. This exquisite coordination might be achieved biochemically, via selective binding of polymerases and depolymerases, or mechanically, because k-fiber microtubules are coupled through a shared load that influences their growth. Here, we use a novel dual laser trap assay to show that microtubule pairs growing in vitro are coordinated by mechanical coupling. Kinetic analyses show that microtubule growth is interrupted by stochastic, force-dependent pauses and indicate persistent heterogeneity in growth speed during non-pauses. A simple model incorporating both force-dependent pausing and persistent growth speed heterogeneity explains the measured coordination of microtubule pairs without any free fit parameters. Our findings illustrate how microtubule growth may be synchronized during mitosis and provide a basis for modeling k-fiber bundles with three or more microtubules, as found in many eukaryotes. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.06.29.546917v1?rss=1 Authors: Leles, S. G., Levine, N. M. Abstract: Phytoplankton are responsible for half of all oxygen production and drive the ocean carbon cycle. Metabolic theory predicts that increasing global temperatures will cause phytoplankton to become more heterotrophic and smaller. Here we uncover the metabolic trade-offs between cellular space, energy, and stress management driving phytoplankton thermal acclimation and how these might be overcome through evolutionary adaptation. We show that the observed relationships between traits such as chlorophyll, lipid content, C:N and size can be predicted based on the metabolic demands of the cell, the thermal dependency of transporters, and changes in membrane lipids. We suggest that many of the observed relationships are not fixed physiological constraints but rather can be altered through adaptation. For example, the evolution of lipid metabolism can favor larger cells with higher lipid content to mitigate oxidative stress. These results have implications for rates of carbon sequestration and export in a warmer ocean. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.06.29.547150v1?rss=1 Authors: Daga, P., Thurakkal, B., Rawal, S., Das, T. Abstract: Mechanical cues from the tissue microenvironment, such as the stiffness of the extracellular matrix, modulate cellular forms and functions. As numerous studies have shown, this modulation depends on the stiffness-dependent remodeling of cytoskeletal elements. In contrast, very little is known about how the intracellular organelles such as mitochondria respond to matrix stiffness and whether their form, function, and localization change accordingly. Here, we performed an extensive quantitative characterization of mitochondrial morphology, subcellular localization, dynamics and membrane tension on soft and stiff matrices. This characterization revealed that while matrix stiffness affected all these aspects, matrix stiffening most distinctively led to an increased perinuclear clustering of mitochondria. Subsequently, we could identify the matrix stiffness-sensitive perinuclear localization of filamin as the key factor dictating this perinuclear clustering. Photo-conversion labeling and fluorescent recovery after photobleaching experiments revealed that perinuclear and peripheral mitochondrial populations differed in their motility on the soft matrix but surprisingly they did not show any difference on the stiff matrix. Finally, perinuclear mitochondrial clustering appeared to be crucial for priming human mesenchymal stem cells towards osteogenesis on the stiff matrix. Taken together, we elucidate a dependence of mitochondrial localization on matrix stiffness, which possibly enables a cell to adapt to its microenvironment. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.06.29.547089v1?rss=1 Authors: Araki, D., Chen, V., Redekar, N., Salisbury-Ruf, C., Luo, Y., Liu, P., Li, Y., Smith, R., Dagur, P., Combs, C., Larochelle, A. Abstract: Granulocyte colony stimulating factor (G-CSF) is commonly used as adjunct treatment to hasten recovery from neutropenia following chemotherapy and autologous transplantation of hematopoietic stem and progenitor cells (HSPCs) for malignant disorders. However, the utility of G-CSF administration after ex vivo gene therapy procedures targeting human HSPCs has not been thoroughly evaluated. Here, we provide evidence that post-transplant administration of G-CSF impedes engraftment of CRISPR-Cas9 gene edited human HSPCs in xenograft models. G-CSF acts by exacerbating the p53-mediated DNA damage response triggered by Cas9-mediated DNA double-stranded breaks. Transient p53 inhibition in culture attenuates the negative impact of G-CSF on gene edited HSPC function. In contrast, post-transplant administration of G-CSF does not impair the repopulating properties of unmanipulated human HSPCs or HSPCs genetically engineered by transduction with lentiviral vectors. The potential for post-transplant G-CSF administration to aggravate HSPC toxicity associated with CRISPR-Cas9 gene editing Cas9 should be considered in the design of ex vivo autologous HSPC gene editing clinical trials. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.06.29.547095v1?rss=1 Authors: Fan, R., Sportelli, C., Lai, Y., Salehe, S., Pinnell, J., Richardson, J. R., Luo, S., Tieu, K. Abstract: Dynamin-related protein 1 (Drp1) is typically known for its role in mitochondrial fission. A partial inhibition of this protein has been reported to be protective in experimental models of neurodegenerative diseases. The protective mechanism has been attributed primarily to improved mitochondrial function. Herein, we provide evidence showing that a partial Drp1-knockout improves autophagy flux independent of mitochondria. First, we characterized in cell and animal models that at low non-toxic concentrations, manganese (Mn), which causes parkinsonian-like symptoms in humans, impaired autophagy flux but not mitochondrial function and morphology. Furthermore, nigral dopaminergic neurons were more sensitive than their neighbouring GABAergic counterparts. Second, in cells with a partial Drp1-knockdown and Drp1+/- mice, autophagy impairment induced by Mn was significantly attenuated. This study demonstrates that autophagy is a more vulnerable target than mitochondria to Mn toxicity. Furthermore, improving autophagy flux is a separate mechanism conferred by Drp1 inhibition independent of mitochondrial fission. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.06.29.547030v1?rss=1 Authors: Muhar, M., Farnung, J., Hofmann, R., Cernakova, M., Sidiropoulos, N. D., Bode, J. W., Corn, J. E. Abstract: During normal cellular homeostasis unfolded and mis-localized proteins are recognized and removed, preventing the build-up of toxic byproducts. When protein homeostasis is perturbed during aging, neurodegeneration or cellular stress, proteins can accumulate several forms of chemical damage through reactive metabolites. Such modifications have been proposed to trigger the selective removal of chemically marked proteins; however, discovering modifications sufficient to induce protein degradation has remained challenging. Using a semi-synthetic chemical biology approach coupled to cellular assays, we found that C-terminal amide-bearing proteins (CTAPs) are rapidly cleared from human cells. A CRISPR screen identified the SCF/FBXO31 ubiquitin ligase as a reader of C-terminal amides, which ubiquitylates CTAPs for subsequent proteasomal degradation. A conserved binding pocket enables FBXO31 to bind almost any C-terminal peptide bearing an amide while retaining exquisite selectivity over non-modified clients. This mechanism facilitates binding and turnover of endogenous CTAPs that are formed following oxidative stress. A dominant human mutation found in neurodevelopmental disorders switches CTAP recognition, such that non-amidated neosubstrates are now degraded and FBXO31 becomes markedly toxic. We propose that CTAPs may represent the vanguard of a largely unexplored class of modified amino acid degrons that could provide a general strategy for selective yet broad surveillance of chemically damaged proteins. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.06.30.547259v1?rss=1 Authors: Dodel, M., Guiducci, G., Dermit, M., Krishnamurthy, S., Stojic, L., Mardakheh, F. K. Abstract: Different regions of RNA molecules can often engage in specific interactions with distinct RNA-binding proteins (RBPs), giving rise to diverse modalities of RNA regulation and function. However, there are currently no methods for unbiased identification of RBPs that interact with specific RNA regions in living cells under endogenous settings. Here, we introduce TREX (Targeted RNase H-mediated extraction of crosslinked RBPs), a highly sensitive approach for identifying proteins that directly bind to specific RNA regions in living cells. We demonstrate that TREX outperforms existing methods in identifying known interactors of U1 snRNA, and reveals endogenous region-specific interactors of NORAD lncRNA. Using TREX, we generated a comprehensive region-by-region interactome for 45S rRNA, uncovering both established and novel interactions that regulate ribosome biogenesis. With its applicability to any RNA in any cell-type, TREX is the first RNA-centric tool for unbiased positional mapping of endogenous RNA-protein interactions in living cells. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.06.29.546885v1?rss=1 Authors: Yang, Z., Johnson, B. A., Meliopoulos, V. A., Ju, X., Zhang, P., Hughes, M. P., Wu, J., Koreski, K. P., Chang, T.-C., Wu, G., Hixon, J., Duffner, J., Wong, K., Lemieux, R., Lokugamage, K. G., Alvardo, R. E., Crocquet-Valdes, P. A., Walker, D. H., Plante, K. S., Plante, J. A., Weaver, S. C., Kim, H. J., Meyers, R., Schultz-Cherry, S., Ding, Q., Menachery, V. D., Taylor, J. P. Abstract: G3BP1/2 are paralogous proteins that promote stress granule formation in response to cellular stresses, including viral infection. G3BP1/2 are prominent interactors of the nucleocapsid (N) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, the functional consequences of the G3BP1-N interaction in the context of viral infection remain unclear. Here we used structural and biochemical analyses to define the residues required for G3BP1-N interaction, followed by structure-guided mutagenesis of G3BP1 and N to selectively and reciprocally disrupt their interaction. We found that mutation of F17 within the N protein led to selective loss of interaction with G3BP1 and consequent failure of the N protein to disrupt stress granule assembly. Introduction of SARS-CoV-2 bearing an F17A mutation resulted in a significant decrease in viral replication and pathogenesis in vivo, indicating that the G3BP1-N interaction promotes infection by suppressing the ability of G3BP1 to form stress granules. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.06.29.547006v1?rss=1 Authors: Jha, M. P., Kumar, V., Sharma, L., Ghosh, A., Mapa, K. Abstract: Sse1 is a cytosolic Hsp110 molecular chaperone of yeast, Saccharomyces cerevisiae. Its multifaceted roles in cellular protein homeostasis as Nucleotide Exchange Factor (NEF), as protein-disaggregase and as a Chaperone linked to Protein Synthesis (CLIPS), are well documented. In the currently study, we show that SSE1 genetically interacts with IRE1 and HAC1, the Endoplasmic Reticulum-Unfolded Protein Response (ER-UPR) sensors implicating its role in ER protein homeostasis. Interestingly, absence of this chaperone imparts unusual resistance to tunicamycin-induced ER stress which depends on the intact Ire1-Hac1 mediated ER-UPR signalling. Furthermore, cells lacking SSE1 show ER-stress-responsive inefficient reorganization of translating ribosomes from polysomes to monosomes and increased monosome content that drive uninterrupted protein translation. In consequence, the kinetics of ER-UPR is starkly different in sse1{Delta} strain where we show that stress response induction and restoration of homeostasis is prominently faster in contrast to the wildtype (WT) cells. Importantly, Sse1 plays a critical role in controlling the ER-stress mediated cell division arrest which is escaped in sse1{Delta} strain during chronic tunicamycin stress. Consequently, sse1{Delta} strain shows significantly higher cell viability in comparison to WT yeast, following short-term as well as long-term tunicamycin stress. In summary, we demonstrate a new role of Sse1 in ER protein homeostasis where the chaperone genetically interacts with ER-UPR pathway, controls the protein translation during ER stress and the kinetics of ER-UPR. More importantly, we show the crtiical role of Sse1 in regulating the ER-stress-induced cell division arrest and cell death during global ER stress by tunicamycin. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.06.29.547007v1?rss=1 Authors: Anders, J., König, C., Lender, C., Hellhund, A., Nehls, S., Shalabi, I., Honecker, B., Lorenzen, S., Meyer, M., Matthiesen, J., Cadar, D., Roeder, T., Metwally, N. G., Lotter, H., Bruchhaus, I. Abstract: Recently, two genes involved in pathogenicity in a mouse model of amoebic liver abscess were identified based on their differential expression between non-pathogenic (A1np) and pathogenic (B2p) clones of the Entamoeba histolytica isolate HM:1-IMSS. While overexpression of a gene encoding the metallopeptidase EhMP8-2 decreases the virulence of the pathogenic clone B2p, overexpression of the gene ehi_127670 (ehhp127), encoding a hypothetical protein, increases the virulence of the non-pathogenic clone A1np, while silencing this gene in B2p decreases virulence. To understand the role of both molecules in determining the pathogenicity of E. histolytica, silencing and overexpression transfectants were characterized in detail. Silencing of ehmp8-2, of the homologous gene ehmp8-1, or of both together in A1np trophozoites significantly altered the transcript levels of 60-350 genes. This strong change in the expression profile caused by the silencing of ehmp8-1 and/or ehmp8-2 implies that these peptidases regulate expression of numerous genes. Consequently, numerous phenotypic characteristics including cytopathic, hemolytic and cysteine peptidase activity were changed in response to their silencing. Silencing of ehhp127 in B2p trophozoites did not affect other genes, whereas overexpression in A1np trophozoites results in an altered expression of approximately 140 genes. EhHP127 appears to be important for trophozoite movement, as silencing negatively affects and overexpression positively affects trophozoite motility. Interestingly, the specific silencing of ehhp127 also impairs cytopathic activity, cysteine peptidase and hemolytic activity. All three molecules of interest, namely EhMP8-1, EhMP8-2, and EhHP127 can be detected in amoeba vesicles. Our results clearly show that the proteins studied here influence the pathogenicity of amoebae in different ways and use entirely different mechanisms to do so. Author summary: The human pathogen Entamoeba histolytica can live asymptomatically in the intestine or become invasive and cause fatal liver abscesses. Approximately 15,000 people die each year as a result of an amoebic infection. Recently, two clones with different pathogenicity (A1np: non-pathogenic; B2p: pathogenic) derived from the E. histolytica isolate HM:1-IMSS were compared at the transcriptome level. Two highly differentially expressed genes (ehhp127 encoding a hypothetical protein and ehmp82 encoding a metallopeptidase) were identified. Analysis of E. histolytica transfectants showed that silencing of ehhp127 and overexpression of ehmp8-2 in B2p trophozoites reduced amoebic liver abscess formation in the mouse model. In this study, we characterized E. histolytica silencing and overexpression transfectants of ehmp8-2, as well as of the homologous gene ehmp8-1 and of ehhp127. It was shown that the altered expression of the metallopeptidase genes has a strong influence on the expression of a large number of genes and that the phenotype is strongly altered as a result. Silencing of ehhp127 does not affect the overall expression profile. However, specific silencing has a negative effect on motility, cysteine peptidase, hemolytic and cytopathic activity. All three molecules were shown to be localized in trophozoite vesicles. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.06.28.544680v1?rss=1 Authors: Vandepas, L. E., Crim, R. N., Gilbertson, E., Yonemitsu, M. A., Unsell, E., Metzger, M. J., Lacy-Hulbert, A., Goetz, F. W. Abstract: Disseminated neoplasia (DN) is a form of cancer in bivalve molluscs that has been reported in some cases to be a transmissible cancer. Neoplastic cells are highly proliferative, and infection is often lethal. Some commercially valuable bivalve species (mussels, cockles, soft-shell clams, oysters) are affected by outbreaks of disseminated neoplasia, making disease diagnosis and mitigation an important issue in aquaculture and ecological restoration efforts. Here we describe a minimally invasive, non-lethal method for high-throughput screening for disseminated neoplasia in basket cockles (Clinocardium nuttallii). Basket cockles are native to the North American Pacific coast from California to Alaska. There is recent concern from some Coast Salish Tribes regarding an observed long-term decline in cockle populations in Puget Sound, WA. This has led to increased interest in monitoring efforts and research to improve our understanding of the mechanisms of observed basket cockle population dynamics, including assessing prevalence of disease, such as disseminated neoplasia. The rapid, non-lethal hemolymph smear screening method presented here to diagnose DN in adult C. nuttallii can be applied at field sites at low financial cost, and in a validation study of 29 animals the results were identical to that of the gold standard method, tissue histology. Due to the similar morphology of DN in different bivalves, this method can likely be generally applied for use in any bivalve species. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.06.28.546913v1?rss=1 Authors: Yap, K. K., Schroeder, J., Gerrand, Y.-W., Kong, A., Fox, A. M., Knowles, B., Banting, S. W., Elefanty, A. K., Stanley, E. G., Yeoh, G. C., Lockwood, G. P., Cogger, V. C., Morrison, W. A., Polo, J. M., Mitchell, G. M. Abstract: Liver sinusoidal endothelial cells (LSECs) play an important role in liver development, regeneration and pathophysiology, but the differentiation process that generates their unique tissue-specific phenotype is poorly understood and difficult to study as primary cells are only available in limited quantities. To address this, we hypothesised that human induced pluripotent stem cell (hiPSC)-derived endothelial cells (iECs) can produce hiPSC-derived LSECs upon transplantation into the livers of Fah-/-/Rag2-/-/Il2rg-/- mice, and serve as a model to study LSEC specification. Progressive and long-term repopulation of the liver vasculature was observed, as iECs expanded along the sinusoids that run between hepatocytes and increasingly produced human factor VIII, indicating differentiation into LSEC-like cells. To chart the developmental profile associated with LSEC specification, the bulk transcriptome of transplanted cells at time-points between 1 and 12 weeks were compared against primary human adult LSECs, which demonstrated a chronological increase in LSEC markers, LSEC differentiation pathways, and zonation. Bulk transcriptome analysis suggested that the transcription factors NOTCH1, GATA4, and FOS play a central role in LSEC specification, interacting with a network of 27 transcription factors. Novel markers associated with this process include EMCN and CLEC14A. Additionally, single cell transcriptomic analysis demonstrated that transplanted iECs at 4 weeks contain zonal subpopulations with a region-specific phenotype. Collectively, this study confirms that hiPSC can adopt LSEC-like features and provides insight into LSEC specification. This humanised xenograft system can be applied to further interrogate LSEC developmental biology and pathophysiology, bypassing current logistical obstacles associated with primary human LSECs. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.06.27.546668v1?rss=1 Authors: Banik, P., Kamps, J., Chen, Q.-Y., Luesch, H., Winklhofer, K. F., Tatzelt, J. Abstract: Mistargeting of secretory proteins to the cytosol can induce formation of aggregation-prone conformers and subsequent proteostasis decline. We have identified a quality control pathway that redirects non-ER-imported prion protein (PrP) to proteasomal degradation in the nucleus to prevent formation of toxic aggregates in the cytosol. Upon aborted ER import, PrP sequentially interacted with VCP/p97 and importins, which kept PrP soluble and promoted its nuclear import. In the nucleus, RNA buffered aggregation of PrP to facilitate ubiquitin-dependent proteasomal degradation. Notably, the cytosolic interaction of PrP with VCP/p97 and its nuclear import were independent of ubiquitination but required the intrinsically unstructured N-terminal domain of PrP. Transient proteotoxic stress promoted the formation of self-perpetuating PrP aggregates in the cytosol, which disrupted further nuclear targeting of PrP and compromised cellular proteostasis. Our study delineates a VCP/p97-dependent nucleus-based quality control pathway of non-ER-imported secretory proteins and emphasizes the important role of the nuclear milieu for the degradation of aggregation-prone proteins. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.06.27.546694v1?rss=1 Authors: Bedir, M., Outwin, E., Colnaghi, R., Bassett, L., Abramowicz, I., O'Driscoll, M. Abstract: We previously reported that non-homologous end-joining (NHEJ)-defective human LIG4-/- pre-B lymphocytes were unexpectedly sensitive to killing by the cyclic peptide Cyclosporin A (CsA), a common component of bone marrow transplantation conditioning and maintenance regimes. We also found that CsA induced DNA double strand breaks (DSBs) in LIG4 syndrome patient fibroblasts, specifically upon transit through S-phase. The molecular basis underlying these CsA impacts has not been described hitherto. We postulated that CsA-induced genomic instability may reflect a direct role of Cyclophilin A (CYPA) in DNA repair, as CYPA is the primary physiological target interactor of CsA. CYPA is the founding member of the Cyclophilin family of peptidyl-prolyl cis-trans isomerases (PPIs). CsA inhibits the PPI activity of CYPA through occupation of the latters enzymatic active site. Using an integrated approach involving CRISPR/Cas9-engineering, siRNA, BioID, co-immunoprecipitation, pathway-specific DNA repair investigations as well as protein expression-interaction analysis, we describe novel impacts of CYPA loss and inhibition of its PPI activity on DNA repair. Prompted by findings from our CYPA-BioID proximity interactome, we validate CYPA interactions with different components of the DNA end resection machinery. Moreover, we characterise a novel and direct CYPA interaction with the NBS1 component of the MRE11-RAD50-NBS1 (MRN) complex, providing evidence that the PPI function of CYPA actively influences DNA repair via direct protein-protein interaction at the level of DNA end resection. Consequently, we demonstrate that CYPA loss or inhibition impairs Homologous Recombination Repair (HRR) following DNA replication fork stalling. Additionally, we define a set of genetic vulnerabilities associated with CYPA loss and inhibition, identifying DNA replication fork protection as an important determinant of viability herein. Leveraging the novel insights into CYPA biology we have uncovered; we explore examples of how CYPA PPI inhibition may be exploited to selectively kill cells from a variety of different cancers with a shared characteristic genomic instability profile. These findings propose a potential new disease application or repurposing strategy for the non-immunosuppressive CsA analogue class of Cyclophilin inhibitors. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.06.27.545208v1?rss=1 Authors: Oikawa, T., Hasegawa, J., Ohnishi, N., Onodera, Y., Hashimoto, A., Sasaki, J., Sasaki, T., Ueda, K., Sabe, H. Abstract: H3.1 is predominantly synthesized and enters the nucleus during the G1/S phase of the cell cycle, although the underlying mechanism remains unknown. Here we show that p53 is involved in this process. CTDNEP1 converts phosphatidic acid (PA) into diacylglycerol, and EZH2 generates H3K27me3. p53 increased CTDNEP1 and decreased EZH2 in the nuclear H3.1 interactome of the G1/S phase. Moreover, H3.1 bound robustly to PA but not to diacylglycerol. p53 deficiency caused perinuclear accumulation of EZH2-modified H3K27me3 of non-nucleosomal histones during the G1/S phase. p53 induced the expression of TMEM255A, which reduced nuclear PA levels by increasing CTDNEP1 levels. Therefore, H3.1 entering the nucleus in the absence of p53 may be trapped near the nuclear envelope (NE) and epigenetically marked as repressive without forming nucleosomes. Our study identified the NE as a novel target of p53, in which p53 downregulates nuclear PA levels to normalize H3.1 nuclear entry and epigenetic modification. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.06.28.546853v1?rss=1 Authors: Alvarez Viar, G., Klena, N., Martino, F., Nievergelt, A. P., Pigino, G. Abstract: Control of ciliary beating is crucial for motility and signaling in eukaryotic cells and requires spatially restricted interactions between axonemal proteins and specific protofilaments within the ciliary microtubules. How these interactions are regulated remains poorly understood, but increasing evidence indicates that tubulin post-translational modifications (tPTMs) are required for proper ciliary motility. The Tubulin Code refers to the concept that tPTMs can modulate the function of individual microtubules in cells. Here we use a combination of immuno-cryo-electron tomography, expansion microscopy and mutant analysis to show that, in motile cilia, tubulin glycylation and polyglutamylation form mutually exclusive protofilament-specific nano-patterns at sub-microtubular scale. We show that these two nano-patterns are consistent with the distributions of axonemal dyneins and nexin-dynein regulatory complexes, respectively, and are required for their regulation during ciliary beating. Our discovery of a tubulin nano-code in cilia highlights the need of higher-resolution studies also in other cellular compartments to understand the molecular role of tPTMs. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.06.27.546688v1?rss=1 Authors: Sutcu, H., Rassinoux, P., Donnio, L.-M., Neuillet, D., Vianna, F., Gabillot, O., MARI, P.-O., Baldeyron, C., Giglia-Mari, G. Abstract: DNA integrity is incessantly confronted to endogenous and exogenous agents inducing DNA lesions, which are harmful for cellular homeostasis. Luckily all organisms are equipped with a network of DNA damage response (DDR) mechanisms that will repair DNA lesions and restore the proper cellular activities. Despite DNA repair mechanisms have been revealed in vitro and in replicating cells, still little is known on how DNA lesions are repaired and consequently how cellular homeostasis is maintained in post-mitotic cells. Muscle fibers are highly specialised post-mitotic cells organized in syncytia and, they are vulnerable to age-related degeneration and atrophy following radiotherapy treatment. We have here studied in detail the DNA repair capacity of muscle fibers nuclei and compared it with the one measured in proliferative myoblasts. We focused on the DNA repair mechanisms that correct ionizing radiation (IR)-induced lesions, namely the base excision repair (BER), the non-homologous end joining (NHEJ) and the homologous recombination (HR). We found that in the most differentiated myogenic cells, myotubes, all of these DNA repair mechanisms present weakened kinetics of recruitment of DNA repair proteins to IR-damaged DNA. For BER and HR, this decline can be linked to reduced steady state levels of key proteins involved in these processes, probably since nuclei within muscle fibers no longer replicate their DNA. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.06.27.546770v1?rss=1 Authors: Freibaum, B. D., Messing, J., Nakamura, H., Yurtsever, U., Wu, J., Kim, H. J., Hixon, J., Lemieux, R., Duffner, J., Huynh, W., Wong, K., White, M., Lee, C., Meyers, R., Parker, R. D., Taylor, J. P. Abstract: Stress granule formation is triggered by the release of mRNAs from polysomes and is promoted by the action of the paralogs G3BP1 and G3BP2. G3BP1/2 proteins bind mRNAs and thereby promote the condensation of mRNPs into stress granules. Stress granules have been implicated in several disease states, including cancer and neurodegeneration. Consequently, compounds that limit stress granule formation or promote their dissolution have potential as both experimental tools and novel therapeutics. Herein, we describe two small molecules, referred to as G3BP inhibitor a and b (G3Ia and G3Ib), designed to bind to a specific pocket in G3BP1/2 that is known to be targeted by viral inhibitors of G3BP1/2 function. In addition to disrupting co-condensation of RNA, G3BP1, and caprin 1 in vitro, these compounds inhibit stress granule formation in cells treated prior to or concurrent with stress, and dissolve pre-existing stress granules when added to cells after stress granule formation. These effects are consistent across multiple cell types and a variety of initiating stressors. Thus, these compounds represent ideal tools to probe the biology of stress granules and hold promise for therapeutic interventions designed to modulate stress granule formation. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.06.27.546744v1?rss=1 Authors: Strickland, J., Pan, D., Godfrey, C., Kim, J. S., Hopke, A., Degrange, M., Villavicencio, B., Mansour, M. K., Zerbe, C. S., Irimia, D., Amir, A., Weiner, O. D. Abstract: Neutrophils exhibit self-amplified swarming to sites of injury and infection. How swarming is controlled to ensure the proper level of neutrophil recruitment is unknown. Using an ex vivo model of infection, we find that human neutrophils use active relay to generate multiple pulsatile waves of swarming signals. Unlike classic active relay systems such as action potentials, neutrophil swarming relay waves are self-extinguishing, limiting the spatial range of cell recruitment. We identify an NADPH-oxidase-based negative feedback loop that is needed for this self-extinguishing behavior. Through this circuit, neutrophils adjust the number and size of swarming waves for homeostatic levels of cell recruitment over a wide range of initial cell densities. We link a broken homeostat to neutrophil over-recruitment in the context of human chronic granulomatous disease. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.06.28.546823v1?rss=1 Authors: Chauve, L., McLysaght, A., McGarry, A., Butler, L. Abstract: Gene duplications play a major evolutionary role by providing raw material for functional innovation. Whole Genome Duplication (WGD), or polyploidization, is a particular case of duplication encompassing the entire genome and has been implicated in evolutionary diversification. In plants, WGD is recognized as a major evolutionary force, and is linked to speciation and the ability to resist periods of stress and of environmental upheaval. In animals, examples of current polyploid species are rarer, but multiple ancient events are known, including the charismatic two rounds (2R) of WGD that occurred during early vertebrate evolution. The conditions favouring the success of polyploid lineages are unclear. One debated hypothesis states that polyploidy is adaptive in the short-term, however this has never been studied in animals. In this study, we investigated the consequences of polyploidy on physiology and stress resistance in Caenorhabditis elegans, where tetraploidy can be artificially constructed. Our results reveal that although tetraploidy reduces fitness by decreasing fertility and lifespan in regular conditions, tetraploid animals exhibit increased resilience under specific stress conditions related to temperature changes. While neotetraploid animals exhibit similar pathogen resistance, their response to heat stress is altered. They exhibit modest improvement in thermotolerance and prolonged hsp (heat shock protein) mRNA induction upon heat shock (HS) accompanied by altered hsp-16.2 nuclear localization upon HS. Most notably, under severe cold stress, gravid neotetraploids animals massively escape cold-induced death, and generate more progeny than diploid animals. These results suggest a potential adaptive value of tetraploidy under cold stress and might help explain recent correlations found between the frequency of extant animal polyploids and regions experiencing recent glaciation and large temperature variation. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.06.27.546809v1?rss=1 Authors: Devarakonda, P. M., Sarmiento, V., Heaslip, A. T. Abstract: Toxoplasma gondii contains an essential plastid organelle called the apicoplast that is necessary for fatty acid, isoprenoid, and heme synthesis. Perturbations affecting apicoplast function or inheritance lead to parasite death. The apicoplast is a single copy organelle and therefore must be divided so that each daughter parasite inherits an apicoplast during cell division. In this study we identify new roles for F-actin and an unconventional myosin motor, TgMyoF, in this process. First, loss of TgMyoF and actin lead to an accumulation of apicoplast vesicles in the cytosol indicating a role for this actomyosin system in apicoplast protein trafficking or morphological integrity of the organelle. Second, live cell imaging reveals that during division the apicoplast is highly dynamic, exhibiting branched, U-shaped and linear morphologies that are dependent on TgMyoF and actin. In parasites where movement was inhibited by the depletion of TgMyoF, the apicoplast fails to associate with the parasite centrosomes. Thus, this study provides crucial new insight into mechanisms controlling apicoplast-centrosome association, a vital step in the apicoplast division cycle, which ensures that each daughter inherits a single apicoplast. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.06.28.546840v1?rss=1 Authors: Ait-Si-Ali, S., Granados, A., Zamperoni, M., Rapone, R., Bouyioukos, C., Del Maestro, L., Joliot, V., Albini, S. Abstract: The histone lysine methyltransferase SETDB1 is involved in muscle differentiation and has been shown as a regulator of the TGF{beta} pathway in cancer contexts. Here, we investigated the role of SETDB1 in Duchenne muscular dystrophy (DMD) myotubes harboring an overactivated TGFbeta pathway. Our data show that challenging healthy myotubes with TGFbeta induces nuclear accumulation of SETDB1, while in DMD myotubes SETDB1 is constantly accumulated in nuclei in TGFbeta-dependent fashion. SETDB1 loss-of-function (LOF) leads to a decrease of the TGFbeta downstream target genes in DMD myotubes, suggesting the involvement of a TGFbeta/SETDB1 axis in DMD physiopathology. Transcriptomics show that many targets of SETDB1 code for secreted factors involved in fibrosis, extracellular matrix remodeling and inflammation and are downregulated when SETDB1 is silenced. Conditioned medium assays show that SETDB1 LOF in DMD myotubes abrogates the deleterious effect of the secretome on myoblast differentiation and impairs their pro-fibrotic response. Together, our findings indicate that SETDB1 potentiates the TGFbeta response in DMD muscles and participates in fibrosis. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.06.28.546942v1?rss=1 Authors: Husser, M. C., Pham, N. P., Law, C., Araujo, F. R. B., Martin, V. J. J., Piekny, A. Abstract: Endogenous tags have become invaluable tools to visualize and study native proteins in live cells. However, generating human cell lines carrying endogenous tags is difficult due to the low efficiency of homology-directed repair. Recently, an engineered split mNeonGreen protein was used to generate a large-scale endogenous tag library in HEK293 cells. Using split mNeonGreen for large-scale endogenous tagging in human iPSCs would open the door to studying protein function in healthy cells and across differentiated cell types. We engineered an iPS cell line to express the large fragment of the split mNeonGreen protein (mNG21-10) and showed that it enables fast and efficient endogenous tagging of proteins with the short fragment (mNG211). We also demonstrate that neural network-based image restoration enables live imaging studies of highly dynamic cellular processes such as cytokinesis in iPSCs. This work represents the first step towards a genome-wide endogenous tag library in human stem cells. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.06.25.546442v1?rss=1 Authors: White, J. S., Su, J. J., Ruark, E. M., Hua, J., Hutson, M. S., Page-McCaw, A. Abstract: Injury is a constant reality of life, and survival requires all organisms to repair wounds. Cell behaviors like proliferation, migration, and invasion replace missing cells and close wounds. However, the contribution of other wound-induced cell behaviors is not understood, including formation of multi-nucleate syncytia. Wound-induced epithelial syncytia were first reported around epidermal puncture wounds in Drosophila larvae and adults with similarities to multinucleation increases in mammalian cardiomyocytes after pressure overload. Although these tissues are post-mitotic, syncytia have more recently been reported in mitotically competent tissues: around laser wounds in Drosophila pupal epidermis and in zebrafish epicardium damaged by endotoxin, microdissection, or laser. Further, injury induces fusion of other cells: after injury, bone marrow derived cells fuse with various somatic cells to promote repair, and after biomaterial implantation, immune cells fuse into multinucleated giant cells associated with rejection . These observations raise the possibility that syncytia offer adaptive benefits, but it is unknown what those benefits are. Here, we utilize in vivo live imaging to analyze wound-induced syncytia in mitotically competent Drosophila pupae. Almost half the epithelial cells near a wound fuse, forming large syncytia. Syncytia migrate rapidly, outpacing diploid cells to complete wound closure. We show syncytia can both pool resources of their component cells to concentrate them toward the wound, and reduce cell intercalation during wound closure, two mechanisms for speeding wound repair. In addition to wound healing, these properties of syncytia are likely to contribute to their roles in development and pathology. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.06.23.546241v1?rss=1 Authors: Ullrich, S., Leidescher, S., Feodorova, Y., Thanisch, K., Fini, J.-B., Kaspers, B., Weber, F., Markova, B., Fuehrer, D., Romitti, M., Krebs, S., Blum, H., Leonhardt, H., Costagliola, S., Heuer, H., Solovei, I. Abstract: Abnormalities are indispensable for studying normal biological processes and mechanisms. In the present work, we draw attention to the remarkable phenomenon of a perpetually and robustly upregulated gene, the thyroglobulin gene (Tg). The gene is expressed in the thyroid gland and, as it has been recently demonstrated, forms so-called transcription loops, easily observable by light microscopy. Using this feature, we show that Tg is expressed at a high level from the moment a thyroid cell acquires its identity and both alleles remain highly active over the entire life of the cell, i.e. for months or years depending on the species. We demonstrate that this high upregulation is characteristic of thyroglobulin genes in all major vertebrate groups. We provide evidence that Tg is not influenced by the thyroid hormone status, does not oscillate round the clock and is expressed during both the exocrine and endocrine phases of thyrocyte activity. We conclude that the thyroglobulin gene represents a valuable model to study the maintenance of a high transcriptional upregulation. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.06.23.546275v1?rss=1 Authors: Shaw, A. B., Tse, H. N., Byford, O., plahe, g., Moon-Walker, A., Saphire, E. O., Whelan, S. P. J., Mankouri, J., Fontana, J., Barr, J. N. Abstract: Lymphocytic choriomeningitis virus (LCMV) is a model arenavirus that causes fatalities within immunocompromised populations. To enter cells, the LCMV envelope fuses with endosomal membranes, for which two requirements are low pH and interaction between LCMV GP spike and receptor CD164. LCMV subsequently uncoats, where genome-associated NP separates from Z matrix. To further examine LCMV entry, an siRNA screen identified K+ channels as important for LCMV infection, and pharmacological inhibition confirmed K+ involvement during entry. We tracked incoming virions along their entry pathway under physiological conditions, where uncoating was signified by separation of NP and Z. In contrast, K+ channel blockade, prevented uncoating, trapping virions within Rab7 and CD164-positive endosomes, identifying K+ as a third LCMV entry requirement. K+ did not increase GP/CD164 binding, thus we suggest K+ mediates uncoating by modulating NP/Z interactions within the virion interior. These results suggest repurposing licensed K+ channel inhibitors represents a potential anti-arenaviral strategy. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.06.24.546381v1?rss=1 Authors: guang, x., jiang, z., chun, z. Abstract: Myocardial cells, fibroblasts and vascular cells in the heart are connected by a complex matrix mainly composed of fibrillar collagen, which helps to protect the integrity and compliance of the heart structure. Previous studies have shown that hypoxia can induce myocardial hypoxia, but the mechanism is still unclear. In this study, we found that hypoxia promotes TGF beta induced collagen deposition and myocardial fibrosis by inducing Endoplasmic reticulum stress and oxidative stress in cardiomyocytes. Moreover, we also found that antioxidant drugs can effectively alleviate hypoxia induced myocardial fibrosis. Therefore, our study provides an experimental basis for the treatment of myocardial fibrosis. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.06.24.546334v1?rss=1 Authors: Bragg, R. M., Coffey, S. R., Cantle, J. P., Hu, S., Singh, S., Legg, S. R. W., McHugh, C. A., Toor, A., Zeitlin, S. O., Kwak, S., Howland, D., Vogt, T. F., Monga, S. P., Carroll, J. B. Abstract: Huntington's disease arises from a toxic gain of function in the huntingtin (HTT) gene. As a result, many HTT-lowering therapies are being pursued in clinical studies, including those that reduce HTT RNA and protein expression in the liver. To investigate potential impacts, we characterized molecular, cellular, and metabolic impacts of chronic HTT lowering in mouse hepatocytes. Lifelong hepatocyte HTT loss is associated with multiple physiological changes, including increased circulating bile acids, cholesterol and urea, hypoglycemia, and impaired adhesion. HTT loss causes a clear shift in the normal zonal patterns of liver gene expression, such that pericentral gene expression is reduced. These alterations in liver zonation in livers lacking HTT are observed at the transcriptional, histological and plasma metabolite level. We have extended these phenotypes physiologically with a metabolic challenge of acetaminophen, for which the HTT loss results in toxicity resistance. Our data reveal an unexpected role for HTT in regulating hepatic zonation, and we find that loss of HTT in hepatocytes mimics the phenotypes caused by impaired hepatic {beta}-catenin function. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.06.24.546372v1?rss=1 Authors: Dasgupta, N., Lei, X., Arnold, R., Teneche, M. G., Miller, K. N., Rajesh, A., Davis, A., Anschau, V., Campos, A. R., Gilson, R., Havas, A., Yin, S., Chua, Z. M., Proulx, J., Alcaraz, M., Rather, M. I., Baeza, J., Schultz, D. C., Berger, S. L., Adams, P. D. Abstract: Cellular senescence, a stable proliferation arrest caused by a range of cellular stresses, is a bona fide cause of cell and tissue aging. As well as proliferation arrest, cell senescence is associated with a potent pro-inflammatory phenotype, the senescence-associated secretory phenotype (SASP). Recent studies have shown the importance of cytoplasmic DNA and chromatin, either reverse transcribed expressed retrotransposons or cytoplasmic chromatin fragments (CCF) expelled from the nucleus, in activation of nuclear SASP gene expression via the cGAS/STING cytoplasmic DNA-sensing pathway. As a source of chronic inflammation, over the long term SASP promotes tissue aging and disease. Thus, it is important to better define the mechanism of SASP activation in senescence. We show here that both the Promyelocytic Leukemia (PML) protein and HIRA histone chaperone are required for SASP expression in senescent cells. PML protein is the key organizer of PML nuclear bodies, nuclear features up to 1 micron in diameter, containing many proteins and previously implicated in diverse cellular processes, including control of cell senescence and cellular intrinsic anti-viral immunity. HIRA is a histone chaperone best known for its ability to incorporate histone variant H3.3 into nuclear chromatin in a DNA replication-independent manner, including in non-proliferating senescent cells. HIRA localizes to PML nuclear bodies in senescent cells. We show that both HIRA and PML are required for activation of NF-kB and SASP. We found that HIRA regulates cytoplasmic NF-kB signaling in senescent cells through the CCF-cGAS-STING-TBK1 pathway. HIRA physically interacts with the autophagy cargo receptor p62 Sequestosome-1 (p62), and HIRA and p62 antagonistically regulate SASP. PML is required to maintain integrity of colocalized HIRA and p62 foci in the cell nucleus. Overall, our findings point to functions for HIRA and PML in coordination of cytoplasmic signalling and nuclear gene expression to regulate inflammation during cell senescence and aging. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.06.25.546446v1?rss=1 Authors: Waite, K. A., Vontz, G., Lee, S. Y., Roelofs, J. Abstract: Stress conditions can cause the relocalization of proteasomes to condensates in yeast and mammalian cells. The interactions that facilitate the formation of proteasome condensates, however, are unclear. Here, we show that the formation of proteasome condensates in yeast depends on long K48-linked ubiquitin chains together with the proteasome shuttle factors Rad23 and Dsk2. These shuttle factors colocalize to these condensates. Strains deleted for the third shuttle factor gene, DDI1, show proteasome condensates in the absence of cellular stress, consistent with the accumulation of substrates with long K48-linked ubiquitin chains that accumulate in this mutant. We propose a model where the long K48-linked ubiquitin chains function as a scaffold for the ubiquitin binding domains of the shuttle factors and the proteasome, allowing for the multivalent interactions that further drive condensate formation. Indeed, we determined different intrinsic ubiquitin receptors of the proteasome (Rpn1, Rpn10, and Rpn13) are critical under different condensate inducing conditions. In all, our data support a model where the cellular accumulation of substrates with long ubiquitin chains, potentially due to reduced cellular energy, allows for proteasome condensate formation. This suggests that proteasome condensates are not simply for proteasome storage, but function to sequester soluble ubiquitinated substrates together with inactive proteasomes. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.06.24.546289v1?rss=1 Authors: Wang, Y., Jefferson, M., Mccoll, J., Verkade, P., Mayer, U., Wileman, T. Abstract: Autophagosomes deliver cytosolic material to lysosomes to provide amino acids during starvation and to degrade damaged proteins and organelles to maintain tissue homeostasis. Delivery to lysosomes requires LC3/ATG8 (LC3), the major membrane protein of the autophagosome, that uses adaptor proteins to capture cargo and recruits tethering and SNARE proteins to promote fusion with lysosomes. LC3 is also recruited to endo-lysosome compartments in response to increases in vacuolar pH to facilitate degradation of material entering cells by endocytosis. A series of ubiquitin-like reactions conjugate LC3 to amino groups exposed by phosphatidylethanolamine (PE) or phosphatidylserine (PS) in target membranes. The E1 and E2-ubiquitin-like activities of ATG7 and ATG3 use thioester bonds to transfer LC3 to the ATG5-ATG12 conjugate. At the same time binding of ATG16L1 to ATG5-ATG12 provides the E3 ubiquitin-ligase like activity necessary to conjugate LC3 to PE or PS. The ATG5-ATG12 conjugate can also bind TECPR1 (tectonin beta propeller repeat-containing protein) which shares an ATG5 interaction (AIR) region with ATG16L1 and can binds LC3 directly through a LC3 interaction region (LIR). In this study we have used cells lacking ATG16L1 to determine if TECPR1 can substitute for ATG16L1 during LC3 conjugation. The results show that ATG16L1-/- MEFS can conjugate LC3 to lysosomes damaged by chloroquine or LLOMes and conjugation is dependent on the ubiquitin-like enzymes ATG3, ATG5 and ATG7 upstream of ATG16L1. TECPR1, ATG5 and galectin 3 are recruited to LAMP positive damaged lysosomes in the absence of ATG16L1 suggesting that TECPR-1 recruits ATG5-ATG12 to conjugate LC3 to damaged lysosomes. This was confirmed when truncation of TECPR1 at the central PH domain required for lysosome binding prevented LC3 conjugation, and LC3 conjugation could be restored by full length TECPR1. Recruitment of TECPR1 to damaged lysosomes required the N-terminal LIR motif and was partially dependent on the central PH domain that binds PI4P exposed during lysosome repair. TECPR1 can therefore conjugate LC3 to damaged lysosomes independently of ATG16L1 by providing E3 ligase-like activity to ATG5-ATG12. Direct conjugation of LC3 by TECPR1 may contribute to the autophagosome tethering functions reported for TECPR1 by increasing recruitment of cargo receptors, tethering proteins and SNARE proteins required for fusion with lysosomes and protein degradation. TECPR1-dependent conjugation of LC3 may also facilitate lysosome repair pathways involving autophagic lysosomal reformation and transcriptional activation of autophagy by TFEB. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.06.23.546357v1?rss=1 Authors: Crapart, C., Scott, Z. C., Konno, T., Sharma, A., Bailey, D. M., Avezov, E., Koslover, E. F. Abstract: The endoplasmic reticulum (ER) forms an interconnected network of tubules stretching throughout the cell. Understanding how ER functionality relies on its structural organization is crucial for elucidating cellular vulnerability to ER perturbations, which have been implicated in several neuronal pathologies. One of the key functions of the ER is enabling Ca2+ signalling by storing large quantities of this ion and releasing it into the cytoplasm in a spatiotemporally controlled manner. Through a combination of physical modeling and live-cell imaging, we demonstrate that alterations in ER shape significantly impact its ability to support efficient local Ca2+ releases, due to hindered transport of luminal content within the ER. Our model reveals that rapid Ca2+ release necessitates mobile luminal buffer proteins with moderate binding strength, moving through a well-connected network of ER tubules. These findings provide insight into the functional advantages of normal ER architecture, emphasizing its importance as a kinetically efficient intracellular Ca2+ delivery system. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.06.25.546318v1?rss=1 Authors: Đukic, N., Stromland, O., Munnur, D., Zhu, K., Schuller, M., Chatrin, C., Kar, P., Rack, J. G. M., Baretic, D., Schüler, H., Wijngaarden, S., Filippov, D. V., Sanyal, S., Smith, R., Ahel, D., Ahel, I. Abstract: PARP14 is a mono-ADP-ribosyl transferase involved in the control of immunity, transcription and DNA replication stress management. However, little is known about the ADP-ribosylation activity of PARP14, including its substrate specificity or how PARP14-dependent ADP-ribosylation is reversed. Here we show that PARP14 is dual function enzyme with both ADP-ribosyl transferase and hydrolase activity acting on both protein and nucleic acid substrates. In particular, we show that the PARP14 macrodomain 1 is an active ADP-ribosyl hydrolase. We also demonstrate hydrolytic activity for the first macrodomain of PARP9. We reveal that expression of a PARP14 mutant with the inactivated macrodomain 1 results in a dramatic increase in mono(ADP-ribosyl)ation of proteins in human cells, including PARP14 itself and antiviral PARP13. Moreover, we demonstrate that the closely related hydrolytically active macrodomain of SARS2 Nsp3, Mac1, efficiently reverses PARP14 ADP-ribosylation in vitro and in cells, supporting the evolution of viral macrodomains to counteract PARP14-mediated antiviral response. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.06.23.546353v1?rss=1 Authors: Nouchi, E., Oka, T., Kataoka, N., Kawano, Y., Gan, B. S. Abstract: The dynamic mechanism of a cellular cytoskeleton is essential for the role of the cell, and its accurate characterization has been a long standing problem for cell scientists. A cytoskeleton vibrations are highly influenced by interactions of filamentous proteins mediated by axial vibration of the stiff microtubules (compressive member) and lateral vibration of F_actin (tensile member). Among various structures in a cell, the cytoplasmic contractile bundles, lamellipodia, and filipodia cells can be modeled by a symmetrical cylinder shaped self equilibrium tensegrity structure with different radii at the top and bottom of the cylinder. The truncated cone-like cylinder model is made to be small in height compared to both radii. This study investigates the tensegrity self vibrational behavior of the cellular cytoskeleton to calculate its natural frequencies, composed of the individual vibration of microtubules and F_actins from measured data. The spectral element method is adopted based on the Wittrick_Williams procedure to solve the vibrational behaviors of the cellular cytoskeleton. Various n_polygon cylindrical truncated cone shaped tensegrity structures to mimic the cellular cytoskeletons are presented to demonstrate the robustness of the present study. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.06.24.546395v1?rss=1 Authors: Tao, H., Dar, H. Y., Tian, C., Banerjee, S., Glazer, E. S., Srinivasan, S., Zhu, L., Pacifici, R., He, P. Abstract: Males show higher incidence and severity than females in hepatic injury and many liver diseases, but the mechanisms are not well understood. Ferroptosis, an iron-mediated lipid peroxidation-dependent death, plays an important role in the pathogenesis of liver diseases. We determined whether hepatocyte ferroptosis displays gender difference, accounting for sexual dimorphism in liver diseases. Compared to female hepatocytes, male hepatocytes were much more vulnerable to ferroptosis by iron and pharmacological inducers including RSL3 and iFSP1. Male but not female hepatocytes exhibited significant increases in mitochondrial Fe2+ and mitochondrial ROS (mtROS) contents. Female hepatocytes showed a lower expression of iron importer transferrin receptor 1 (TfR1) and mitochondrial iron importer mitoferrin 1 (Mfrn1), but a higher expression of iron storage protein ferritin heavy chain 1 (FTH1). It is well known that TfR1 expression is positively correlated with ferroptosis. Herein, we showed that silencing FTH1 enhanced while knockdown of Mfrn1 decreased ferroptosis in HepG2 cells. Removing female hormones by ovariectomy (OVX) did not dampen but rather enhanced hepatocyte resistance to ferroptosis. Mechanistically, OVX potentiated the decrease in TfR1 and increase in FTH1 expression. OVX also increased FSP1 expression in ERK-dependent manner. Elevation in FSP1 suppressed mitochondrial Fe2+ accumulation and mtROS production, constituting a novel mechanism of FSP1-mediated inhibition of ferroptosis. In conclusion, differences in hepatocellular iron handling between male and female account, at least in part, for sexual dimorphism in induced ferroptosis of the hepatocytes. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.06.24.546411v1?rss=1 Authors: Yang, N. V., Rogers, S., Guerra, R., Pagliarini, D. J., Theusch, E., Krauss, R. M. Abstract: Background: Statins are the drugs most commonly used for lowering plasma low-density lipoprotein (LDL) cholesterol levels and reducing cardiovascular disease risk. Although generally well tolerated, statins can induce myopathy, a major cause of non-adherence to treatment. Impaired mitochondrial function has been implicated as a cause of statin-induced myopathy, but the underlying mechanism remains unclear. We have shown that simvastatin downregulates transcription of TOMM40 and TOMM22, genes that encode major subunits of the translocase of outer mitochondrial membrane (TOM) complex which is responsible for importing nuclear-encoded proteins and maintaining mitochondrial function. We therefore investigated the role of TOMM40 and TOMM22 in mediating statin effects on mitochondrial function, dynamics, and mitophagy. Methods: Cellular and biochemical assays and transmission electron microscopy were used to investigate effects of simvastatin and TOMM40 and TOMM22 expression on measures of mitochondrial function and dynamics in C2C12 and primary human skeletal cell myotubes. Results: Knockdown of TOMM40 and TOMM22 in skeletal cell myotubes impaired mitochondrial oxidative function, increased production of mitochondrial superoxide, reduced mitochondrial cholesterol and CoQ levels, disrupted mitochondrial dynamics and morphology, and increased mitophagy, with similar effects resulting from simvastatin treatment. Overexpression of TOMM40 and TOMM22 in simvastatin-treated muscle cells rescued statin effects on mitochondrial dynamics, but not on mitochondrial function or cholesterol and CoQ levels. Moreover, overexpression of these genes resulted in an increase in number and density of cellular mitochondria. Conclusion: These results confirm that TOMM40 and TOMM22 are central in regulating mitochondrial homeostasis and demonstrate that downregulation of these genes by statin treatment mediates disruption of mitochondrial dynamics, morphology, and mitophagy, effects that may contribute to statin-induced myopathy. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.06.25.545688v1?rss=1 Authors: Tian, Y.-M., Holdship, P., To, T. Q., Ratcliffe, P. J., Keeley, T. P. Abstract: In animals, adaptation to changes in cellular oxygen levels is coordinated largely by the 2-oxoglutarate dependent prolyl-hydroxylase domain (PHD) dioxygenase family, which regulate the stability of their hypoxia-inducible factor (HIF) substrates to promote expression of genes that adapt cells to hypoxia. Recently, 2-aminoethanethiol dioxygenase (ADO) was identified as a novel O2-sensing enzyme in animals. Through N-terminal cysteine dioxygenation and the N-degron pathway, ADO regulates the stability of a set of non-transcription factor substrates; the regulators of G-protein signalling 4, 5 and 16, and interleukin-32. Here, we set out to compare and contrast the in cellulo characteristics of ADO and PHD enzymes in an attempt to better understand their co-evolution in animals. We find that ADO operates to regulate the stability of its substrates rapidly and with similar O2-sensitivity to the PHD/HIF pathway. ADO appeared less sensitive to iron chelating agents or transition metal exposure than the PHD enzymes, possibly due to tighter catalytic-site Fe2+ coordination. Unlike the PHD/HIF pathway, the ADO/N-degron pathway was not subject to feedback by hypoxic induction of ADO and induction of ADO substrates was well sustained in response to prolonged hypoxia. The data also reveal strong interactions between proteolytic regulation of targets by ADO and transcriptional induction of those targets, that shape integrated cellular responses to hypoxia. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.06.23.546291v1?rss=1 Authors: Mellentine, S. Q., Ramsey, A. S., Li, J., Brown, H. N., Tootle, T. Abstract: A key regulator of collective cell migration is prostaglandin (PG) signaling. However, it remains largely unclear whether PGs act within the migratory cells or their microenvironment to promote migration. Here we use Drosophila border cell migration as a model to uncover the cell-specific roles of two PGs in collective migration. Prior work shows PG signaling is required for on-time migration and cluster cohesion. We find that the PGE2 synthase cPGES is required in the substrate, while the PGF2 synthase Akr1B is required in the border cells for on-time migration. Akr1B acts in both the border cells and their substrate to regulate cluster cohesion. One means by which Akr1B regulates border cell migration is by promoting integrin-based adhesions. Additionally, Akr1B limits myosin activity, and thereby cellular stiffness, in the border cells, whereas cPGES limits myosin activity in both the border cells and their substrate. Together these data reveal that two PGs, PGE2 and PGF2, produced in different locations, play key roles in promoting border cell migration. These PGs likely have similar migratory versus microenvironment roles in other collective cell migrations. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.06.25.546468v1?rss=1 Authors: Bracey, K., Noguchi, P., Edwards, C., Cario, A., Gu, G., Kaverina, I. Abstract: In pancreatic islet beta cells, molecular motors use cytoskeletal polymers microtubules as tracks for intracellular transport of insulin secretory granules. Beta-cell microtubule network has a complex architecture and is non-directional, which provide insulin granules at the cell periphery for rapid secretion response, yet to avoid over-secretion and subsequent hypoglycemia. We have previously characterized a peripheral sub-membrane microtubule array, which is critical for withdrawal of excessive insulin granules from the secretion sites. Microtubules in beta cells originate at the Golgi in the cell interior, and how the peripheral array is formed is unknown. Using real-time imaging and photo-kinetics approaches in clonal mouse pancreatic beta cells MIN6, we now demonstrate that kinesin KIF5B, a motor protein with a capacity to transport microtubules as cargos, slides existing microtubules to the cell periphery and aligns them to each other along the plasma membrane. Moreover, like many physiological beta-cell features, microtubule sliding is facilitated by a high glucose stimulus. These new data, together with our previous report that in high glucose sub-membrane MT array is destabilized to allow for robust secretion, indicate that MT sliding is another integral part of glucose-triggered microtubule remodeling, likely replacing destabilized peripheral microtubules to prevent their loss over time and beta-cell malfunction. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.06.25.546476v1?rss=1 Authors: Xie, Z., Chai, Y., Zhu, Z., Shen, Z., Zhao, Z., Xiao, L., Du, Z., Ou, G., Li, W. Abstract: Asymmetric cell divisions (ACDs) generate two daughter cells with identical genetic information but distinct cell fates through epigenetic mechanisms. However, the process of partitioning different epigenetic information into daughter cells remains unclear. Here, we demonstrate that the nucleosome remodeling and deacetylase (NuRD) complex is asymmetrically segregated into the surviving daughter cell rather than the apoptotic one during ACDs in Caenorhabditis elegans. The absence of NuRD triggers apoptosis via the EGL-1-CED-9-CED-4-CED-3 pathway, while an ectopic gain of NuRD enables apoptotic daughter cells to survive. We identify the vacuolar H+-adenosine triphosphatase (V-ATPase) complex as a crucial regulator of NuRD's asymmetric segregation. V-ATPase interacts with NuRD and is asymmetrically segregated into the surviving daughter cell. Inhibition of V-ATPase disrupts cytosolic pH asymmetry and NuRD asymmetry. We suggest that asymmetric segregation of V-ATPase may cause distinct acidification levels in the two daughter cells, enabling asymmetric epigenetic inheritance that specifies their respective life-versus-death fates. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.06.26.546585v1?rss=1 Authors: Wu, H., Zhou, L.-Y., Jin, C.-X., Wang, W.-X., Song, L., Shin, J.-B., Du, T.-T. Abstract: The MRTF-SRF pathway has been extensively studied for its crucial role in driving the expression of a large number of genes involved in actin cytoskeleton of various cell types. However, the specific contribution of MRTF-SRF in hair cells remains unknown. In this study, we showed that hair cell-specific deletion of Srf or Mrtfb, but not Mrtfa, leads to similar defects in the development of stereocilia dimensions and the maintenance of cuticular plate integrity. We used FACS-based hair cell RNA-seq analysis to investigate the mechanistic underpinnings of the changes observed in Srf and Mrtfb mutants, respectively. Interestingly, the transcriptome analysis revealed distinct profiles of genes regulated by Srf and Mrtfb, suggesting different transcriptional regulation mechanisms of actin cytoskeleton activities mediated by Srf and Mrtfb. Exogenous delivery of calponin 2 using Adeno-associated virus transduction in Srf mutants partially rescued the impairments of stereocilia dimensions and the F-actin intensity of cuticular plate, suggesting the involvement of Cnn2, as an Srf downstream target, in regulating the hair bundle morphology and cuticular plate actin cytoskeleton organization. Our study uncovers, for the first time, the unexpected differential transcriptional regulation of actin cytoskeleton mediated by Srf and Mrtfb in hair cells, and also demonstrates the critical role of SRF-CNN2 in modulating actin dynamics of the stereocilia and cuticular plate, providing new insights into the molecular mechanism underlying hair cell development and maintenance. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.06.26.546513v1?rss=1 Authors: Spits, C., Lei, Y., Al Delbany, D., Krivec, N., Regin, M., Couvreu de Deckersberg, E., Janssens, C., Ghosh, M., Sermon, K. D. Abstract: Human pluripotent stem cell (hPSC) cultures are prone to genetic drift, as cells that have acquired specific genetic abnormalities experience a selective advantage in vitro. These abnormalities are highly recurrent in hPSC lines worldwide, but currently their functional consequences in differentiating cells are scarcely described. An accurate assessment of the risk associated with these genetic variants in both research and clinical settings is therefore lacking. In this work, we established that one of these recurrent abnormalities, the loss of chromosome 18q, impairs neuroectoderm commitment and affects the cardiac progenitor differentiation of hESCs. We show that downregulation of SALL3, a gene located in the common 18q loss region, is responsible for failed neuroectodermal differentiation. Knockdown of SALL3 in control lines impaired differentiation in a manner similar to the loss of 18q, while transgenic overexpression of SALL3 in hESCs with 18q loss rescued the differentiation capacity of the cells. Finally, we show by gene expression analysis that loss of 18q and downregulation of SALL3 leads to changes in the expression of genes involved in pathways regulating pluripotency and differentiation, including the WNT, NOTCH, JAK-STAT, TGF-beta and NF-kB pathways, suggesting that these cells are in an altered state of pluripotency. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.06.26.546598v1?rss=1 Authors: Shangguan, J., Rock, R. S. Abstract: Myosin 10 (Myo10) is a vertebrate-specific motor protein well known for its role in filopodia formation. Although Myo10-driven filopodial dynamics have been characterized, there is no information about the numbers of Myo10 in filopodia. To better understand molecular stoichiometries and packing restraints in filopodia, we measured Myo10 abundance in these structures. Here we combined SDS-PAGE analysis with epifluorescence microscopy to quantitate HaloTag-labeled Myo10 in U2OS cells. About 6% of total intracellular Myo10 localizes to filopodia, where it tends to be enriched at opposite ends of the cell. Hundreds of Myo10 are found in a typical filopodium, and their distribution across filopodia is log-normal. Some filopodial tips even contain more Myo10 than accessible binding sites on the actin filament bundle. Our estimates of Myo10 molecules in filopodia provide insight into the physics of packing Myo10, its cargo, and other filopodia-associated proteins in narrow membrane deformations in addition to the numbers of Myo10 required for filopodia initiation. Our protocol provides a framework for future work analyzing Myo10 abundance and distribution upon perturbation. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.06.26.546565v1?rss=1 Authors: Hoyer, M. J., Smith, I. R., Paoli, J. C., Jiang, Y., Paulo, J. A., Harper, W. Abstract: The endoplasmic reticulum (ER) has a vast proteomic landscape to perform many diverse functions including protein and lipid synthesis, calcium ion flux, and inter-organelle communication. The ER proteome is remodeled in part through membrane-embedded receptors linking ER to degradative autophagy machinery (selective ER-phagy). A refined tubular ER network is formed in neurons within highly polarized dendrites and axons. Autophagy-deficient neurons in vivo display axonal ER accumulation within synaptic ER boutons, and the ER-phagy receptor FAM134B has been genetically linked with human sensory and autonomic neuropathy. However, mechanisms, including receptor selectivity, that define ER remodeling by autophagy in neurons are limited. Here, we combine a genetically tractable induced neuron (iNeuron) system for monitoring extensive ER remodeling during differentiation with proteomic and computational tools to create a quantitative landscape of ER proteome remodeling via selective autophagy. Through analysis of single and combinatorial ER-phagy receptor mutants, we delineate the extent to which each receptor contributes to both magnitude and selectivity of ER clearance via autophagy for individual ER protein cargos. We define specific subsets of ER curvature-shaping proteins or lumenal proteins as preferred clients for distinct receptors. Using spatial sensors and flux reporters, we demonstrate receptor-specific autophagic capture of ER in axons, which correlates with aberrant ER accumulation in axons of ER-phagy receptor or autophagy-deficient neurons. This molecular inventory of ER proteome remodeling and versatile genetic toolkit provides a quantitative framework for understanding contributions of individual ER-phagy receptors for reshaping ER during cell state transitions. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.06.26.546470v1?rss=1 Authors: Wu, H., Lv, P., Wang, J., Bennett, B., Wang, J., Li, P., Peng, Y., Hu, G., Lin, J. Abstract: Epigenetic regulators present novel opportunities for both ischemic stroke research and therapeutic interventions. While previous work has implicated that they may provide neuroprotection by potentially influencing coordinated sets of genes and pathways, most of them remains largely uncharacterized in ischemic conditions. In this study, we used the oxygen-glucose deprivation (OGD) model in the immortalized mouse hippocampal neuronal cell line HT-22 and carried out an RNAi screen on epigenetic regulators. We identified Prmt5 as a novel negative regulator of neuronal cell survival after OGD, which presented a phenotype of translocation from the cytosol to the nucleus upon oxygen and energy depletion both in vitro and in vivo. Prmt5 bound to the chromatin and a large number of promoter regions to repress downstream gene expression. Silencing Prmt5 significantly dampened the OGD-induced changes for a large-scale of genes, and gene ontology analysis showed that Prmt5-target genes were highly enriched for Hedgehog signaling. Encouraged by the above observation, we treated mice with middle cerebral artery occlusion (MCAO) with the Prmt5 inhibitor EPZ015666 and found that Prmt5 inhibition sustain protection against neuronal death in vivo. Together, our findings revealed a novel epigenetic mechanism of Prmt5 in cerebral ischemia and uncovered a potential target for neuroprotection. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.06.26.543767v1?rss=1 Authors: Laundos, T. L., Li, S., Cheang, E., De Santis, R., Piccolo, F. M., Brivanlou, A. H. Abstract: Huntington's disease (HD) remains an incurable and fatal neurodegenerative disease long after CAG-expansion mutation in the huntingtin gene (HTT) was identified as the cause. The underlying pathological mechanism, whether HTT loss of function or gain of toxicity results from mutation, remains a matter of debate. In this study, we genetically modulated wild-type or mutant HTT expression levels in isogenic human embryonic stem cells to systematically investigate their contribution to HD-specific phenotypes. Using highly reproducible and quantifiable in vitro micropattern-based assays, we observed comparable phenotypes with HD mutation and HTT depletion. However, halving endogenous wild-type HTT levels did not strongly recapitulate the HD phenotypes, arguing against a classical loss of function mechanism. Remarkably, expression of CAG-expanded HTT in non-HD cells induced HD-like phenotypes akin to HTT depletion. By corollary, these results indicate a dominant negative effect of mutated HTT on its wild-type counterpart. Complementation with additional copies of wild-type HTT ameliorated the HD-associated phenotypes, strongly supporting a classical dominant negative mechanism. Understanding the molecular basis of this dominant negative effect will guide the development of efficient clinical strategies to counteract the deleterious impact of mutant HTT on the wild-type protein. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.06.26.546610v1?rss=1 Authors: Goncalves, D. L., Peffer, S., Morano, K. A. Abstract: Cells employ multiple systems to maintain homeostasis when experiencing environmental stress. For example, the folding of nascent polypeptides is exquisitely sensitive to proteotoxic stressors including heat, pH and oxidative stress, and is safeguarded by a network of protein chaperones that concentrate potentially toxic misfolded proteins into transient assemblies to promote folding or degradation. The redox environment itself is buffered by both cytosolic and organellar thioredoxin and glutathione pathways. How these systems are linked is poorly understood. Here, we determine that specific disruption of the cytosolic thioredoxin system resulted in constitutive activation of the heat shock response in Saccharomyces cerevisiae and accumulation of the sequestrase Hsp42 into an exaggerated and persistent juxtanuclear quality control (JUNQ) compartment. Terminally misfolded proteins also accumulated in this compartment in thioredoxin reductase (TRR1)-deficient cells, despite apparently normal formation and dissolution of transient cytoplasmic quality control (CytoQ) bodies during heat shock. Notably, cells lacking TRR1 and HSP42 exhibited severe synthetic slow growth exacerbated by oxidative stress, signifying a critical role for Hsp42 under redox-challenged conditions. Finally, we demonstrated that Hsp42 localization patterns in trr1{triangleup} cells mimic those observed in chronically aging and glucose-starved cells, linking nutrient depletion and redox imbalance with management of misfolded proteins via a mechanism of long-term sequestration. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.06.23.546309v1?rss=1 Authors: Xuan, W., Cheng, F., Han, X., Tipparaju, S., Ashraf, M. Abstract: Background: Extensive studies have been conducted in skeletal muscle and myocardium affected by Duchenne Muscular Dystrophy (DMD) disease but there is a significant gap of research in the role of vascular smooth muscle cells (VSMCs) in DMD. Here, we investigated the role of dystrophin deficiency in the maintenance of VSMCs contractile phenotype. Methods: 12-14 months old mdx mice and DMD induced pluripotent stem cells (iPSC) derived VSMCs were used as disease models. Morphological and immunohistochemistry analyses were performed to determine histological changes and the expression of contractile markers. Transmission Electron Microscopy (TEM) was used to assess ultrastructural changes in the VSMCs. Mito-tracker staining and TUNEL staining were performed to determine mitochondria fission-fusion and apoptosis respectively. mRNA Sequencing for normal iPSC derived VSMCs (WT-VSMCs) and DMD iPSC derived VSMCs (DMD-VSMCs) with or without oxidative stress was performed. KEGG signaling pathway enrichment, Go function enrichment and Gene set enrichment analysis (GESA) were conducted to explore the potential mechanism responsible for these changes. In addition, transcription factor enrichment analysis was performed to unravel mechanistic pathways of regulatory networks. Results: Spontaneous abnormal VSMCs proliferation, loss of vascular structure and degenerative changes occurred in VSMCs in aorta from 12-14 months old mdx mice. The DMD-VSMCs showed maturation defect, loss of mitochondrial hemostasis, and increased vulnerability to oxidative stress compared with WT-VSMCs. Transcriptome analysis revealed dysregulation of smooth muscle proliferation, differentiation, and vascular development in DMD-VSMCs. Transcriptional factor, target, and motif discovery analysis of the dysregulated gene set suggested potential contributions of transcriptional factors GADD45A, SOX9, TIA1, RBBP9 and FOXM to the phenotypes of DMD-VSMCs. Under oxidative stress, initiation of apoptotic process was significantly enhanced in DMD-VSMCs while their response to hypoxia and oxidative stress was downregulated. Conclusions: Dystrophin deficiency induced VSMCs phenotype switching and disrupted mitochondrial metabolism. The findings in this study underscore the importance of vascular dysfunction in DMD disease and therapeutic interventions to restore VSMC phenotype may ameliorate the propensity of disease progression. It is suggested that the transcriptome analysis may allow the discovery of potential signaling pathways involved in the dysregulation of transcription factors. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.06.23.546317v1?rss=1 Authors: Tharp, K., Park, S., Timblin, G. A., Richards, A., Berg, J., Twells, N., Riley, N. M., Peltan, E., Shon, D. J., Stevenson, E., Tsui, C. K., Palomba, F., Lefebvre, A. E., Soens, R., Ayad, N., ten Hoeve-Scott, J., Healy, K., Digman, M., Dillin, A., Bertozzi, C., Mahal, L. K., Swaney, D., Cantor, J. R., Paszek, M., Weaver, V. Abstract: Efforts to identify anti-cancer therapeutics and understand tumor-immune interactions are built with in vitro models that do not match the microenvironmental characteristics of human tissues. Using in vitro models which mimic the physical properties of healthy or cancerous tissues and a physiologically relevant culture medium, we demonstrate that the chemical and physical properties of the microenvironment regulate the composition and topology of the glycocalyx. Remarkably, we find that cancer and age-related changes in the physical properties of the microenvironment are sufficient to adjust immune surveillance via the topology of the glycocalyx, a previously unknown phenomenon observable only with a physiologically relevant culture medium. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.06.23.546346v1?rss=1 Authors: Neelakshi, K., Caruso, A. P., Prokopiou, N., Logue, J. S. Abstract: Migrating cells must adapt to the mechanochemical properties of the microenvironment. Here, we demonstrate that intracellular [Ca2+] in melanoma cells rises in response to 2D confinement. Upon chelating Ca2+ or inhibiting Piezo1, cells were less likely to phenotypically transition from mesenchymal to fast amoeboid migration. In VCAM-1 coated microchannels, melanoma cells adopt a hybrid mode of migration, which has hallmarks of mesenchymal and amoeboid migration. In the absence of Piezo1 activity, cells predominantly adopted a mesenchymal mode of migration. In undulating channels, however, melanoma cells are almost entirely amoeboid. In agreement with a curvature induced increase in plasma membrane tension, this effect could be blocked by inhibiting Piezo1 or chelating Ca2+. In addition to ROCK2, we found that activation of inverted formin-2 (INF2) downstream of Piezo1/Ca2+ to be required for amoeboid migration. Thus, the activation of INF2 by Piezo1/Ca2+ constitutes an essential mechanism by which cells respond to confining environments. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.06.23.546123v1?rss=1 Authors: Cairns, G., Thumiah-Mootoo, M., Abbasi, M. R., Racine, J., Lariovov, N., Prola, A., Khacho, M., Burelle, Y. Abstract: Maintenance of optimal mitochondrial function plays a crucial role in the regulation of muscle stem cell (MuSC) behavior, but the underlying maintenance mechanisms remain ill defined. In this study, we explored the importance of mitophagy, as a mitochondrial quality control regulator, in MuSCs and the role this process plays in maintaining optimal muscle regenerative capacity. Here we show that MuSCs exhibit dynamic alterations in mitophagy under different physiological myogenic states. In particular, quiescent MuSCs exhibit high levels of PINK1/Parkin-dependent mitophagy, which is rapidly decreased upon transition to an early activation state. Genetic disruption of this pathway using Pink1 knockout mice reduced mitophagy in quiescent MuSCs, which was accompanied by increased mitochondrial ROS release and mitochondrial network fragmentation. These abnormalities led to hampered self-renewal of MuSCs which ultimately translated in a progressive loss of muscle regeneration following repetitive injury. However, proliferation and differentiation capacity were unaltered in the absence of PINK1, indicating that altered fate decisions is the main mechanism underlying impaired muscle regeneration. Impaired fate decisions in PINK1 deficient MuSCs could be restored by scavenging excess mitochondrial ROS. Together, these data shed new light on the regulation of mitophagy during MuSC state transitions and position the PINK1-dependent pathway as an important regulator of MuSC mitochondrial properties and fate decisions. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.06.22.545817v1?rss=1 Authors: Altrieth, A. L., Suarez, E., Nelson, D. A., Gabunia, S., Larsen, M. Abstract: Vascular endothelial cells have important functions in fibrosis via direct and indirect methods and in regeneration through secretion of tissue-specific, paracrine-acting angiocrine factors. In the salivary gland, endothelial cells are required for proper development, but their roles within adult glands are largely unknown. The goal of this work was to identify ligand-receptor interactions between endothelial cells and other cell types that are important during homeostasis, fibrosis, and regeneration. To model salivary gland fibrosis and regeneration, we utilized a reversible ductal ligation. To induce injury, a clip was applied to the primary ducts for 14 days, and to induce a regenerative response, the clip was subsequently removed for 5 days. To identify endothelial cell-produced factors, we used single-cell RNA-sequencing of stromal-enriched cells from adult salivary glands. Transcriptional profiles of homeostatic salivary gland endothelial cells were compared to endothelial cells of other organs. Salivary gland endothelial cells were found to express some unique genes and some that were similar to other fenestrated endothelial cells from the colon and small intestine. Comparison of the 14-day ligated, mock ligated, and 5-day deligated stromal-enriched transcripts and lineage tracing were used to identify evidence for a partial endoMT phenotype, which was observed in a small number of endothelial cell subsets with ligation. CellChat was used to predict changes in ligand-receptor interactions in response to ligation and deligation. CellChat predicted that endothelial cells are sources of protein tyrosine phosphatase receptor type m, tumor necrosis factor ligand superfamily member 13, and myelin protein zero signaling and targets for tumor necrosis factor signaling following ligation. CellChat also predicted that endothelial cells are sources of angiocrine factors, chemokine (C-X-C motif) ligand, and EPH signaling to promote regenerative responses following deligation. These studies will inform future endothelial cell-based regenerative therapies. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.06.23.546220v1?rss=1 Authors: Perrin, S., Wotawa, C.-A., Luka, M., Coulpier, F., Masson, C., Menager, M., Colnot, C. Abstract: Bone regeneration is a highly efficient process allowing bones to fully regenerate after fracture. Skeletal stem/progenitor cells (SSPCs) allowing bone regeneration are recruited from several sources, including the periosteum located at the outer surface of bones. Periosteal SSPCs are major actors of bone healing and display the unique property to form both bone and cartilage after fracture. Yet, the steps of periosteal SSPCs activation and differentiation leading to osteogenesis and chondrogenesis remain poorly understood. Here, we generated a single-nuclei atlas of the periosteum at steady-state and in response to bone fracture. We described the heterogeneity of the uninjured periosteum and identified a population of periosteal SSPCs expressing stemness markers, such as Pi16 and Ly6a (Sca1). After fracture, we observed major changes in cell populations with a reduction of the SSPC population, a concomitant increase in injury-induced fibrogenic cells and immune cells, followed by the appearance of chondrocytes and osteoblasts. We show that periosteal SPPCs respond to injury via a unique differentiation trajectory. Cells first leave their stem/progenitor state to adopt an injury-activated fibrogenic fate, prior to undergoing either osteogenesis or chondrogenesis. Overall, our study provides a complete temporal topography of the fracture healing environment and the dynamic response of periosteal SSPC to injury, redefining our knowledge on the early steps of bone regeneration. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.06.23.544496v1?rss=1 Authors: Sirbu, A., Bathe-Peters, M., Inoue, A., Lohse, M., Annibale, P. Abstract: G protein-coupled receptors conformational landscape can be reportedly affected by their local, microscopic interactions within the cell plasma membrane. A pleiotropic stimulus to alter the cortical environment within intact cells, namely osmotic swelling, is employed here to monitor the response in terms of receptor function and downstream signaling. We observe that in osmotically swollen cells the beta2-Adrenergic receptor, a prototypical GPCR, favors an active conformation, resulting in cAMP transient responses to adrenergic stimulation that have increased amplitude. The results are validated in primary cell types such as adult cardiomyocytes, a relevant model where swelling occurs upon ischemia-reperfusion injury. Our results suggest that receptors function is finely modulated by their biophysical context, and specifically that osmotic swelling acts as a potentiator of downstream signaling, not only for the beta2-Adrenergic receptor, but also for other receptors, hinting at a more general regulatory mechanism. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.06.23.546223v1?rss=1 Authors: Valenzisi, P., Marabitti, V., Pichierri, P., Franchitto, A. Abstract: R-loops are non-canonical DNA structures that form during transcription and play diverse roles in various physiological processes. Disruption of R-loop homeostasis can lead to genomic instability and replication impairment, contributing to several human diseases, including cancer. Although the molecular mechanisms that protect cells against such events are not fully understood, recent research has identified the fork protection factors and the DNA damage response proteins as regulators of R-loop dynamics. Here, we identify the Werner helicase-interacting protein 1 (WRNIP1) as a novel factor that counteracts transcription-associated DNA damage upon replication perturbation. Loss of WRNIP1 leads to R-loop accumulation, resulting in collisions between the replisome and transcription machinery. We observe co-localization of WRNIP1 with transcription/replication complexes and R-loops after replication perturbation, suggesting its involvement in resolving transcription-replication conflicts. Moreover, WRNIP1-deficient cells show impaired replication restart from transcription-induced fork stalling. Notably, transcription inhibition and RNase H1 overexpression rescue all the defects caused by loss of WRNIP1. Importantly, our findings highlight the critical role of WRNIP1 ubiquitin-binding zinc finger (UBZ) domain in preventing pathological persistence of R-loops and limiting DNA damage, thereby safeguarding genome integrity. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.06.22.545997v1?rss=1 Authors: DiGiovanni, G. T., Han, W., Sherrill, T., Taylor, C. J., Nichols, D. S., Geis, N. M., Singha, U. K., Calvi, C. L., McCall, A. S., Dixon, M. M., Lui, Y., Jang, J.-H., Gutor, S. S., Polosukhin, V. V., Blackwell, T. S., Kropski, J. A., Gokey, J. J. Abstract: A hallmark of idiopathic pulmonary fibrosis (IPF) and other interstitial lung diseases is dysregulated repair of the alveolar epithelium. The Hippo pathway effector transcription factors YAP and TAZ have been implicated as essential for type 1 and type 2 alveolar epithelial cell (AT1 and AT2) differentiation in the developing lung, yet aberrant activation of YAP/TAZ is a prominent feature of the dysregulated alveolar epithelium in IPF. In these studies, we sought to define the functional role of YAP/TAZ activity during alveolar regeneration. We demonstrate that Yap and Taz are normally activated in AT2 cells shortly after injury, and deletion of Yap/Taz in AT2 cells led to pathologic alveolar remodeling, failure of AT2 to AT1 cell differentiation, increased collagen deposition, exaggerated neutrophilic inflammation, and increased mortality following injury induced by a single dose of bleomycin. Loss of Yap/Taz activity prior to a LPS injury prevented AT1 cell regeneration, led to intra-alveolar collagen deposition, and resulted in persistent innate inflammation. Together these findings establish that AT2 cell Yap/Taz activity is essential for functional alveolar epithelial repair and prevention of fibrotic remodeling. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.06.21.546000v1?rss=1 Authors: Sharninghausen, R., Hwang, J., Dennison, D., Baldridge, R. Abstract: Degrons are the minimal features that target proteins for degradation. In most cases, degrons allow recognition by components of the cytosolic ubiquitin proteasome system. Currently, every degron that has been identified only functions within the cytosol. Using Saccharomyces cerevisiae, we identified the first short linear sequences that function as degrons from the endoplasmic reticulum (ER) lumen. We show that when these degrons are transferred to proteins, they facilitate degradation through the ERAD system at the cytosolic proteasome. These degrons enable degradation of both luminal and integral membrane ER proteins, expanding the types of proteins that can be targeted for degradation both in budding yeast and in mammalian tissue culture. This discovery provides a framework to target proteins for degradation from the previously unreachable ER lumen and enables novel therapeutic approaches that exploit the highly-conserved ERAD system. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.06.21.545868v1?rss=1 Authors: Ehrlich, W., Kuehn, F. J. P. Abstract: Channel function of human transient receptor potential melastatin type 2 (hsTRPM2) essentially depends on its C-terminal domain NUDT9H, which is homologous to the human Nudix hydrolase NUDT9. This cytosolic enzyme specifically binds and cleaves adenosine 5'-diphosphate ribose (ADPR), which in turn represents the principal agonist of TRPM2. For hsTRPM2 the experimental data strongly suggest, that binding of ADPR to NUDT9H as well as to a separate N-terminal binding pocket induces channel gating. Recent cryogenic electron microscopy (cryo-EM) analyses have provided the first concrete clues as to how NUDT9H interacts with the channel domain. In the present study we take an alternative approach by testing co-expression of NUDT9H together with a C-terminally truncated non-functional variant of hsTRPM2. Our data obtained from co-immunoprecipitation and proximity ligation assays reveal that NUDT9H and channel domain also specifically interact when co-expressed as independent proteins. Most importantly, calcium imaging as well as whole-cell patch-clamp recordings demonstrate that this in-trans interaction restores channel function, after stimulation either with intracellular ADPR or with extracellular hydrogen peroxide. Moreover, point mutation N1326D within the NUDT9H domain previously shown to be essential for TRPM2 function significantly reduces co-immunoprecipitation of NUDT9H as well as ADPR-dependent channel activity. These findings open up new possibilities to identify the molecular determinants of this crucial inter-domain interaction. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.06.21.546015v1?rss=1 Authors: Hanaoka, K., Nishikawa, K., Ikeda, A., Schlarmann, P., Yamashita, S., Nakaji, A., Fujii, S., Funato, K. Abstract: Membrane contact sites (MCSs) are junctures that perform important roles including coordinating lipid metabolism. Previous studies have indicated that vacuolar fission/fusion processes are coupled with modifications in the membrane lipid composition. However, it has been still unclear whether MCS-mediated lipid metabolism controls the vacuolar morphology. Here we report that deletion of tricalbins (Tcb1, Tcb2, Tcb3), tethering proteins at endoplasmic reticulum (ER)-plasma membrane (PM) and ER-Golgi contact sites, alters fusion/fission dynamics and causes vacuolar fragmentation in the yeast Saccharomyces cerevisiae. In addition, we show that the sphingolipid precursor phytosphingosine accumulates in tricalbin-deleted cells, triggering the vacuolar division. Detachment of the nucleus vacuole junction (NVJ), an important contact site between the vacuole and the perinuclear ER, restored vacuolar morphology in both cells subjected to high exogenous phytosphingosine and Tcb3-deleted cells, supporting that phytosphingosine transport across the NVJ induces vacuole division. Thus, our results suggest that vacuolar morphology is maintained by MCSs through the metabolism of sphingolipids. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.06.21.545928v1?rss=1 Authors: Alvarez-Villanueva, D., Galan, L., Bertran, j., Floor, M., Sole, L., Lobo-Jarne, T., Maqueda, M., Rajbhandari, R., Marruecos, L., Villa, J., Bredel, M., Bigas, A., Espinosa, L. Abstract: We previously demonstrated that the NF-{kappa}B inhibitor I{kappa}B binds the chromatin together with PRC2 to regulate a subset of developmental- and stem cell-related genes. This alternative function has been elusive in both physiological and disease conditions because of the predominant role of I{kappa}B as a negative regulator of NF-{kappa}B. We here uniquely characterize specific residues of I{kappa}B that allow the generation of separation-of-function (SOF) mutants that are defective for either NF-{kappa}B-related (SOF{Delta}NF-{kappa}B) or chromatin-related (SOF{Delta}H2A,H4) activities. Expression of I{kappa}B SOF{Delta}NF-{kappa}B, but not SOF{Delta}H2A/H4, is sufficient to negatively regulate a specific stemness program in intestinal cells, thus rescuing the differentiation blockage imposed by I{kappa}B deficiency. In contrast, full I{kappa}B activity is required for regulating clonogenic/tumor-initiating activity of colorectal cancer cells. Our data indicate that SOF mutants represent an exclusive tool for studying I{kappa}B functions in physiology and disease, and identified I{kappa}B as a robust prognosis biomarker for human cancer. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.06.21.545957v1?rss=1 Authors: Hernandez-Ono, A., Zhao, Y. P., Murray, J. M., Ostlund, C. A., Lee, M. J., Ginsberg, H. N., Worman, H. J., Shin, J.-Y. Abstract: TorsinA is an atypical ATPase that lacks intrinsic activity unless it is bound to its activators lamina-associated polypeptide 1 (LAP1) in the perinuclear space or luminal domain-like LAP1 (LULL1) throughout the endoplasmic reticulum. However, the interaction of torsinA with LAP1 and LULL1 has not yet been shown to modulate a defined physiological process in mammals in vivo. We previously demonstrated that depletion of torsinA from mouse hepatocytes leads to reduced liver triglyceride secretion and marked steatosis, whereas depletion of LAP1 had more modest similar effects. We now show that depletion of LULL1 alone does not significantly decrease liver triglyceride secretion or cause steatosis. However, simultaneous depletion of both LAP1 and LULL1 from hepatocytes leads to defective liver triglyceride secretion and marked steatosis similar to that observed with depletion of torsinA. Our results demonstrate that torsinA and its activators dynamically regulate a physiological process in mammals in vivo. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.06.21.545899v1?rss=1 Authors: Turiel, G., Desgeorges, T., Masschelein, E., Birrer, M., Zhang, J., Engelberger, S., De Bock, K. Abstract: BackgroundPeripheral artery disease (PAD) is caused by atherosclerosis and chronic narrowing of lower limb arteries leading to decreased muscle perfusion and oxygenation. Current guidelines for treating PAD include endovascular strategies or bypass surgery but long-term outcomes have been suboptimal. This is likely due to our limited understanding of the contribution of the microvasculature as well as other cell types, in particular macrophages, to PAD skeletal muscle pathophysiology. We used single cell sequencing to investigate cellular and transcriptional heterogeneity of the skeletal muscle microenvironment in PAD. MethodsSamples from the medial head of the gastrocnemius muscle of individuals undergoing either lower limb aneurysm surgery (controls) or PAD bypass surgery (PAD) were collected. Samples were either frozen for histological evaluation (control: n=4; PAD: n=6) or were immediately processed for single cell RNA sequencing of mononuclear cells (control: n=4; PAD: n= 4). Bioinformatic tools were used to annotate cell types and their subpopulations, to study transcriptional changes and to analyze cellular interactions. ResultsWe generated a dataset comprised of 106,566 high-quality, deep-sequenced cells that compose the muscle microenvironment. Focusing on endothelial cells (ECs) and macrophages, we confirmed the presence of ATF3/4+ ECs with angiogenic and immune regulatory capacities in human muscle and found that their transcriptional profile profoundly alters during PAD. Also, capillary ECs display features of endothelial to mesenchymal transition. Furthermore, we identified LYVE1hiMHCIIlow resident macrophages as the dominant macrophage population in human muscle, even under a chronic inflammatory condition such as PAD. During PAD, LYVE1hiMHCIIlow macrophages get activated and acquire a more pro-inflammatory profile. Finally, we map strong intercellular communication in the muscle microenvironment, which is significantly altered in PAD. ConclusionsThe dataset we present here provides a highly valuable resource for gaining deeper insights into the critical roles that cells in the muscle microenvironment may play in PAD skeletal muscle pathology. We propose that targeting the crosstalk between ECs and macrophages could provide novel insights for developing effective treatments against this disease. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.06.19.545600v1?rss=1 Authors: Pierron, M., Woglar, A., Busso, C., Jha, K., Mikeladze-Dvali, T., Croisier, M., Gönczy, P. Abstract: Centrioles are lost during oogenesis in most metazoans, ensuring that the zygote is endowed with the correct number of two centrioles, which are paternally contributed. How centriole architecture is dismantled during oogenesis is not understood. Here, we analyze with unprecedent detail the ultrastructural and molecular changes during oogenesis centriole elimination in C. elegans. Centriole elimination begins with loss of the so-called central tube and organelle widening, followed by microtubule disassembly. The resulting cluster of centriolar proteins then disappears gradually, usually moving in a microtubule- and dynein-dependent manner to the plasma membrane. Moreover, we find that neither Polo-like kinases nor the PCM, which modulate oogenesis centriole elimination in Drosophila, do so in C. elegans. Furthermore, we demonstrate that the central tube protein SAS-1 normally departs first from the organelle, which loses integrity earlier in sas-1 mutants. Overall, our work provides novel mechanistic insights regarding the fundamental process of oogenesis centriole elimination. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.06.20.545672v1?rss=1 Authors: Lacroix, B., Vigneron, S., Labbé, J. C., Pintard, L., Labesse, G., Castro, A., Lorca, T. Abstract: Entry into mitosis has been classically attributed to the activation of cyclin B/cdk1 amplification loop by a partial pool of this kinase that becomes active at the end of G2. However, how this pool is activated is still unknown. Here we discovered a new role of the recently identified PP2A-B55 inhibitor FAM122A in triggering mitotic entry. Accordingly, the depletion of the orthologue of FAM122A in C. elegans, prevents entry into mitosis in germline stem cells. Moreover, our data in Xenopus egg extract strongly supports that FAM122A29 dependent inhibition of PP2A-B55 could be the initial event promoting mitotic entry. The inhibition of this phosphatase allows the subsequent phosphorylation of first mitotic substrates by cyclin A/cdk resulting in cyclin B/cdk1 and Greatwall (Gwl) activation. However, interestingly, from Gwl activation, Arpp19/ENSA become phosphorylated and compete with FAM122A promoting its dissociation from PP2A-B55 and taking over its inhibition until the end of mitosis. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.06.20.545791v1?rss=1 Authors: Simpson, M. S., De Luca, H., Cauthorn, S., Luong, P., Udeshi, N., Svinkina, T., Schmeider, S. S., Carr, S. A., Grey, M. J., Lencer, W. I. Abstract: IRE1 is an endoplasmic reticulum sensor that recognizes misfolded proteins to activate the unfolded protein response (UPR). We used cholera toxin (CTx), which activates IRE1 in cells, to understand how unfolded proteins are recognized. In vitro, the A1 subunit of CTx (CTxA1) bound IRE1 lumenal domain (IRE1LD). Global unfolding was not required. Instead, IRE1LD recognized a 7-residue motif within a metastable region of CTxA1 that was also found in microbial and host proteins involved in IRE1 activation. Binding mapped to a pocket on IRE1LD normally occupied by a segment of the IRE1 C-terminal flexible loop implicated in IRE1 regulation. Mutation of the recognition motif blocked CTx-induced IRE1 activation in live cells. These findings describe a mechanism for substrate recognition by IRE1 that induces the UPR. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.06.21.545896v1?rss=1 Authors: Wuenkhaus, D., Tang, R., Nyame, K., Laqtom, N. N., Schweizer, M., Rosato, A. S., Kroegsaeter, E. K., Wollnik, C., Abu-Remaileh, M., Grimm, C., Hermey, G., Kuhn, R., Gruber-Schoffnegger, D., Markmann, S. Abstract: Mutations in the lysosomal membrane protein CLN3 cause Juvenile Neuronal Ceroid Lipofuscinosis (JNCL). Activation of the lysosomal ion channel TRPML1 has previously been shown to be beneficial in several neurodegenerative disease models. Here, we tested whether TRPML1 activation rescues disease-associated phenotypes in CLN3-deficient retinal pigment epithelial (ARPE-19 CLN3-KO) cells. ARPE-19 CLN3-KO cells accumulate LAMP1 positive organelles and show lysosomal storage of mitochondrial ATPase subunit C (SubC), globotriaosylceramide (Gb3), and glycerophosphodiesters (GPDs), whereas lysosomal bis(monoacylglycero)phosphate (BMP/LBPA) lipid levels were significantly decreased. Activation of TRPML1 reduced lysosomal storage of Gb3 and SubC but failed to restore BMP levels in CLN3-KO cells. TRPML1-mediated decrease of storage was TFEB-independent, and we identified TRPML1-mediated enhanced lysosomal exocytosis as a likely mechanism for clearing storage including GPDs. Therefore, ARPE-19 CLN3-KO cells represent a human cell model for CLN3 disease showing many of the described core lysosomal deficits, some of which can be improved using TRPML1 agonists. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.06.22.546046v1?rss=1 Authors: He, K., Sun, X., Chen, C., Luc, S., Hao, J., Zhang, Y., Huang, Y., Wang, H., Ling, K., Hu, J. Abstract: Tubulin polyglutamylation is a post-translational modification that occurs primarily along the axoneme of cilia. Defective axoneme polyglutamylation impairs cilia function and has been correlated with ciliopathies, including Joubert Syndrome (JBTS). However, the precise mechanisms regulating proper axoneme polyglutamylation remain vague. Here, we show that Cyclin-Dependent Kinase 6 (CDK6), but not its paralog CDK4, localizes to cilia base and suppresses axoneme polyglutamylation by phosphorylating RAB11-interacting protein FIP5 at site S641, a critical regulator of cilia import of glutamylases. S641 phosphorylation disrupts the ciliary recruitment of FIP5 by impairing its association with RAB11, thereby reducing the ciliary import of glutamylases. Interestingly, significant upregulation of CDK6 and defective axoneme polyglutamylation were observed in Autosomal dominant polycystic kidney disease (ADPKD) cells. Encouragingly, the FDA-approved CDK4/6 inhibitor Abemaciclib can effectively restore cilia function in JBTS and ADPKD cells with defective glutamylation and suppresses renal cystogenesis in an ex vivo ADPKD model. In summary, our study elucidates regulatory mechanisms governing axoneme polyglutamylation and suggests developing CDK6-specific inhibitors could be a promising therapeutic strategy to enhance cilia function in ciliopathy patients. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.06.21.545954v1?rss=1 Authors: Thompson, J., Boisvert, F.-M., Salsman, J., Dellaire, G., Ridgway, N. D. Abstract: When exposed to excess fatty acids, specific cell types produce nuclear lipid droplets (nLDs) that associate with promyelocytic leukemia (PML) protein to form Lipid Associated PML Structures (LAPS) that are enriched in lipid biosynthetic enzymes but deficient in canonical proteins associated with PML nuclear bodies (PML NBs). To identify the PML interactome during lipid stress, we employed proximity-dependent biotin identification (BioID) in U2OS cells expressing PMLI and PMLII fused to the ascorbate peroxidase APEX2 and cultured in the absence or presence of oleate to enhance lipid droplet formation. The resulting interactome included proteins enriched under oleate-treated conditions, such mitogen activated protein kinase-activated protein kinase 2 (MK2), ESCRT proteins and the COPII vesicle transport proteins Sec23B, Sec24A and USO1. COPII proteins co-localized with both PML-NBs and LAPS but were selectively enriched in PML-NBs following oleate treatment. The nuclear localization of USO1 was uniquely dependent on PML expression. Thus, the APEX2-PML proximity interactome implicates PML domains in the nuclear function of a non-canonical network of COPII vesicle trafficking proteins. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.06.21.545854v1?rss=1 Authors: Gebert, M., Bartoszewska, S., Opalinski, L., Collawn, J., Bartoszewski, R. Abstract: The unfolded protein response is a survival signaling pathway that is induced during various types of ER stress. Here we focus on the IRE1 pathway to determine IRE1 role in miRNA regulation during ER stress. During induction of ER stress in human bronchial epithelial cells, we utilized next generation sequencing to demonstrate that pre-miR-301a and pre-miR-106b, were significantly increased in the presence of an IRE1 inhibitor. Conversely, using nuclear-cytosolic fractionation on ER stressed cells, we found that these three pre-miRNAs were decreased in the nuclear fractions without the IRE1 inhibitor. We also found that miR-301a-3p targets the proapoptotic UPR factor, growth arrest and DNA-damage-inducible alpha (GADD45A). Inhibiting miR-301a-3p levels or blocking its predicted miRNA binding site in GADD45A 3 UTR with a target protector increased GADD45A mRNA expression. An elevation of XBP1s expression had no effect on GADD45A mRNA expression. We also demonstrated that the introduction of a target protector for the miR-301a-3p binding site in GADD45A mRNA during ER stress promoted cell death in the airway epithelial cells. These results indicated that IRE1 endonuclease activity is a two-edged sword that splices XBP1 mRNA for survival and degrades pre-miR-301a to elevate the mRNA expression of a pro-apoptotic gene, GADD45A. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.06.22.546088v1?rss=1 Authors: Mikhova, M., Heyza, J. R., Meek, K., Schmidt, J. C. Abstract: Non-homologous end joining (NHEJ) is the predominant path-way that repairs DNA double-stranded breaks (DSBs) in vertebrates. The DNA termini of many DSBs must be processed to allow ligation while minimizing genetic changes that result from break repair. Emerging models propose that DNA termini are first synapsed approximately 115[A] apart in one of two long-range synaptic complexes. The first long-range complex can be formed with only the KU70/80 heterodimer and DNA-PKcs while the second long-range complex also includes XRCC4, XLF, and Ligase 4. Both long-range complexes inefficiently progress to short-range synaptic complexes that juxtapose DNA ends to facilitate ligation. Here we perform singlemolecule analyses of the recruitment of Halo-tagged NHEJ factors to DSBs. Our results provide direct evidence for stepwise maturation of NHEJ complex and precisely define kinetics of core NHEJ factor binding to DSBs in living cells. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.06.22.546089v1?rss=1 Authors: Yu, F., Courjaret, R., Elmi, A., Hammad, A. S., Fisher, M., Terasaki, M., Machaca, K. Abstract: Membrane contact sites between various organelles define specialized spatially defined signaling hubs, they are of great interest to better understand inter-organelle communication and its implications on cellular physiology. ER-mitochondria contact sites (ERMCS) are one of the best studied and mediate Ca2+ signaling that regulates mitochondrial bioenergetics. However, little is known about ERMCS during mitosis. Here we show that ERMCS expand during mitosis using transmission electron microscopy, serial EM coupled to 3D reconstruction, and ERMCS markers. ERMCS expansion in mitosis is functionally significant as it is associated with enhanced Ca2+ coupling between the ER and mitochondria resulting in heightened activation of mitochondrial dehydrogenases. Our data suggest that ERMCS remodeling in mitosis is important to meet the increased energy needs during cell division. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.06.22.546109v1?rss=1 Authors: Tillery, M. M. L., Zheng, C., Zheng, Y., Megraw, T. L. Abstract: Ninein (Nin) is a microtubule (MT) anchor at the subdistal appendages of mother centrioles and the pericentriolar material (PCM) of centrosomes that also functions to organize microtubules at non-centrosomal microtubule-organizing centers (ncMTOCs). In humans, the NIN gene is mutated in Seckel syndrome, an inherited developmental disorder. Here we dissect the protein domains involved in Nin's localization and interactions with dynein and ensconsin (ens/MAP7) and show that the association with ens cooperatively regulates microtubule assembly in Drosophila fat body cells. We define domains of Nin responsible for its localization to the ncMTOC on the fat body cell nuclear surface, localization within the nucleus, and association with Dynein light intermediate chain (Dlic) and ens, respectively. We show that Nin's association with ens synergistically regulates MT assembly. Together, these findings reveal novel features of Nin function and its regulation of a ncMTOC. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.06.22.546073v1?rss=1 Authors: Jablonski, M., Luque, G. M., Gomez-Elias, M., Sanchez-Cardenas, C., Xu, X., de la Vega-Beltran, J. L., Corkidi, G., Linares, A., Abonza, V., Krapf, D., Krapf, D., Darszon, A., Guerrero, A., Buffone, M. G. Abstract: Mammalian sperm delve into the female reproductive tract to fertilize the female gamete. Within the midpiece of the sperm flagellum lies a cortical network of actin that is arranged as a double helix sheltering the mitochondrial sheath. This work demonstrates that the actin network of the midpiece undergoes structural changes that result in motility cessation. This structural modification is accompanied by a decrease in diameter of the midpiece and is driven by intracellular calcium changes that occur concomitant with a reorganization of the actin helicoidal cortex. Although midpiece contraction may occur in a subset of cells that undergo acrosomal exocytosis, live-cell imaging during in vitro fertilization showed that the midpiece contraction is required for motility cessation during the fusion process. These findings provide the first evidence of the F-actin network's role in regulating sperm motility, adapting its function to meet specific cellular requirements during fertilization. These findings provide the first evidence of the F-actin network's role in regulating sperm motility, adapting its function to meet specific cellular requirements during fertilization, and highlighting the broader significance of understanding sperm motility. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.06.22.546020v1?rss=1 Authors: Beale, A. D., Rzechorzek, N. M., Mihut, A., Zeng, A., Smyllie, N. J., Pilorz, V., Richardson, R., Bertlesen, M. F., James, N. R., Fazal, S. V., Voysey, Z., Pelletier, J., Crosby, P., Peak-Chew, S. Y., Lancaster, M. A., Hut, R. A., O'Neill, J. S. Abstract: Early mammals were nocturnal until the Cretaceous-Paleogene extinction facilitated their rapid expansion into daytime niches. Diurnality subsequently evolved multiple times, independently, but the mechanisms facilitating this switch are unknown. We found that physiological daily temperature shifts oppositely affect circadian clock rhythms in nocturnal versus diurnal mammals. This occurs through a cell-intrinsic signal inverter, mediated by global differences in protein phosphorylation, and effected at the level of bulk protein synthesis rates, with diurnal translation rate being less thermosensitive than nocturnal. Perturbations that reduce translational initiation or mTOR activity are sufficient to trigger the nocturnal-to-diurnal switch at the cellular, tissue, and organismal scale. Our results suggest a convergent selection pressure to attain diurnality by reducing the effect of temperature-dependent changes in protein synthesis on circadian clocks. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.06.22.546081v1?rss=1 Authors: Rochon, K., Bauer, B. L., Roethler, N. A., Buckley, Y., Su, C.-C., Huang, W., Ramachandran, R., Stoll, M. S. K., Yu, E. W., Taylor, D. J., Mears, J. A. Abstract: Mitochondrial fission is crucial for distributing cellular energy throughout cells and for isolating damaged regions of the organelle that are targeted for degradation. This multistep process is initiated by the enhanced recruitment and oligomerization of dynamin-related protein 1 (Drp1) at the surface of mitochondria. As such, Drp1 is essential for inducing mitochondrial division in mammalian cells, and homologous proteins are found in all eukaryotes. De novo missense mutations in the Drp1 gene, DNM1L, are associated with severe neurodevelopmental diseases in patients, and no effective treatments are available. As a member of the dynamin superfamily of proteins (DSPs), controlled Drp1 self-assembly into large helical polymers stimulates its GTPase activity to promote membrane constriction. Still, little is known about the regulatory mechanisms that determine when and where Drp1 self-assembles, and proper mitochondrial dynamics requires correct spatial and temporal assembly of the fission machinery. Here we present a cryo-EM structure of a full-length, native Drp1 dimer in an auto-inhibited state. This dimer reveals two key conformational rearrangements that must be unlocked through intermolecular interactions to achieve the assembly competent state previously observed in crystal and filament structures. Specifically, the G domain is closed against the stalk domain and occludes intermolecular interactions necessary for self-assembly beyond a dimer. Similarly, adjacent stalks in the dimer form a more continuous interface that further occludes conserved intermolecular contact sites. This structural insight provides a novel mechanism for regulated self-assembly of the mitochondrial fission machinery. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.06.22.546113v1?rss=1 Authors: Vieira, P., Kantor, M. R., Jansen, A., Handoo, Z., Eisenback, J. D. Abstract: The beech leaf disease nematode, Litylenchus crenatae subsp. mccannii, is recognized as a newly emergent nematode species that causes beech leaf disease (BLD) in beech trees (Fagus spp.) in North America. Changes of leaf morphology induced by BLD can provoke dramatic effects into the leaf architecture and consequently to tree performance and development. The initial symptoms of BLD appear as dark green interveinal banding patterns of the leaf. Despite the fast progression of this disease, the cellular mechanisms leading to the formation of such type of aberrant leaf phenotype remains totally unknown. To understand the cellular basis of BLD, we employed several microscopy approaches to provide an exhaustive characterization of nematode-infected buds and leaves. Histological sections revealed a dramatic cell change composition of these nematode-infected tissues. Diseased bud scale cells were typically hypertrophied and showed a high variability of size. Moreover, while altered cell division had no influence on leaf organogenesis, induction of cell proliferation on young leaf primordia led to a dramatic change in cell layer architecture. Hyperplasia and hypertrophy of the different leaf cell layers, coupled with an abnormal proliferation of chloroplasts specially in the spongy mesophyll cells, resulted in the typical interveinal leaf banding. These discrepancies in leaf cell structure were depicted by an abnormal rate of cellular division of the leaf interveinal areas infected by the nematode, promoting significant increase of cell size and leaf thickness. The formation of symptomatic BLD leaves is therefore orchestrated by distinct cellular processes, to enhance the value of these feeding sites and to improve their nutrition status to the nematode. These results revealed a high specialized mode of parasitism of L. crenatae subsp. mccannii. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.06.19.545587v1?rss=1 Authors: Ancel, S., Michaud, J., Sizzano, F., Tauzin, L., Oliveira, M., Migliavacca, E., Dammone, G., Karaz, S., Sanchez-Garcia, J. L., Metairon, S., Jacot, G., Bentzinger, F. C., Feige, J. N., Stuelsatz, P. Abstract: Increasing evidence suggests that muscle stem cells (MuSCs) are a heterogeneous population. In particular, a rare subset of Pax7 positive MuSCs that has never expressed the myogenic regulatory factor Myf5 has been shown to have superior self-renewal and engraftment characteristics when compared to lineage positive cells. However, their scarcity and the limited availability of protein markers make the characterization of this MuSC subpopulation challenging. Complementing lineage tracing approaches, we developed the StemRep in vivo model with reporter alleles allowing to monitor Pax7 and Myf5 protein levels based on equimolar amounts of dual nuclear fluorescence. Using transcriptomic profiling and ex vivo cellular assays, we demonstrate that levels of Pax7 and Myf5 protein delineate subpopulations of MuSCs with distinct molecular signatures and dynamics of activation, proliferation, and commitment. MuSCs expressing high levels of Pax7 protein (Pax7high) and low levels of Myf5 (Myf5Low) retain phenotypic features and molecular signatures of quiescence, and transiently remodel during regeneration. The Pax7 reporter tracks the decline of the MuSC pool during aging, while the Myf5 reporter enables live monitoring of the age-related loss of quiescence via skewing of the MuSC population towards Myf5High. Altogether, we characterize Pax7highMyf5Low MuSCs as an uncommitted subpopulation in deep quiescence and establish the StemRep line as a novel versatile tool for the physiological study of stem cell dynamics in skeletal muscle. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.06.18.545506v1?rss=1 Authors: McNeill, M. C., Chee, F. L., Ebrhimighaei, R., Sala Newby, G. B., Newby, A. C., Hathway, T., Annaiah, A. S., Joseph, S., Carrabba, M., Bond, M. Abstract: 1.0BackgroundVascular calcification (VC) is a prevalent independent risk factor for adverse cardiovascular events and is associated with diabetes, hypertension, chronic kidney disease, and atherosclerosis. However, the mechanisms regulating the osteogenic differentiation of vascular smooth muscle cells (VSMC) are not fully understood. MethodsUsing hydrogels of tuneable stiffness and lysyl oxidase-mediated stiffening of human saphenous vein ex vivo,we investigated the role of extracellular matrix (ECM) stiffness in the regulation of VSMC calcification ResultsWe demonstrate that increased ECM stiffness enhances VSMC osteogenic differentiation and VSMC calcification. We show that the effects of ECM stiffness are mediated via a reduction in the level of actin monomer within the nucleus. We show that in cells interacting with soft ECM, elevated levels of nuclear actin monomer repress osteogenic differentiation and calcification by repressing YAP-mediated activation of both TEA Domain transcription factor (TEAD) and RUNX Family Transcription factor 2 (RUNX2). ConclusionThis work highlights for the first time the role of nuclear actin in mediating ECM stiffness-dependent VSMC calcification and the dual role of YAP-TEAD and YAP-RUNX2 transcriptional complexes. 2.0 GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=140 SRC="FIGDIR/small/545506v1_ufig1.gif" ALT="Figure 1" greater than View larger version (24K): [email protected]@619f4borg.highwire.dtl.DTLVardef@1212f97org.highwire.dtl.DTLVardef@1bb9766_HPS_FORMAT_FIGEXP M_FIG C_FIG 9.0 HIGHLIGHTSO_LIIncreased ECM stiffness promotes VSMC calcification. C_LIO_LIIncreased ECM stiffness reduces levels of nuclear actin monomer. C_LIO_LIOn physiological soft ECM, high levels of nuclear actin monomer inhibits calcification by repressing YAP activation. C_LIO_LIYAP activation promotes calcification by stimulating the activity of TEAD and RUNX2. C_LI Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.06.19.545545v1?rss=1 Authors: Kandasamy, K., Fan, X., Haribalaganesh, R., Bharanidharan, D., Sharmila, R., Krishnadas, R., Muthukkaruppan, V., Willoughby, C. E., Srinivasan, S. Abstract: The present study aimed to understand the role of miRNAs in differential glucocorticoid (GC) responsiveness in human trabecular meshwork (HTM) cells using small RNA sequencing. For this, total RNA was extracted from cultured HTM cells with known GC responsiveness using Human organ-cultured anterior segment (HOCAS) (GC-responder GC-R; n=4) and GC-non-responder (GC-NR; n=4) after treatment with either 100nM dexamethasone (DEX) or ethanol (ETH) for 7 days. Differentially expressed miRNAs (DEMIRs) were compared among 5 groups and validated by RT-PCR. There were 13 and 21 DEMIRs identified in Group #1 (ETH vs DEX-treated GC-R) and Group #2 (ETH vs DEX-treated GC-NR) respectively. Seven miRNAs were found as common miRNAs dysregulated in both GC-R and GC-NR (Group #3). There were 6 and 14 unique DEMIRs were identified in GC-R (Gropu#4) and GC-NR (Group#5) HTM cells respectively. Ingenuity Pathway Analysis identified enriched pathways and biological processes associated with differential GC responsiveness in HTM cells. Integrative analysis of miRNA-mRNA of the same set of HTM cells revealed several molecular regulators for GC non-responsiveness. This is the first study revealed a unique miRNA signature between GC-R and GC-NR HTM cells which raises the possibility of developing new molecular targets for the management of steroid-OHT/glaucoma. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.06.19.545547v1?rss=1 Authors: Shaba, E., Landi, C., Marzocchi, C., Vantaggiato, L., Bini, L., Ricci, C., Cantara, S. Abstract: The genome sequencing of the tardigrade Ramazzottius varieornatus revealed a unique nucleosome-binding protein, named Damage Suppressor (Dsup), which resulted to be crucial for the extraordinary abilities of tardigrades in surviving extreme stresses, such as UV. Evidence in Dsup transfected human cells, suggests that Dsup mediates an overall response of DNA damage signaling, DNA repair and cell cycle regulation resulting in an acquired resistance to stress. Given these promising outcomes, our study attempts to provide a wider comprehension of the molecular mechanisms modulated by Dsup in human cells, and to explore the Dsup-activated molecular pathways under stress. We performed a differential proteomic analysis of Dsup-transfected and control human cells, under basal condition and at 24-hour recovery after exposure to UV-C. We demonstrate by enrichment and network analyses, for the first time, that even in the absence of external stimuli and more significantly after stress, Dsup activates mechanisms involved with the Unfolded Protein Response, the mRNA processing and stability, cytoplasmic stress granules, the DNA Damage Response and the telomere maintenance. In conclusion, our results shed new light on Dsup-mediated protective mechanisms, and increase our knowledge of the molecular machineries of extraordinary protection against UV-C stress. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.06.19.545304v1?rss=1 Authors: Heydasch, M., Hinderling, L., van Unen, J., Dobrzynski, M., Pertz, O. Abstract: Tightly regulated spatio-temporal Rho GTPase activity patterns regulate morphogenetic processes such as cell migration. Emerging evidence suggests that binding of Guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs) to the cytoskeleton or adhesions mediate feedback regulation to spatio-temporal Rho GTPase activation. To explore such feedback regulation, we study the Rho specific GAP Deleted in Liver Cancer 1 (DLC1) which binds to focal adhesions (FAs) through mechanosensitive interactions. Using a FRET biosensor, we show that DLC1 loss of function leads to global increase in Rho activity and contractility throughout the cell without affecting a striking lamellar RhoA activity band in fibroblasts. To interrogate the Rho GTPase signaling flux, we build a genetic circuit consisting of an optogenetic actuator to control Rho activity, and a Rho activity biosensor. In spreading cells at steady state, optogenetic manipulation of Rho activity reveals that DLC1 controls the rate of Rho activation rather than duration, both at FAs and at the plasma membrane (PM). Local and reversible optogenetic control of contractility shows that DLC1 associates/dissociates with FAs during their reinforcement/relaxation. This might provide positive feedback that locally increases the rate of Rho activation at FAs that experience local tension to facilitate FA disassembly. Our results indicate that DLC1 operates both at the PM and at FAs to regulate global Rho activity levels at steady state, or to amplify local Rho activity at FAs experiencing a strong mechanical input, presumably to induce robust FA disassembly. This provides new insights in the complexity of spatio-temporal Rho GTPase signaling. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.05.05.539649v1?rss=1 Authors: Morales, E. A., Tyska, M. J. Abstract: Actin bundling proteins crosslink filaments into polarized structures that shape and support membrane protrusions including filopodia, microvilli, and stereocilia. In the case of epithelial microvilli, mitotic spindle positioning protein (MISP) is an actin bundler that localizes specifically to the basal rootlets, where the pointed ends of core bundle filaments converge. Previous studies established that MISP is prevented from binding more distal segments of the core bundle by competition with other actin binding proteins. Yet whether MISP holds a preference for binding directly to rootlet actin remains an open question. Using in vitro TIRF microscopy assays, we found that MISP exhibits a clear binding preference for filaments enriched in ADP-actin monomers. Consistent with this, assays with actively growing actin filaments revealed that MISP binds at or near their pointed ends. Moreover, although substrate attached MISP assembles filament bundles in parallel and antiparallel configurations, in solution MISP assembles parallel bundles consisting of multiple filaments exhibiting uniform polarity. These discoveries highlight nucleotide state sensing as a mechanism for sorting actin bundlers along filaments and driving their accumulation near filament ends. Such localized binding might drive parallel bundle formation and/or locally modulate bundle mechanical properties in microvilli and related protrusions. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.05.05.539436v1?rss=1 Authors: Smythe, E., Alshahrani, F., Zhu, Z., Ferreira, F., Maib, H., Pruzina, S., Robinson, D., Murray, D. H., McComb, A. Abstract: Epidermal growth factor receptor (EGFR) signalling results in a variety of cell behaviours, including cell proliferation, migration and apoptosis, which depend on cell context. Here we have explored how the Rab5GEF, Rme-6, regulates EGFR signalling by modulating endocytic flux. We demonstrate that Rme-6, which acts early in the endocytic pathway, regulates EGFR trafficking through an endocytic compartment that is competent for ERK1/2 signalling. While overexpression of Rme-6 results in enhanced ERK1/2 nuclear localisation and c-Fos activation, loss of Rme-6 results in aberrant ERK1/2 signalling with increased cytoplasmic ERK1/2 phosphorylation (Thr202/Tyr204) but decreased ERK1/2 nuclear translocation and c-Fos activation, the latter leading to decreased cell proliferation. Phosphorylation of ERK1/2 by protein kinase 2 (CK2) is required for its nuclear translocation and our data support a model whereby Rme-6 provides a scaffold for a population of CK2 which is required for efficient nuclear translocation of ERK1/2. Rme-6 is itself a substrate for CK2 on Thr642 and Ser996 and phosphorylation on these sites can activate its Rab5GEF activity and endocytic trafficking of EGFR. Together our results indicate that that Rme-6 co-ordinates EGFR trafficking and signalling to regulate the assembly and disassembly of an ERK1/2 signalosome. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.05.04.539490v1?rss=1 Authors: Pimm, M. L., Marcin, A. G., Haarer, B. K., Eligio, M. A., Henty-Ridilla, J. L. Abstract: Cell processes require precise regulation of actin polymerization at filament plus ends to execute normal functions. The detailed mechanisms used to control filament assembly at plus ends in the presence of diverse and often opposing regulators is not clear. Here we explore and identify residues important for the plus-end related activities of IQGAP1. In multi-wavelength TIRF assays, we directly visualize dimers of IQGAP1, mDia1, and capping protein (CP) on filament ends alone and as a multicomponent end binding complex. IQGAP1 promotes the turnover of end-binding proteins, reducing the dwell times of CP, mDia1, or mDia1-CP "decision complexes" by 8-18-fold. Loss of these activities in cells disrupts actin filament arrays, morphology, and migration. Together, our results reveal a new role for IQGAP1 in promoting protein turnover on filament ends and provide new insights into how actin assembly is regulated in cells. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.05.04.539347v1?rss=1 Authors: Green, D. C., Reynard, L., Henstock, J., Reppe, S., Gutvik, K., Peffers, M., Shanley, D., Clegg, P. D., Canty-Laird, E. G. Abstract: Background: Age is a significant risk factor for functional decline and disease of the musculoskeletal system, yet few biomarkers exist to facilitate ageing research in musculoskeletal tissues. Multivariate models based on DNA methylation, termed epigenetic clocks, have shown promise as markers of biological age. However, the accuracy of existing epigenetic clocks in musculoskeletal tissues are no more, and often less accurate than a randomly sampled baseline model. Results: We developed a highly accurate epigenetic clock, MskAge, that is specific to tissues and cells of the musculoskeletal system. MskAge was built using a penalised genetic algorithm islands model that addresses multi-tissue clock bias. The final model was trained on the transformed principal components of CpGs selected by the genetic algorithm, which are significantly enriched for pathways terms related to the skeletal system and mesenchyme development. We show that MskAge tracks epigenetic ageing ex vivo and in vitro. Epigenetic age estimates are rejuvenated to zero with cellular reprogramming and are accelerated at a rate of 0.45 years per population doubling. Remarkably, MskAge explains more variance associated with in vitro ageing of fibroblasts than the purpose-developed skin and blood clock. Conclusion: The precision of MskAge and its ability to capture perturbations in biological ageing make it a promising research tool for musculoskeletal and ageing biologists. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.05.04.539340v1?rss=1 Authors: Morozumi, Y., Mahayot, F., Nakase, Y., Soong, J. X., Yamawaki, S., Sofyantoro, F., Imabata, Y., Oda, A. H., Tamura, M., Kofuji, S., Akikusa, Y., Ohta, K., Shiozaki, K. Abstract: Cells cease to proliferate above their growth-permissible temperatures, a ubiquitous phenomenon generally attributed to protein denaturing and heat damage to other cellular macromolecules. We here report that, in the presence of the macrolide compound rapamycin, the fission yeast Schizosaccharomyces pombe can proliferate at high temperatures that normally arrest its growth. Rapamycin is a potent inhibitor of the protein kinase complex TOR Complex 1 (TORC1), and consistently, mutations to the TORC1 subunit RAPTOR/Mip1 and the TORC1 substrate Sck1 significantly improve cellular heat resistance. These results suggest that TORC1, a well-established growth promoter, restricts the high-temperature growth of fission yeast and that compromised TORC1 signaling allows cell proliferation at higher temperatures. Aiming for a more comprehensive understanding of the negative regulation of high-temperature growth, we conducted genome-wide screens in S. pombe, which identified Sck1 and additional factors that appear to suppress cell proliferation at high temperatures. Our study has uncovered unexpected mechanisms of growth restraint even below the temperatures deleterious to cell physiology. Thus, growth arrest at high temperatures may not directly result from heat damage to cellular components essential for proliferation and viability. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.05.05.539547v1?rss=1 Authors: Dupuy, L., Mimault, M., Ptashnyk, M. Abstract: Movement is critical for bacterial species inhabiting soils because nutrient availability is limited and heterogeneously distributed both in space and time. Recent live microscopy experiments show that bacteria form flocks when navigating through porous medium, and complex cell-cell interactions may be required to maintain such flocks. Here we propose a non-local model to study how peer attraction can affect flocking patterns in a porous medium. We establish the existence and uniqueness of the solution of the problem, propose a numerical scheme for simulations of the non-local convection-diffusion equation, and investigate the numerical convergence of the scheme. Numerical simulations showed that the strength of peer attraction is critical to control the size, shape, and nature of movement of the flocks in a porous network. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.05.04.539483v1?rss=1 Authors: DiPeso, L., Pendyala, S., Huang, H. Z., Fowler, D. M., Hatch, E. M. Abstract: Micronuclei are aberrant nuclear compartments that trap a portion of a cell's chromatin in a distinct organelle separate from the nucleus and are drivers of inflammation, DNA damage, chromosome instability, and chromothripsis. Many of the consequences of micronucleus formation stem from micronucleus rupture: the sudden loss of micronucleus compartmentalization, resulting in mislocalization of nuclear factors and the exposure of chromatin to the cytosol for the remainder of interphase. Micronuclei form primarily from segregation errors during mitosis, errors that also give rise to other, non-exclusive phenotypes, including aneuploidy and chromatin bridges. The stochastic formation of micronuclei and phenotypic overlap confounds the use of population-level assays or hypothesis discovery, requiring labor-intensive techniques to visually identify and follow micronucleated cells individually. In this study, we present a novel technique for automatically identifying and isolating micronucleated cells generally and cells with ruptured micronuclei specifically using a de novo neural net combined with Visual Cell Sorting. As a proof of concept, we compare the early transcriptomic responses to micronucleation and micronucleus rupture with previously published responses to aneuploidy, revealing micronucleus rupture to be a potential driver of the aneuploidy response. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.05.05.539557v1?rss=1 Authors: Christofidou, E., Tomazou, M., Voutouri, C., Michael, C., Stylianopoulos, T., Spyrou, G. M., Strati, K. Abstract: Oct4 is a pioneer transcription factor regulating pluripotency. However, it is poorly known whether Oct4 has an impact on somatic cells. We generated OCT4 knockout clonal cell lines using immortalized human skin keratinocytes to identify a functional role for the protein. Here we report that Oct4-deficient cells transitioned into a mesenchymal-like phenotype with enlarged size and shape, exhibited accelerated migratory behavior, decreased adhesion and appeared arrested at G2/M cell cycle checkpoint. Oct4 absence had a profound impact on cortical actin organization, with loss of microfilaments from cell periphery, increased puncta deposition in the cytoplasm and stress fiber formation. E-cadherin, beta-catenin and ZO1 were almost absent from cell-cell contacts while fibronectin deposition was markedly increased in ECM. Mapping of the transcriptional and chromatin profiles of Oct4-deficient cells revealed that Oct4 controls the levels of cytoskeletal, ECM and differentiation related genes, whereas epithelial identity is preserved through transcriptional and non-transcriptional mechanisms. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.05.04.539499v1?rss=1 Authors: ZHANG, C., Hu, J., SHI, Y., FENG, Y., LI, Z., ZHANG, T., HONG, L., DONG, Z., TANG, Y., WANG, Z., NING, G., Huang, G.-R. Abstract: Microparticles (MPs) are convenient for clinical diagnosis, and have functional roles in signal transduction. Although the importance of MPs is being increasingly recognized, the diversity of isolated protocols for MPs results in a heterogeneous population of their unknown origins, even expands to uncertain functions. Here we systematically studied the composition of MPs at different centrifugal speed intervals, and found that 3000g was a critical centrifugation speed in determining new MPs composition. The platelet-derived particles accounted for more than 80% under 3000g, while only about 20% in MPs obtained over 3000g. Furthermore, we found that the function of new MPs was significantly different from that of traditional ones, such as procoagulation activity, anti-inflammation and clinical diagnosis etc. Thus, our work optimized the method of MPs isolation, clarified some characteristics and physiological functions that should belong to platelets rather than MPs, which will derive new conceptual MPs for its composition and function. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.05.04.539104v1?rss=1 Authors: Salois, M. N., Gugger, J. A., Webb, S., Sheldon, C. E., Parraga, S. P., Lewitt, G. M., Grange, D. K., Koch, P. J., Koster, M. I. Abstract: The goal of this study was to investigate the molecular mechanisms responsible for the formation of skin erosions in patients affected by Ankyloblepharon-ectodermal defects-cleft lip/palate syndrome (AEC). This ectodermal dysplasia is caused by mutations in the TP63 gene, which encodes several transcription factors that control epidermal development and homeostasis. We generated induced pluripotent stem cells (iPSC) from AEC patients and corrected the TP63 mutations using genome editing tools. Three pairs of the resulting conisogenic iPSC lines were differentiated into keratinocytes (iPSC-K). We identified a significant downregulation of key components of hemidesmosomes and focal adhesions in AEC iPSC-K compared to their gene-corrected counterparts. Further, we demonstrated reduced iPSC-K migration, suggesting the possibility that a process critical for cutaneous wound healing might be impaired in AEC patients. Next, we generated chimeric mice expressing a TP63-AEC transgene and confirmed a downregulation of these genes in transgene-expressing cells in vivo. Finally, we also observed these abnormalities in AEC patient skin. Our findings suggest that integrin defects in AEC patients might weaken the adhesion of keratinocytes to the basement membrane. We propose that reduced expression of extracellular matrix adhesion receptors, potentially in conjunction with previously identified desmosomal protein defects, contribute to skin erosions in AEC. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.05.04.538989v1?rss=1 Authors: Nanes, B. A., Bhatt, K., Azarova, E., Rajendran, D., Isogai, T., Dean, K. M., Danuser, G. Abstract: Keratin intermediate filaments form strong mechanical scaffolds that confer structural stability to epithelial tissues, but the reason this function requires a protein family with fifty-four isoforms is not understood. During skin wound healing, a shift in keratin isoform expression alters the composition of keratin filaments. How this change modulates cellular function to support epidermal remodeling remains unclear. We report an unexpected effect of keratin isoform variation on kinase signal transduction. Increased expression of wound-associated keratin 6A, but not of steady-state keratin 5, potentiated keratinocyte migration and wound closure without compromising epidermal stability by activating myosin motors. This pathway depended on isoform-specific interaction between intrinsically disordered keratin head domains and non-filamentous vimentin shuttling myosin-activating kinases. These results substantially expand the functional repertoire of intermediate filaments from their canonical role as mechanical scaffolds to include roles as signaling scaffolds that spatiotemporally organize signal transduction cascades depending on isoform composition. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.05.05.539603v1?rss=1 Authors: Seager, R., Shree Ramesh, N., Cross, S., Guo, C., Wilkinson, K., Henley, J. Abstract: Mitochondrial fission regulates mitochondrial morphology, function, mitophagy and apoptosis. Fission is mediated by the GTPase dynamin related protein-1 (DRP1) and its recruitment to the outer mitochondrial membrane by DRP1 receptors. Mitochondrial fission factor (MFF) is considered the major pro-fission receptor, whereas the mitochondrial dynamics proteins (MiD49/51) sequester inactive DRP1 and facilitate the MFF-DRP1 interaction by forming a trimeric DRP1-MiD-MFF complex. Here, we identify MFF as a target of poly-SUMOylation at a single residue (Lys151). Following bioenergetic stress, AMPK phosphorylates MFF to promote its SUMOylation, a critical step in stress-induced fragmentation. MFF SUMOylation is not required for DRP1 recruitment from the cytosol but causes a rearrangement of the trimeric fission complex to displace MiD proteins. This alleviates MiD inhibition of DRP1 to facilitate formation of a fission-competent complex. Thus, our data demonstrate that MFF SUMOylation fine-tunes the ratio of MiD to DRP1 for the dynamic control of stress-induced mitochondrial fragmentation. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.05.05.539599v1?rss=1 Authors: Varela, L., van de Lest, C., Boere, J., Libregts, S., Lozano-Andres, E., van Weeren, R., Wauben, M. Abstract: Inflammation is the hallmark of most joint disorders. However, the precise regulation of induction, perpetuation, and resolution of joint inflammation is not entirely understood. Since extracellular vesicles (EVs) are critical for intercellular communication, we aim to unveil their role in these processes. Here, we investigated the EVs' dynamics and phospholipidome profile from synovial fluid (SF) of healthy equine joints and from horses with lipopolysaccharide (LPS)-induced synovitis. LPS injection triggered a sharp increase of SF-EVs at 5-8hr post-injection, which started to decline at 24h post-injection. Importantly, we identified significant changes in the lipid profile of SF-EVs after synovitis induction. Compared to healthy joint-derived SF-EVs (0h), SF-EVs collected at 5, 24, and 48h post-LPS injection were strongly increased in hexosylceramides. At the same time, phosphatidylserine, phosphatidylcholine, and sphingomyelin were decreased in SF-EVs at 5h and 24h post-LPS injection. Based on the lipid changes during acute inflammation, we composed specific lipid profiles associated with healthy and inflammatory state-derived SF-EVs. The sharp increase in SF-EVs during acute synovitis and the correlation of specific lipids with either healthy or inflamed states-derived SF-EVs are findings of potential interest for unveiling the role of SF-EVs in joint inflammation, as well as for the identification of EV-biomarkers of joint inflammation. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.05.05.539527v1?rss=1 Authors: Alves Nicolau, C., Renaud, C., Maghe, C., Trillet, K., Jardine, J., Escot, S., David, N. B., Gavard, J., Bidere, N. Abstract: (PCM1) protein, that gravitate as particles around the centrosome. Centriolar satellites have been linked to key cellular processes including the formation of primary cilia and GABARAP-mediated autophagy. However, to date, there is no pharmacological inhibitor of centriolar satellites. Here, we report that necrosulfonamide (NSA), a compound previously shown to target the cell death effector MLKL, induced non-degradative ubiquitination of PCM1. This also caused a defect in ciliogenesis and an accumulation of autophagy markers such as P62 and GABARAPL1, although the overall architecture and distribution of centriolar satellites were maintained. We further found that the depletion of PCM1 lessened the impact of NSA on autophagy. Altogether, NSA may be a valuable tool to study centriolar satellites and provide further insights into their interplay with autophagy. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.05.04.537313v1?rss=1 Authors: Hellweg, L., Pfeifer, M., Chang, L., Tarnawski, M., Bergner, A., Kress, J., Hiblot, J., Reinhardt, J., Johnsson, K., Leippe, P. Abstract: Aspartate is a limiting metabolite in proliferating cells with its production closely linked to glutamine and mitochondrial metabolism. To date, measuring aspartate concentrations in live cells was deemed impossible. We present iAspSnFR, a genetically-encoded biosensor for intracellular aspartate, engineered by displaying and screening biosensor libraries in HEK293 cells. In live cells, iAspSnFR exhibits a dynamic range of 130% fluorescence change and detects reduced aspartate levels upon glutamine deprivation or glutaminase inhibition. Furthermore, iAspSnFR tracks aspartate uptake by excitatory amino acid transporters, or of asparagine after co-expression of an asparaginase. Importantly, iAspSnFR reports aspartate depletions upon electron transport chain inhibition, and therefore it can serve as a proxy for mitochondrial respiration. Consequently, iAspSnFR can dissect the major cellular pathways of aspartate production, offering immediate applications, particularly in cancer biology, such as screening small molecules targeting aspartate and glutamine metabolism. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.05.03.539245v1?rss=1 Authors: Pani, A. M., Favichia, M., Goldstein, B. Abstract: Wnt signaling performs critical functions in development, homeostasis, and disease states. Wnt ligands are secreted signaling proteins that often move between cells to activate signaling across a range of distances and concentrations. In different animals and developmental contexts, Wnts utilize distinct mechanisms for intercellular transport including diffusion, cytonemes and exosomes [1]. Mechanisms for intercellular Wnt dispersal remain controversial in part due to technical challenges with visualizing endogenous Wnt proteins in vivo, which has limited our understanding of Wnt transport dynamics. As a result, the cell-biological bases for long-range Wnt dispersal remain unknown in most instances, and the extent to which differences in Wnt transport mechanisms vary by cell type, organism, and/or ligand remain uncertain. To investigate processes underlying long-range Wnt transport in vivo, we utilized C. elegans as an experimentally tractable model where it is possible to tag endogenous Wnts with fluorescent proteins without disrupting signaling [2]. Live imaging of two endogenously tagged Wnt homologs revealed a novel mode for long-distance Wnt movement in axon-like structures that may complement Wnt gradients generated by diffusion and highlighted cell type-specific Wnt transport processes in vivo. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.05.03.539306v1?rss=1 Authors: den Toom, W. T. F., van Soest, D. M. K., Polderman, P. E., van Triest, M. H., Bruurs, L. J. M., Burgering, B. M. T., De Henau, S., Dansen, T. B. Abstract: Reactive Oxygen Species (ROS) in the form of H2O2 can act both as physiological signaling molecules as well as damaging agents, depending on its concentration and localization. The downstream biological effects of H2O2 were often studied making use of exogenously added H2O2, generally as a bolus and at supraphysiological levels. But this does not mimic the continuous, low levels of intracellular H2O2 production by for instance mitochondrial respiration. The enzyme D-Amino Acid Oxidase (DAAO) catalyzes H2O2 formation using D-amino acids, which are absent from culture media, as a substrate. Ectopic expression of DAAO has recently been used in several studies to produce inducible and titratable intracellular H2O2. However, a method to directly quantify the amount of H2O2 produced by DAAO has been lacking, making it difficult to assess whether observed phenotypes are the result of physiological or artificially high levels of H2O2. Here we describe a simple assay to directly quantify DAAO activity by measuring the oxygen consumed during H2O2 production. The oxygen consumption rate of DAAO can directly be compared to the basal mitochondrial respiration in the same assay, allowing to estimate whether the ensuing level of H2O2 production is within the range of physiological mitochondrial ROS production. We show that the assay can also be used to select clones that express differently localized DAAO with the same absolute level of H2O2 production to be able to discriminate the effects of H2O2 production at different subcellular locations from differences in total oxidative burden. This method therefore greatly improves the interpretation and applicability of DAAO-based models, thereby moving the redox biology field forward. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.05.03.539259v1?rss=1 Authors: Garadi Suresh, H., Bonneil, E., Albert, B., Dominique, C., Costanzo, M., Pons, C., Masinas, M. P. D., Shuteriqi, E., Shore, D., Henras, A. K., Thibault, P., Boone, C., Andrews, B. J. Abstract: Ribosome assembly requires precise coordination between the production and assembly of ribosomal components. Mutations in ribosomal proteins that inhibit the assembly process or ribosome function are often associated with Ribosomopathies, some of which are linked to defects in proteostasis. In this study, we examine the interplay between several yeast proteostasis enzymes, including deubiquitylases (DUBs), Ubp2 and Ubp14, and E3 ligases, Ufd4 and Hul5, and we explore their roles in the regulation of the cellular levels of K29-linked unanchored polyubiquitin (polyUb) chains. Accumulating K29-linked unanchored polyUb chains associate with maturing ribosomes to disrupt their assembly, activate the Ribosome assembly stress response (RASTR), and lead to the sequestration of ribosomal proteins at the Intranuclear Quality control compartment (INQ). These findings reveal the physiological relevance of INQ and provide insights into mechanisms of cellular toxicity associated with Ribosomopathies. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.05.03.539131v1?rss=1 Authors: McCormick, L. A., Cleary, J. M., Hancock, W. O., Rice, L. M. Abstract: GTP-tubulin is preferentially incorporated at growing microtubule ends, but the biochemical mechanism by which the bound nucleotide regulates the strength of tubulin:tubulin interactions is debated. The "self-acting" (cis) model posits that the nucleotide (GTP or GDP) bound to a particular tubulin dictates how strongly that tubulin interacts, whereas the "interface-acting" (trans) model posits that the nucleotide at the interface of two tubulin dimers is the determinant. We identified a testable difference between these mechanisms using mixed nucleotide simulations of microtubule elongation: with self-acting nucleotide plus- and minus-end growth rates decreased in the same proportion to the amount of GDP-tubulin, whereas with interface-acting nucleotide plus-end growth rates decreased disproportionately. We then experimentally measured plus- and minus-end elongation rates in mixed nucleotides and observed a disproportionate effect of GDP-tubulin on plus-end growth rates. Simulations of microtubule growth were consistent with GDP-tubulin binding at and "poisoning" plus-ends but not at minus-ends. Quantitative agreement between simulations and experiments required nucleotide exchange at terminal plus-end subunits to mitigate the poisoning effect of GDP-tubulin there. Our results indicate that the interfacial nucleotide determines tubulin:tubulin interaction strength, thereby settling a longstanding debate over the effect of nucleotide state on microtubule dynamics. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.05.03.539271v1?rss=1 Authors: Amosu, M., Gregory, A. J., Murtagh, J. D., Pavin, N., Meyers, C. T., Grano de Oro Fernandez, J., Moore, K., Maisel, K. Abstract: The use of single cell analysis methods has grown rapidly in the last two decades and has led to rapid discoveries in cell biology and beyond. Single cell analysis requires complex systems like tissues to be dissociated, separating individual cells from extracellular tissue materials. This requires manual processing of tissues and materials through chopping, pipetting, and suspension with enzymes for degradation of the structural elements of the tissue. Manual processing can be time consuming and lead to variability between scientists. Automating this process through motorized dissociation could thus improve reproducibility of research and reduce time of cell manipulation prior to analysis. Here, we have designed a low-cost, customizable automatic tissue dissociator device that can be easily assembled by research groups for individual use. Our device allows for customizable programmed dissociation protocols for ease of use and reproducibility between researchers and can be placed into heat or cold environments based on the protocol need. We have found this device comparable in cell viability and reproducibility to manual dissociation, while significantly reducing time spent and even enhancing cells extracted from more fibrous tissues. Broad dissemination and use of this device could enhance single cell analysis reproducibility and provide a time-saving alternative to the currently used manual dissociation protocols. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.05.03.539216v1?rss=1 Authors: Galas, S., Le Goff, E., Cazevieille, C., Tanaka, A., Cuq, P., Baghdiguian, S., Kunieda, T., Godefroy, N., Richaud, M. Abstract: Tardigrades can survive hostile environments such as desiccation by adopting a state of anhydrobiosis. Numerous tardigrade species have been described thus far, and recent genome and transcriptome analyses revealed that several distinct strategies were employed to cope with harsh environments depending on the evolutionary lineages. Detailed analyses at the cellular and subcellular levels are essential to complete these data. In this work, we analyzed a tardigrade species that can withstand rapid dehydration, Ramazzottius varieornatus. Surprisingly, we noted an absence of the anhydrobiotic-specific extracellular structure previously described for the Hypsibius exemplaris species. Both Ramazzottius varieornatus and Hypsibius exemplaris belong to the same evolutionary class of Eutardigrada. Nevertheless, our observations reveal discrepancies in the anhydrobiosis mechanisms between these two species. Interestingly, these discrepancies are correlated with their variations in dehydration resistance. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.05.03.539242v1?rss=1 Authors: Wilken, M. B., Maguire, J. A., Dungan, L. V., Gagne, A., Osorio-Quintero, C., Waxman, E. A., Chou, S. T., Gadue, P., French, D. L., Thom, C. S. Abstract: The CHOPWT17_TPM1KOc28 iPSC line was generated to interrogate the functions of Tropomyosin 1 (TPM1) in primary human cell development. This line was reprogrammed from a previously published wild type control iPSC line. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.05.02.539064v1?rss=1 Authors: Laporte, D., Massoni-Laporte, A., LEfranc, C., Dompierre, J., Mauboules, D., Nsamba, E. T., Royou, A., Gal, L., Schuldiner, M., Gupta, M., Sagot, I. Abstract: Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.05.02.539089v1?rss=1 Authors: Vural, A., Lanier, S. M. Abstract: Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.05.02.538744v1?rss=1 Authors: Collins, B. C., Shapiro, J. B., Scheib, M. M., Musci, R. V., Verma, M., Kardon, G. Abstract: Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.05.02.539076v1?rss=1 Authors: Kwon, Y.-Y., Hui, S. Abstract: Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.05.02.539080v1?rss=1 Authors: Queen, K. A., Cario, A., Berger, C. L., Stumpff, J. Abstract: Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.05.01.539004v1?rss=1 Authors: Yeh, H.-I., Tien, T.-Y., Wu, Y.-J., Su, C.-H., Hsieh, C.-L., Wang, B.-J., Lee, Y.-N., Su, Y. Abstract: Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.05.01.538962v1?rss=1 Authors: Zhang, M., Fang, Z., Li, X., Yang, F., Xiaoli, A. M. Abstract: Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.05.02.539023v1?rss=1 Authors: Zhang, H., Zhu, D. S., Zhu, J. Abstract: Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.05.01.538977v1?rss=1 Authors: Zhu, B., Liu, S. Abstract: Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.05.01.538722v1?rss=1 Authors: Sahan, A. Z., Youngblood, E., Das, S., Schmitt, D. L., Zhang, J. Abstract: Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.05.02.538835v1?rss=1 Authors: Hazari, Y., Urra, H., Lopez, V. A. G., Diaz, J., Tamburini, G., Milani, M., Pihan, P., Durand, S., Aprahamia, F., Baxter, R., Huang, M., Dong, X. C., Vihinen, H., Gonzalez, A. B., Godoy, P., Criollo, A., Ratziu, V., Foufelle, F., Hengstler, J. G., Jokitalo, E., Maitre, B. B., Maiers, J. L., Plate, L., Kroemer, G., Hetz, C. Abstract: Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.05.01.538513v1?rss=1 Authors: Gautam, S., Verma, N., Manvati, S., Dhar, P. K. Abstract: Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.05.01.538871v1?rss=1 Authors: Ferrero, R., Rainer, P., Russeil, J., Zachara, M., Pezoldt, J., van Mierlo, G., Gardeux, V., Saelens, W., Alpern, D., Favre, L., Mantziari, S., Zingg, T., Pitteloud, N., Suter, M., Matter, M., Canto, C., Deplancke, B. Abstract: Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.05.01.538914v1?rss=1 Authors: Yang, Q., Yang Loureiro, Z., Desai, A., DeSouza, T., Li, K., Wang, H., Nicoloro, S. M., Solivan-Rivera, J., Corvera, S. Abstract: Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.05.01.538990v1?rss=1 Authors: Zheng, C., Weinstein, L. D., Nguyen, K. K., Grewal, A., Gurevich, E. V., Gurevich, V. V. Abstract: Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.05.01.538972v1?rss=1 Authors: Gergely, Z., Jones, M. H., Zhou, B., Cash, C., McIntosh, R., Betterton, M. Abstract: Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.30.538878v1?rss=1 Authors: Mizobata, H., Tomita, K., Yonezawa, R., Hayashi, K., Kinoshita, S., Yoshitake, K., Asakawa, S. Abstract: Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.28.538787v1?rss=1 Authors: Raju, S., Botts, S. R., Blaser, M. C., Prajapati, K., Ho, T. W. W., Ching, C., Galant, N. J., Fiddes, L., Wu, R., Clift, C. L., Pham, T., Lee, W. L., Singh, S. A., Aikawa, E., Fish, J. E., Howe, K. L. Abstract: Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.28.538740v1?rss=1 Authors: Windham, I. A., Ragusa, J. V., Wallace, E. D., Wagner, C. H., White, K. K., Cohen, S. Abstract: Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.29.538828v1?rss=1 Authors: Isola, J., Ocanas, S. R., Hubbart, C., Ko, S., Ali Mondal, S., Hense, J., Schneider, A., Kovats, S., Freeman, W. M., Stout, M. Abstract: Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.29.538819v1?rss=1 Authors: Woodard, T. K., Rioux, D. J., Prosser, D. C. Abstract: Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.29.538793v1?rss=1 Authors: Kooltheat, N., Chaiissarapap, A., Jeanthaisong, O., Molek, N., Kawinwanalai, W., Nokkaew, N. Abstract: Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.29.538796v1?rss=1 Authors: Oyamada, Y., Iizasa, E., Usa, A., Otomaru, K. Abstract: Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.27.538651v1?rss=1 Authors: Xiao, C., Sun, Y., Huang, H., Song, Z., Yue, X., David, T., Xu, S. Abstract: Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.28.538636v1?rss=1 Authors: Purnell, C., Heebner, J., Swulius, M. T., Hylton, R., Kabonick, S., Grillo, M., Grigoryev, S., Heberle, F. A., Waxham, N., Swulius, M. T. Abstract: Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.27.538629v1?rss=1 Authors: Han, S., Mueller, C., Wuebbolt, C., Kilcullen, S., Nayyar, V., Calle Gonzalez, B., Mahdavi Fard, A., Floss, J., Morales, M. J., Patel, S. P. Abstract: Fuchs endothelial corneal dystrophy (FECD) results from genetic and environmental factors triggering mitochondrial and oxidative stress in corneal endothelial cells (CEnCs) leading to CEnC death and corneal opacification. FECD is more common in women than men, but the basis for this observation is unknown. Because FECD is commonly diagnosed around the time of the menopausal transition in women when estrogen levels decrease precipitously, we studied the effects of the potent estrogen,17-{beta} estradiol (E2) on growth, oxidative stress, and metabolism in primary cultures of human CEnCs (HCEnCs) under conditions of physiologic 2.5% O2 ([O2]2.5) and under hyperoxic stress ([O2]A: room air + 5% CO2). We hypothesized that E2 would counter the stresses of the hyperoxic environment in HCEnCs. HCEnCs were treated {+/-}10 nM E2 for 7-10 days at [O2]2.5 and [O2]A followed by measurements of cell density, viability, reactive oxygen species (ROS), mitochondrial morphology, oxidative DNA damage, ATP levels, mitochondrial respiration (O2 consumption rate [OCR]), and glycolysis (extracellular acidification rate [ECAR]). There were no significant changes in HCEnC density, viability, ROS levels, oxidative DNA damage, OCR, and ECAR in response to E2 under either O2 condition. We found that E2 disrupted mitochondrial morphology in HCEnCs from female donors but not male donors at the [O2]A condition. ATP levels were significantly higher at [O2]2.5 compared to [O2]A in HCEnCs from female donors only, but were not affected by E2. Our findings demonstrate the overall resilience of primary HCEnCs against hyperoxic stress. The selective detrimental effects of hyperoxia and estradiol on HCEnCs from female but not male donors suggests mechanisms of toxicity based upon cell-sex in addition to hormonal environment. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.27.538580v1?rss=1 Authors: Lee, S., Zhan, H. Abstract: Thrombopoietin (TPO) and its receptor MPL play crucial roles in hematopoietic stem cell (HSC) function and platelet production. However, the precise effects of TPO/MPL signaling on HSC regulation in different hematopoietic niches remain unclear. Here, we investigated the effects of TPO/MPL ablation on marrow and splenic hematopoiesis in TPO-/- and MPL-/- mice during aging. Despite severe thrombocytopenia, TPO-/- and MPL-/- mice did not develop marrow failure during a 2-year follow-up. Marrow and splenic HSCs exhibited different responses to TPO/MPL ablation and exogenous TPO treatment. Splenic niche cells compensated for marrow HSC loss in TPO-/- and MPL-/- mice by upregulating CXCL12 levels. These findings provide new insights into the complex regulation of HSCs by TPO/MPL and reveal a previously unknown link between TPO and CXCL12, two key growth factors for HSC maintenance. Understanding the distinct regulatory mechanisms between marrow and spleen hematopoiesis will help develop novel therapeutic approaches for hematopoietic disorders. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.27.538597v1?rss=1 Authors: Rotty, J., Stinson, M., Laurenson, A. Abstract: Macrophages are indispensable for proper immune surveillance and inflammatory regulation. They also exhibit dramatic phenotypic plasticity and are highly responsive to their local microenvironment, which includes the extracellular matrix (ECM). The present work demonstrates that two fibrous ECM glycoproteins, fibronectin (FN) and laminin (LAM), elicit distinct morphological and migratory responses to macrophages in 2D environments. Laminin 111 inhibits macrophage cell spreading, but drives them to migrate rapidly and less persistently compared to cells on fibronectin. Differential integrin engagement and ROCK/myosin II organization helps explain why macrophages alter their morphology and migration character on these two ECM components. The present study also demonstrates that laminin 111 exerts a suppressive effect toward fibronectin, as macrophages plated on a LAM/FN mixture adopt a morphology and migratory character almost identical to LAM alone. This suggests that distinct responses can be initiated in the cell downstream of receptor-ECM engagement, and that one component of the microenvironment may affect the ability to sense another. Overall, macrophages appear intrinsically poised to rapidly switch between distinct migratory modes based on their ECM environments. The role of ECM composition in dictating motile and inflammatory responses in 3D and in vivo contexts warrants further study. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.27.538577v1?rss=1 Authors: Colizzi, F. S., Veenstra, J. A., Lazzaro, G., Helfrich-Foerster, C., Martinez-Torres, D. Abstract: The neuropeptide Pigment-Dispersing Factor (PDF) plays a pivotal role in the circadian clock of most Ecdysozoa and is additionally involved in the timing of seasonal responses of several photoperiodic species. The pea aphid, Acyrthosiphon pisum, is a paradigmatic photoperiodic species with an annual life cycle tightly coupled to the seasonal changes in day length. Nevertheless, PDF could not be identified in A. pisum so far. In the present study, we identified a PDF-coding gene that has undergone significant changes in the otherwise highly conserved insect C-terminal amino acid sequence. A newly generated aphid-specific PDF antibody stained four neurons in each hemisphere of the aphid brain that co-express the clock protein Period and have projections to the pars lateralis that are highly plastic and change their appearance in a daily and seasonal manner, resembling those of the fruit fly PDF neurons. Most intriguingly, the PDF terminals overlap with dendrites of the insulin-like peptide (ILP) positive neurosecretory cells in the pars intercerebralis and with putative terminals of Cryptochrome (CRY) positive clock neurons. Since ILP has been previously shown to be crucial for seasonal adaptations and CRY might serve as a circadian photoreceptor vital for measuring day length, our results suggest that PDF plays a critical role in aphid seasonal timing. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.27.538564v1?rss=1 Authors: Waehler, H. A., Labba, N. A., Paulsen, R. E., Sandve, G. K., Eskeland, R. Abstract: Background Imaging of in vitro neuronal differentiation and measurements of cell morphologies has led to novel insights into neuronal development. Live-cell imaging techniques and large datasets of images has increased the demand for automated pipelines for quantitative analysis of neuronal morphological metrics. Results We present ANDA, an analysis workflow for quantification of various aspects of neuronal morphology from high-throughput live-cell imaging screens. This tool automates the analysis of neuronal cell numbers, neurite lengths and neurite attachment points. We used rat, chicken and human in vitro models for neuronal differentiation and have demonstrated the accuracy, versatility, and efficiency of the tool. Conclusions ANDA is an open-source tool that is easy to use and capable of automated processing from time-course measurements of neuronal cells. The strength of this pipeline is the capability to analyse high-throughput imaging screens. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.27.538587v1?rss=1 Authors: Toth, A. D., Szalai, B., Kovacs, O. T., Garger, D., Prokop, S., Balla, A., Inoue, A., Varnai, P., Turu, G., Hunyady, L. Abstract: The varying efficacy of biased and balanced agonists is generally explained by the stabilization of different active receptor conformations. In this study, systematic profiling of transducer activation of AT1 angiotensin receptor agonists revealed that the extent and kinetics of {beta}-arrestin binding exhibit substantial ligand-dependent differences, which however completely disappear upon the inhibition of receptor internalization. Even weak partial agonists for the {beta}-arrestin pathway acted as full or near full agonists, if receptor endocytosis was prevented, indicating that receptor conformation is not an exclusive determinant of {beta}-arrestin recruitment. The ligand-dependent variance in {beta}-arrestin translocation at endosomes was much larger than it was at the plasma membrane, showing that ligand efficacy in the {beta}-arrestin pathway is spatiotemporally determined. Experimental investigations and mathematical modeling demonstrated how multiple factors concurrently shape the effects of agonists on endosomal receptor-{beta}-arrestin binding and thus determine the extent of bias. Among others, ligand dissociation rate and G protein activity have particularly strong impact on receptor-{beta}-arrestin interaction, and their effects are integrated at endosomes. Our results highlight that endocytosis forms a key spatiotemporal platform for biased GPCR signaling and can aid the development of more efficacious functionally-selective compounds. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.27.538600v1?rss=1 Authors: Cullati, S. N., Zhang, E., Shan, Y., Guillen, R. X., Chen, J.-S., Navarrete-Perea, J., Elmore, Z. C., Ren, L., Gygi, S. P., Gould, K. L. Abstract: The CK1 family are conserved serine/threonine kinases with numerous substrates and cellular functions. The fission yeast CK1 orthologues Hhp1 and Hhp2 were first characterized as regulators of DNA repair, but the mechanism(s) by which CK1 activity promotes DNA repair had not been investigated. Here, we found that deleting Hhp1 and Hhp2 or inhibiting CK1 catalytic activities in yeast or in human cells activated the DNA damage checkpoint due to persistent double-strand breaks (DSBs). The primary pathways to repair DSBs, homologous recombination and non-homologous end joining, were both less efficient in cells lacking Hhp1 and Hhp2 activity. In order to understand how Hhp1 and Hhp2 promote DSB repair, we identified new substrates using quantitative phosphoproteomics. We confirmed that Arp8, a component of the INO80 chromatin remodeling complex, is a bona fide substrate of Hhp1 and Hhp2 that is important for DSB repair. Our data suggest that Hhp1 and Hhp2 facilitate DSB repair by phosphorylating multiple substrates, including Arp8. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.28.538646v1?rss=1 Authors: Hall, T. E., Ariotti, N., Lo, H. P., Ferguson, C. E., Martel, N., Lim, Y.-W., Giacomotto, J., Parton, R. G. Abstract: Plasma membrane rupture can result in catastrophic cell death. The skeletal muscle fibre plasma membrane, the sarcolemma, provides an extreme example of a membrane subject to mechanical stress since these cells specifically evolved to generate contraction and movement. A quantitative model correlating ultrastructural remodelling of surface architecture with tissue changes in vivo is required to understand how membrane domains contribute to the shape changes associated with tissue deformation in whole animals. We and others have shown that loss of caveolae, small invaginations of the plasma membrane particularly prevalent in the muscle sarcolemma, renders the plasma membrane more susceptible to rupture during stretch. While it is thought that caveolae are able to flatten and be absorbed into the bulk membrane to buffer local membrane expansion, a direct demonstration of this model in vivo has been unachievable since it would require measurement of caveolae at the nanoscale combined with detailed whole animal morphometrics under conditions of perturbation. Here, we describe the development and application of the ''active trapping model'' where embryonic zebrafish are immobilised in a curved state that mimics natural body axis curvature during an escape response. The model is amenable to multiscale, multimodal imaging including high-resolution whole animal three-dimensional quantitative electron microscopy. Using the active trapping model, we demonstrate the essential role of caveolae in maintaining sarcolemmal integrity and quantify the specific contribution of caveolar-derived membrane to surface expansion. We show that caveolae directly contribute to an increase in plasma membrane surface area under physiologically relevant membrane deformation conditions. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.27.538576v1?rss=1 Authors: Morral, C., Ayyaz, A., Kuo, H.-C., Fink, M., Verginadis, I., Daniel, A. R., Burner, D. N., Driver, L. M., Satow, S., Hasapis, S., Ghinnagow, R., Luo, L., Ma, Y., Attardi, L. D., Koumenis, C., Minn, A. J., Wrana, J. L., Lee, C.-L., Kirsch, D. G. Abstract: Ionizing radiation induces cell death in the gastrointestinal (GI) epithelium by activating p53. However, p53 also prevents animal lethality caused by radiation-induced GI injury. Through single-cell RNA-sequencing of the irradiated mouse intestine, we find that p53 target genes are specifically enriched in stem cells of the regenerating epithelium, including revival stem cells that promote animal survival after GI damage. Accordingly, in mice with p53 deleted specifically in the GI epithelium, ionizing radiation fails to induce revival stem cells. Using intestinal organoids, we show that transient p53 expression is required for the induction of revival stem cells that is controlled by an Mdm2-mediated negative feedback loop. These results suggest that p53 suppresses severe radiation-indued GI injury by promoting intestinal epithelial cell reprogramming. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.28.538645v1?rss=1 Authors: Cheng, L., Wei, C., Dong, L., Xiong, S., Yu, P., Zhou, R. Abstract: Extensive research has been conducted on the heterogenicity of immune cells within the tumor microenvironment, like cancer cell heterogenicity, particularly with the emergence of single cell analysis. While inducing factors have been used to artificially alter immune cell fate in vitro and dynamic cancer cell plasticity has been recently discovered, it remains unknown whether tumor infiltrating immune cells acquire plasticity and dynamics that contribute to heterogenicity. In this study, we explored mitochondrial DNA mutation combining with chromosome single nucleotide polymorphism to construct phylogenetic trees of immune cells within multiple solid tumors, together with precise cell type and subtype definition based on single cell RNA sequencing data. Based on these lineage tracing landscapes, we systematically identified cell state transitions and fate changes among different immune cell subtypes and types within multiple solid tumors. Interestingly, immune cells demonstrated a high level of plasticity for transitioning between different states, transdifferentiating from one type to another or dedifferentiating to a progenitor stage, in varying frequencies across different cancers. Moreover, most of these cell state transitions and cell fate changes discovered here were previously unknown. The cell changes may arise from extrinsic growth factors and cytokines secreted by tumor microenvironment cells, but intrinsic genetic mutations, particularly those related to ribosomes, may also be involved. Our data reveal that immune cell complexity extends beyond heterogenicity and also encompasses plasticity similar to that of cancer cells. Understanding the underlying mechanism of these cell changes will help elucidate the role of immune cells in cancer development and manipulating the cell change direction may ultimately enhance the efficiency of current immunotherapy. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.26.538432v1?rss=1 Authors: So, E., Hayat, S., Kadambat Nair, S., Wang, B., Haibe-Kains, B. Abstract: Cell-cell interactions coordinate various functions across cell-types in health and disease. Novel single-cell techniques allow us to investigate cellular crosstalk at single-cell resolution. Cell-cell interactions are mediated by underlying gene-gene networks, however most current methods are unable to account for complex inter-connections within the cell as well as incorporate the effect of pathway and protein complexes on interactions. Therefore, to utilise relations of cells to ligands and receptors as well as multiple annotations, we present GraphComm - a new graph-based deep learning method for predicting cell-cell communication in single-cell RNAseq datasets. By learning off of a prior model and fine-tuning a network on single-cell transcriptomic data, GraphComm is able to predict cell-cell communication (CCC) activity across cells, and its impact on downstream pathways, spatially adjacent cells and changes due to drug perturbations. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.26.538448v1?rss=1 Authors: Chen, R., Qiu, K., Han, G., Kundu, B. K., Ding, G., Sun, Y., Diao, J. Abstract: Detecting cell viability is crucial in research involving the precancerous discovery of abnormal cells, the evaluation of treatments, and drug toxicity testing. Although conventional methods afford cumulative results regarding cell viability based on a great number of cells, they do not permit investigating cell viability at the single-cell level. In response, we rationally designed and synthesized a fluorescent probe, PCV-1, to visualize cell viability under the super-resolution technology of structured illumination microscopy. Given its sensitivity to mitochondrial membrane potential and affinity to DNA, PCV-1's ability to stain mitochondria and nucleoli was observed in live and dead cells, respectively. During cell injury induced by drug treatment, PCV-1's migration from mitochondria to the nucleolus was dynamically visualized at the single-cell level. By extension, harnessing PCV-1's excellent photostability and signal-to-noise ratio and by comparing the fluorescence intensity of the two organelles, mitochondria and nucleoli, we developed a powerful analytical assay named organelle ratiometric probing (ORP) that we applied to quantitatively analyze and efficiently assess the viability of individual cells, thereby enabling deeper insights into the potential mechanisms of cell death. In ORP analysis with PCV-1, we identified 0.3 as the cutoff point for assessing whether adding a given drug will cause apparent cytotoxicity, which greatly expands the probe's applicability. To the best of our knowledge, PCV-1 is the first probe to allow visualizing cell death and cell injury under super-resolution imaging, and our proposed analytical assay using it paves the way for quantifying cell viability at the single-cell level. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.27.538407v1?rss=1 Authors: Khalil, J. S., Saldanha, P. A., Blair, C. M., Ling, J., Ji, W., Baillie, G. S., Naseem, K. M., Nikitenko, L. L., Rivero, F. Abstract: The activity of myosin light chain phosphatase (MLCP) is fine-tuned by the phosphorylation status of the MLCP target subunit 1 (MYPT1), which is determined by the antagonistic effects of Rho kinase (ROCK) and cAMP/cGMP-dependent protein kinases (PKA and PKG). PKA is composed of two regulatory (PKA-R, of which four variants exist) and two catalytic (PKAcat) subunits. PKA is targeted to the vicinity of its substrates by binding to A kinase anchoring proteins (AKAPs). MYPT1 is part of a complex signaling node that includes kinases and other enzymes involved in signal transduction. We hypothesized that MYPT1 might function as an AKAP to target PKA to the MLCP signaling node. Using a combination of immunoprecipitation, affinity pulldown and in situ proximity ligation assay (PLA) in human platelets and endothelial cells, we show that MYPT1 directly interacts with all four PKA-R variants and mapped the interaction to a 200 residues long central region of MYPT1. The interaction does not involve the docking and dimerization domain of PKA-R typically required for binding to AKAPs. Using peptide array overlay we identified K595, E676 and the PKA/ROCK kinase substrate motif R693/R694/S695/T696 as critical for the interaction. Substitution of S695, T696 or both by aspartic acid or the corresponding phosphorylated residue abolished binding. Our findings reveal that MYPT1 functions as a non-canonical AKAP to anchor PKA to the vicinity of non-phosphorylated S695/T696, where PKA-R would prevent PKAcat, and potentially also ROCK, from interacting with and phosphorylating MYPT1. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.27.538533v1?rss=1 Authors: Ehmke, L., Hause, G., Klosgen, R. B., Bennewitz, B. Abstract: For many metabolites, the major barrier between cytosol and mitochondrial matrix is the inner envelope membrane of mitochondria, the site of the respiratory electron transport chain. In consequence, it houses numerous transporters which facilitate the controlled exchange of metabolites, ions, and even proteins between these cellular compartments. While their import into the organelle can be studied with isolated mitochondria or mitoplasts, the analysis of their export from the matrix into the intermembrane space or even the cytosol demands for more sophisticated approaches. Among those, inside-out inner membrane vesicles are particularly useful, since they allow the direct presentation of the potential export substrates to the membrane without prior import into the organelle. Here we present a protocol for the isolation of such inside-out vesicles of the inner envelope membrane of plant mitochondria based on repeated freeze/thaw-cycles of freshly prepared mitoplasts. Electron microscopy and Western analysis could show that the majority of the vesicles have single envelope membranes in an inside-out topology. The vesicles are furthermore physiologically active, as demonstrated by assays measuring the enzymatic activities of Complex I (NADH dehydrogenase), Complex V (ATP synthase) and the mitochondrial processing peptidase (MPP) associated with Complex III. Hence, the method presented here provides a good basis for further studies of the inner mitochondrial envelope membrane and mitochondrial export processes. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.27.538553v1?rss=1 Authors: Golombek, M., Tsigaras, T., Schaumkessel, Y., Haensch, S., Weidtkamp-Peters, S., Anand, R., Reichert, A. S., Kondadi, A. K. Abstract: Cristae membranes have been recently shown to undergo intramitochondrial merging and splitting events. Yet, the metabolic and bioenergetic factors regulating them are unclear. Here we investigated whether and how cristae membrane remodelling is dependent on oxidative phosphorylation (OXPHOS) complexes, the mitochondrial membrane potential ({Delta}{Psi}m), and the ADP/ATP nucleotide translocator. Advanced live-cell STED nanoscopy combined with in-depth quantification were employed to analyse cristae morphology and dynamics after treatment of mammalian cells with rotenone, antimycin A, oligomycin A and CCCP. This led to formation of enlarged mitochondria along with reduced cristae density but did not change the number of cristae remodelling events. CCCP treatment leading to {Delta}{Psi}m abrogation even enhanced the cristae dynamics showing their {Delta}{Psi}m-independent nature. Inhibition of OXPHOS complexes was accompanied by reduced ATP levels but did not affect cristae dynamics. However, inhibition of ADP/ATP exchange led to aberrant cristae morphology and impaired cristae dynamics in a mitochondrial subset. In sum, we provide quantitative data of cristae membrane remodelling under different conditions supporting an important interplay between OXPHOS, metabolite exchange and cristae membrane dynamics. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.27.538411v1?rss=1 Authors: Groh, A.-C., Kleimann, S., Nedvestky, P., Behrens, M., Moeller-Kerutt, A., Hoeffken, V., Ghosh, S., Hansen, U., Krahn, M. P., Ludwig, A., Ebnet, K., Pavenstaedt, H., Weide, T. Abstract: The evolutionarily conserved Crumbs (CRB) polarity complex, which consists of the core components CRB3a, PALS1 and PATJ, plays a key role in epithelial cell-cell contact formation and cell polarization. Recently we observed that deletion of one Pals1 allele in mice results in functional haploinsufficiency characterized by renal cysts. To address the role of PALS1 at the cellular level, we generated PALS1 knockout MDCKII cell lines using the CRISPR/Cas9 system. The loss of PALS1 resulted in increased paracellular permeability indicative of an epithelial barrier defect. This barrier defect was associated with a redistribution of several tight junction-associated proteins from bicellular cell-cell contacts to tricellular junctions. The regulation of tight junction protein localization at bicellular junctions by PALS1 was dependent on its interaction with PATJ. Together, our data uncover a critical role of PALS1 in the correct positioning of tight junction proteins to bicellular junctions. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.26.538419v1?rss=1 Authors: Willet, A. H., Ren, L., Chen, J.-S., Gould, K. Abstract: Myosin-1s are monomeric actin-based motors that function at membranes. Myo1 is the single myosin-1 isoform in Schizosaccharomyces pombe that works redundantly with Wsp1-Vrp1 to activate the Arp2/3 complex for endocytosis. Here, we identified Ank1 as an uncharacterized cytoplasmic Myo1 binding partner. We found that in ank1{Delta} cells, Myo1 dramatically redistributed from endocytic patches to decorate the entire plasma membrane and endocytosis was defective. Biochemical analysis and structural predictions suggested that the Ank1 ankyrin repeats bind the Myo1 lever arm and the Ank1 acidic tail binds the Myo1 TH1 domain to prevent TH1-dependent Myo1 membrane binding. Indeed, Ank1 over-expression precluded Myo1 membrane localization and recombinant Ank1 blocked purified Myo1 liposome binding in vitro. Based on biochemical and cell biology analyses, we propose budding yeast Ank1 and human OSTF1 are functional Ank1 orthologs and that cytoplasmic sequestration by small ankyrin repeat proteins is a conserved mechanism regulating myosin-1s in endocytosis. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.26.538408v1?rss=1 Authors: Bhat, S. A., Malla, A. B., Oddi, V., Sen, J., Bhandari, R. Abstract: Inositol hexakisphosphate kinases (IP6Ks) are enzymes that catalyse the synthesis of the inositol pyrophosphate 5-IP7 which is involved in the regulation of many physiological processes in mammals. The IP6K paralog IP6K1 is expressed at high levels in the mammalian testis, and its deletion leads to sterility in male mice. Here, we show that the loss of IP6K1 in mice causes a delay in the first wave of spermatogenesis. Testes from juvenile Ip6k1 knockout mice show downregulation of transcripts that are involved in cell adhesion and formation of the testis-specific inter-Sertoli cell impermeable junction complex known as the blood-testis barrier (BTB). We demonstrate that loss of IP6K1 in the mouse testis causes BTB disruption associated with transcriptional misregulation of the tight junction protein claudin 3, and subcellular mislocalization of the gap junction protein connexin 43. In addition to BTB disruption, we also observe loss of germ cell adhesion in the seminiferous epithelium of Ip6k1 knockout mice, ultimately resulting in premature sloughing of round spermatids into the epididymis. Mechanistically, we show that loss of IP6K1 in the testis enhances cofilin activity due to increased AKT/ERK and integrin signalling, resulting in destabilization of the actin-based cytoskeleton in Sertoli cells and germ cell loss. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.26.538491v1?rss=1 Authors: Kim, S., Phan, S., Shaw, T. R., Ellisman, M. H., Veatch, S. L., Barmada, S. J., Pappas, S. S., Dauer, W. T. Abstract: Nuclear pore complexes (NPCs) regulate information transfer between the nucleus and cytoplasm. NPC defects are linked to several neurological diseases, but the processes governing NPC biogenesis and spatial organization are poorly understood. Here, we identify a temporal window of strongly upregulated NPC biogenesis during neuronal maturation. We demonstrate that the AAA+ protein torsinA, whose loss of function causes the neurodevelopmental movement disorder DYT-TOR1A (DYT1) dystonia, coordinates NPC spatial organization during this period without impacting total NPC density. Using a new mouse line in which endogenous Nup107 is Halo-Tagged, we find that torsinA is essential for correct localization of NPC formation. In the absence of torsinA, the inner nuclear membrane buds excessively at sites of mislocalized, nascent NPCs, and NPC assembly completion is delayed. Our work implies that NPC spatial organization and number are independently regulated and suggests that torsinA is critical for the normal localization and assembly kinetics of NPCs. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.26.538303v1?rss=1 Authors: Quintanilla, M. A., Patel, H., Wu, H., Sochacki, K. A., Akamatsu, M., Rotty, J. D., Korobova, F., Bear, J. E., Taraska, J. W., Oakes, P. W., Beach, J. R. Abstract: The ability to dynamically assemble contractile networks is required throughout cell physiology, yet the biophysical mechanisms regulating non-muscle myosin 2 filament assembly in living cells are lacking. Here we use a suite of dynamic, quantitative imaging approaches to identify deterministic factors that drive myosin filament appearance and amplification. We find that actin dynamics regulate myosin assembly, but that the actin architecture plays a minimal direct role. Instead, remodeling of actin networks modulates the local myosin monomer levels and facilitates assembly through myosin:myosin driven interactions. Using optogenetically controlled myosin, we demonstrate that locally concentrating myosin is sufficient to both form filaments and jump-start filament amplification and partitioning. By counting myosin monomers within filaments, we demonstrate a myosin-facilitated assembly process that establishes sub-resolution filament stacks prior to partitioning into clusters that feed higher-order networks. Together these findings establish the biophysical mechanisms regulating the assembly of non-muscle contractile structures that are ubiquitous throughout cell biology. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.27.538084v1?rss=1 Authors: Megarioti, A. H., Athanasopoulos, A., Koulouris, D., Esch, B. M., Makridakis, M., Lygirou, V., Samiotaki, M., Zoidakis, J., Sophianopoulou, V., Andre, B., Froehlich, F., Gournas, C. Abstract: Quiescence is a common cellular state, required for stem-cell maintenance and microorganismal survival under stress conditions or starvation. However, the mechanisms promoting quiescence maintenance remain poorly known. Plasma membrane components segregate into distinct microdomains, yet the role of this compartmentalization in quiescence remains unexplored. Here, we show that Flavodoxin-like proteins (FLPs), ubiquinone reductases of the yeast eisosome membrane compartment, protect quiescent cells from lipid peroxidation and ferroptosis. Eisosomes and FLPs expand specifically in respiratory-active quiescent cells, and mutants lacking either show accelerated aging, defective quiescence maintenance, and accumulate peroxidized phospholipids with monounsaturated or polyunsaturated fatty acids (PUFA). FLPs are essential for the extra-mitochondrial regeneration of the lipophilic antioxidant ubiquinol. FLPs, alongside the Gpx1/2/3 glutathione peroxidases, prevent iron-driven, PUFA-dependent ferroptotic cell death. Our work is the first description of ferroptosis-protective mechanisms in yeast and introduces plasma membrane compartmentalization as an important factor for the long-term survival of quiescent cells. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.26.538447v1?rss=1 Authors: Monaverfeshani, A., Yan, W., Pappas, C., Odenigbo, K. A., He, Z., van Zyl, T., Segre, A., Hageman, G., Sanes, J. R. Abstract: Although the visual system extends through the brain, most vision loss originates from defects in the eye. Its central element is the neural retina, which senses light, processes visual signals, and transmits them to the rest of the brain through the optic nerve (ON). Surrounding the retina are numerous other structures, conventionally divided into anterior and posterior segments. Here we used high-throughput single nucleus RNA sequencing (snRNA-seq) to classify and characterize cells in the extraretinal components of the posterior segment: ON, optic nerve head (ONH), peripheral sclera, peripapillary sclera (PPS), choroid, and retinal pigment epithelium (RPE). Defects in each of these tissues are associated with blinding diseases, for example, glaucoma (ONH and PPS), optic neuritis (ON), retinitis pigmentosa (RPE), and age-related macular degeneration (RPE and choroid). From ~151,000 single nuclei, we identified 37 transcriptomically distinct cell types, including multiple types of astrocytes, oligodendrocytes, fibroblasts, and vascular endothelial cells. Our analyses revealed a differential distribution of many cell types among distinct structures. Together with our previous analyses of the anterior segment and retina, the new data complete a Version 1 cell atlas of the human eye. We used this atlas to map the expression of greater than 180 genes associated with the risk of developing glaucoma, which is known to involve ocular tissues in both anterior and posterior segments as well as neural retina. Similar methods can be used to investigate numerous additional ocular diseases, many of which are currently untreatable. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.26.538394v1?rss=1 Authors: Wu, J., Raas, M. W. D., Alcaraz, P. S., Vos, H. R., Tromer, E. C., Snel, B., Kops, G. J. Abstract: Correct chromosome segregation during cell division depends on proper connections between spindle microtubules and kinetochores. During prometaphase, kinetochores are temporarily covered with a dense protein meshwork known as the fibrous corona. Formed by oligomerization of ROD/ZW10/ZWILCH-SPINDLY (RZZ-S) complexes, the fibrous corona promotes spindle assembly, chromosome orientation and spindle checkpoint signaling. The molecular requirements for formation of the fibrous corona are not fully understood. Here we show that the fibrous corona depends on the mitotic kinesin CENP-E, and that poorly expanded fibrous coronas after CENP-E depletion are functionally compromised. This previously unrecognized role for CENP-E does not require its motor activity but instead is driven by farnesyl modification of its C-terminal kinetochore- and microtubule-binding domain. We show that in cells CENP-E interacts with RZZ-S complexes in a farnesyl-dependent manner. CENP-E is recruited to kinetochores following RZZ-S, and - while not required for RZZ-S oligomerization per se - promotes subsequent fibrous corona expansion. Our comparative genomics analyses suggest that the farnesylation motif in CENP-E orthologs emerged alongside the full RZZ-S module in an ancestral lineage close to the fungi-animal split (Obazoa), revealing potential conservation of the mechanisms for fibrous corona formation. Our results show that proper spindle assembly has a potentially conserved non-motor contribution from the kinesin CENP-E through stabilization of the fibrous corona meshwork during its formation. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.24.538138v1?rss=1 Authors: Wu, H., Huang, X., Zhong, W., Li, W., Liu, Z., Zhao, M., Xi, X., Cao, B., Pu, Y., Kong, X., Zhao, H., Zhang, R., Lai, K., Lv, X., Lv, Y., Bao, J., Wang, M., Xiong, Y., Dong, L., Zhang, J., Zhang, G., Hu, Y., Xu, J., Chen, Y. E., Chen, S. Abstract: MicroRNAs (miRNAs) are a class of short noncoding RNAs that regulate gene expression through the binding of their 5'-end to mRNA. However, the biological effects of miRNA's 3'-end binding to mRNA remain unclear. Here we discover that the pairing of miRNA's 3'-end with RNA could serve as a primer to initiate the production of miRNA-derived RNAs (midRs), in the opposite direction of its originating RNA. midRs could consequently translate into miRNA-derived proteins (midPs). Starting from 2,656 Homo Sapiens miRNAs, we predicted 11,453 and 1,239 unique midRs and midPs for humans using a 15-nucleotide-pairing threshold. We verified the bona-fide existence of example midRs and midPs in human cells. Of clinical relevance, we demonstrate that midP0188 is highly expressed in human lung and breast cancer tissues and cells and that midP0188 and its encoding midRs represent novel anti-cancer targets. Our findings propose a miRNA[->]midR[->]midP axis that expands the central dogma and reveals thousands of novel RNAs and proteins that have immense potential for playing crucial biological and pathological roles in human cells, as well as other biological systems. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.24.538172v1?rss=1 Authors: Cremin, K., Meloni, G., Valavanis, D., soyer, o. s., Unwin, P. R. Abstract: Ultramicroelectrode (UME), or - equivalently - microelectrode, probes are increasingly used for single-cell measurements of cellular properties and processes, including physiological activity, such as metabolic fluxes and respiration rates. Major challenges for the sensitivity of such measurements include: (i) the relative magnitude of cellular and UME fluxes (manifested in the current); and (ii) issues around the stability of the UME response over time. To explore the extent to which these factors impact the precision of electrochemical cellular measurements, we undertake a systematic analysis of measurement conditions and experimental parameters for determining single cell respiration rates, via the oxygen consumption rate (OCR) at single HeLa cells. Using scanning electrochemical microscopy (SECM), with a platinum UME as the probe, we employ a self-referencing measurement protocol, rarely employed in SECM, whereby the UME is repeatedly approached from bulk solution to a cell, and a short pulse to oxygen reduction reaction (ORR) potentials is performed near the cell and in bulk solution. This approach enables the periodic tracking of the bulk UME response to which the near-cell response is repeatedly compared (referenced), and also ensures that the ORR near the cell is performed only briefly, minimizing the effect of the electrochemical process on the cell. SECM experiments are combined with a finite element method (FEM) modeling framework, to simulate oxygen diffusion and the UME response. Taking a realistic range of single cell OCR to be 10e-18 to 10e-16 mol s-1, results from the combination of FEM simulations and self-referencing SECM measurements show that these OCR values are at - or below - the present detection sensitivity of the technique. We provide a set of model-based suggestions for improving these measurements in the future, but highlight that extraordinary improvements in the stability and precision of SECM measurements will be required if single cell OCR measurements are to be realized. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.25.538235v1?rss=1 Authors: Chen, Y., Briant, K., Camus, M. D., Brodsky, F. M. Abstract: To identify functional differences between vertebrate clathrin light chains (CLCa or CLCb), phenotypes of mice lacking genes encoding either isoform were characterised. Mice without CLCa displayed 50% neonatal mortality, reduced body weight, reduced fertility, and ~40% of aged females developed uterine pyometra. Mice lacking CLCb displayed a less severe weight reduction phenotype compared to those lacking CLCa, and had no survival or reproductive system defects. Analysis of female mice lacking CLCa that developed pyometra revealed ectopic expression of epithelial differentiation markers (FOXA2 and K14) and a reduced number of endometrial glands, indicating defects in the luminal epithelium. Defects in lumen formation and polarity of epithelial cysts derived from uterine or gut cell lines were also observed when either CLCa or CLCb were depleted, with more severe effects from CLCa depletion. In cysts, the CLC isoforms had different distributions relative to each other, while they converge in tissue. Together, these findings suggest differential and cooperative roles for CLC isoforms in epithelial lumen formation, with a dominant function for CLCa. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.25.538276v1?rss=1 Authors: Yasuda, H., Fukusumi, Y., Zhang, Y., Kawachi, H. Abstract: Adaptor protein 14-3-3s have isoform-specific binding partners and roles. We reported 14-3-3{beta} interacts with FKBP12 and synaptopodin to maintain the structure of actin fibers in podocytes. However, differential roles of 14-3-3 isoforms in kidneys are unclear. Herein, we showed that 14-3-3{beta} was dominantly co-localized with FKBP12 in foot processes and was partially co-localized with Par3 at slit diaphragm in podocytes. 14-3-3{beta} interacted with Par3, and FKBP12 bound to 14-3-3{beta} competitively with Par3. Although deletion of 14-3-3{beta} enhanced the interaction of Par3-Par6, it altered actin fiber structure and processes. 14-3-3{beta} and synaptopodin were downregulated in podocyte injury models. 14-3-3{sigma} in podocytes interacted with vimentin in primary processes but not with the actin-associated proteins in foot processes. Deletion of 14-3-3{sigma} altered vimentin fiber structure and processes. 14-3-3{sigma} and vimentin were upregulated in the early phase of podocyte injury models but were decreased in the end stage. Together, the precise localization of 14-3-3{beta} at actin cytoskeleton plays a role in maintaining foot processes and Par complex in podocytes. 14-3-3{sigma} at vimentin cytoskeleton is essential for maintaining primary processes. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.25.538102v1?rss=1 Authors: Guan, Y., Fang, Z., Hu, A., Roberts, S., Johansson, P. K., Heilshorn, S. C., Enejder, A., Peltz, G. Abstract: Due to the limitations of available in vitro systems and animal models, we lack a detailed understanding of the pathogenetic mechanisms and have minimal treatment options for liver fibrosis. To overcome this barrier, we engineered a live cell imaging system that identifies collagen producing cells in a human multi-lineage hepatic organoid. This system was adapted for use as a microwell-based platform (i.e., microHOs) where exposure to PDGF or TGFb1 induced the formation of thick collagen fibers. Transcriptomic analysis revealed that TGFb1 exposure converted mesenchymal cells into myofibroblast-like cells with a significantly altered pattern of production of proteases and anti-proteases, which contribute to the development of liver fibrosis. When pro-fibrotic intracellular signaling pathways were examined using pharmacological probes, the anti-fibrotic effect of receptor-specific tyrosine kinase inhibitors was limited to the fibrosis induced by the corresponding growth factor, which indicates that their antifibrotic efficacy would be limited to fibrotic diseases that were solely mediated by that growth factor. In contrast, GSK3b or p38 MAPK inhibitors could prevent TGFb1- or PDGF-induced fibrosis in microHOs because they block intracellular signaling pathways that are commonly utilized by the TGFb1 and PDGF receptors. Hence, these studies identified GSK3b and p38 MAPK inhibitors as potential new broad-spectrum therapies for liver fibrosis, and it is likely that other new therapies could subsequently be identified using this microHO system. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.25.538200v1?rss=1 Authors: Peng, Y., Zhang, Y., Fang, R., Jiang, H., Lan, G., Liu, Y., Nie, Z., Zhang, S.-D., Ma, Y., Yang, P., Wang, F., Ge, H.-H., Zhang, W.-D., Luo, C., Li, A., He, W. Abstract: Centromere protein A (CENP-A) is a centromere-specific protein that determines kinetochore positioning and the accuracy of chromosome segregation. Despite its recognized importance in maintaining mitotic fidelity, the molecular details of CENP-A regulation in mitosis are still obscure. We performed a structure-activity relationship (SAR) study of the cell cycle-arresting indole terpenoid mimic JP18 and identified two more potent analogues, (+)-6-Br-JP18 and (+)-6-Cl-JP18. Tubulin was identified as a potential protein target of these two halogenated analogues by using the drug affinity responsive target stability (DARTS) based method. X-ray crystallographic analysis determined that (+)-6-Br-JP18 and (+)-6-Cl-JP18 bind to the colchicine-binding site of beta-tubulin. We further found that treatment of cancer cells with microtubule-targeting agents (MTAs), including these two compounds, upregulated CENP-A by destabilizing Cdh1, an E3 ubiquitin ligase component. The mechanistic study revealed that Cdh1 mediates proteolysis of CENP-A in mitosis, specifying the role of Cdh1 in maintaining mitotic fidelity. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.25.538313v1?rss=1 Authors: Yang, J., Sahu, A., Adams, A., Yin, S., Gu, F., Mayes, H. B., Nielsen, E. Abstract: Plant cell expansion is a dynamic process that is physically constrained by the deposition of new cell wall components. During tip growth, new cell wall materials are delivered at a restricted plasma membrane domain which results in a highly polarized expansion within that specified domain. Previous studies demonstrated that this process requires the activities of members of the Cellulose Synthase-Like D (CSLD) subfamily of cell wall synthases. CSLD3 displays {beta}-1,4 glucan synthase activity, but whether other members of CSLD subfamily share this conserved biochemical activity, and whether CSLD proteins form higher-order complexes to perform {beta}-1,4 glucan synthase activities have not been determined. Here, we use genetic methods to demonstrate that CSLD2 and CSLD3 functions are interchangeable during root hair elongation and cell plate formation, while CSLD5 provides a unique and irreplaceable function in the formation of cell plates. Importantly, genetic analysis with inactivated versions of CSLD3 show that, unlike CESA proteins, CSLDs do not require the simultaneous presence of different isoforms to perform catalytic cell wall synthase activities. In vitro biochemical activity experiments confirmed that CSLD2, CSLD3, and CSLD5 proteins displayed {beta}-1,4 glucan synthases activities. Taken together, these results indicated that while all three vegetatively expressed CSLD proteins possess conserved {beta}-1,4 glucan synthase activities, that CSLD5 has a more complicated and specialized role during cell plate formation. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.24.538205v1?rss=1 Authors: Hirano, S., Udagawa, O., Kato-Udagawa, A. Abstract: Promyelocytic leukemia-nuclear bodies (PML-NBs) are dot-like protein assemblies and implicated in the pathogenesis of leukemia and viral infection. PML is the scaffold protein of PML-NB and its client proteins such as SUMO, DAXX, and Sp100 reside in PML-NBs. It is known that a short exposure to trivalent arsenic (As3+) induces the solubility change and the subsequent SUMOylation of PML, and the SUMO interacting motif (SIM) is not necessary for these biochemical changes. However, it has not been well studied how As3+ initiates or enhances the association of SUMO with PML and the other PML-NB client proteins. Here, we report that As3+ enhanced non-covalent association of PML with SUMO via the SUMO-SIM interaction which is dispensable for the solubility change and SUMOylation of PML. We also report that the As3+-induced solubility change of PML was not affected by ML792, a SUMO E1 enzyme inhibitor, even though the nuclear localization of SUMO2/3 and protein SUMOylation were halted by ML792. As3+ did not change the solubility of DAXX and SUMOylation enzymes such as SAE1, UBA2, and UBC9. In contrast, As3+ induced SUMOylation of Sp100 with a concomitant loss of its solubility like PML in human leukemia cell lines. Our current results indicate that both covalent and non-covalent associations of SUMO with PML are increased in As3+-exposed cells, and Sp100 may play a role in the maintenance of PML-NBs. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.24.538195v1?rss=1 Authors: Akhter, M. Z., Yazbeck, P., Tauseef, M., Anwar, M., Hossen, F., Datta, S., Vellingiri, V., Joshi, J. C., Srivastava, N., Lenzini, S., Zhou, G., Lee, J., Jain, M. K., Shin, J.-W., Mehta, D. Abstract: Vascular endothelium forms a restrictive barrier to defend the underlying tissue against uncontrolled influx of circulating protein and immune cells. Mechanisms that mediate the transition from restrictive to leaky endothelium, a hallmark of tissue injury exemplified by acute lung injury (ALI), remain elusive. Using endothelial cell (EC)-Fak-/- mice, we show that FAK sensing and transmission of mechanical tension to the EC nucleus governs cell fate. In FAK-deleted EC, increased EC tension induced by Rho kinase caused tyrosine phosphorylation of nuclear envelope protein, emerin at Y74/Y95, and its localization in a nuclear cap. Activated emerin stimulated DNMT3a activity and methylation of the KLF2 promoter, impairing the restrictive EC transcriptome, including S1PR1. Inhibiting emerin phosphorylation or DNMT3a activity enabled KLF2 transcription of S1PR1, rescuing the restrictive EC phenotype in EC-Fak-/- lungs. Thus, FAK sensing of tension transmission to the nucleus is crucial for maintaining a restrictive EC fate and lung homeostasis. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.25.538137v1?rss=1 Authors: Pandey, P., Wall, P. K., Lopez, S. R., Dubuisson, O. S., Zunica, E. R. M., Dantas, W. S., Kirwan, J. P., Axelrod, C. L., Johnson, A. E. Abstract: Sleep loss typically imposes negative effects on animal health. However, humans with a rare genetic mutation in the dec2 gene (dec2P384R) present an exception; these individuals sleep less without the usual effects associated with sleep deprivation. Thus, it has been suggested that the dec2P384R mutation activates compensatory mechanisms that allows these individuals to thrive with less sleep. To test this directly, we used a Drosophila model to study the effects of the dec2P384R mutation on animal health. Expression of human dec2P384R in fly sleep neurons was sufficient to mimic the short sleep phenotype and, remarkably, dec2P384R mutants lived significantly longer with improved health despite sleeping less. The improved physiological effects were enabled, in part, by enhanced mitochondrial fitness and upregulation of multiple stress response pathways. Moreover, we provide evidence that upregulation of pro-health pathways also contributes to the short sleep phenotype, and this phenomenon may extend to other pro-longevity models. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.25.538222v1?rss=1 Authors: Platania, A., Erb, C., Barbieri, M., Molcrette, B., Grandgirard, E., de Kort, M. A., Meaburn, K., Taylor, T., Shchuka, V. M., Kocanova, S., Monteiro Oliveira, G., Mitchell, J. A., Soutoglou, E., Lenstra, T. L., Molina, N., Papantonis, A., Bystricky, K., Sexton, T. Abstract: The spatiotemporal configuration of genes with distal regulatory elements, and the impact of chromatin mobility on transcription, remain unclear. Loop extrusion is an attractive model for bringing genetic elements together, but how this functionally interacts with transcription is also largely unknown. We combine live tracking of genomic loci and nascent transcripts with molecular dynamics simulations to assess the 4D arrangement of the Sox2 gene and its enhancer, in response to a battery of perturbations. We find that alterations in chromatin mobility, not promoter-enhancer distance, is more informative about transcriptional status. Active elements display more constrained mobility, consistent with confinement within specialized nuclear sites, and alterations in enhancer mobility distinguish poised from transcribing alleles. Strikingly, we find that whereas loop extrusion and transcription factor-mediated clustering contribute to promoter-enhancer proximity, they have antagonistic effects on chromatin dynamics. This provides an experimental framework for the underappreciated role of chromatin dynamics in genome regulation. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.24.538105v1?rss=1 Authors: Gasparski, A. N., Moissoglu, K., Pallikkuth, S., Meydan, S., Guydosh, N. R., Mili, S. Abstract: Numerous proteins are targeted to two or multiple subcellular destinations where they exert distinct functional consequences. The balance between such differential targeting is thought to be determined post-translationally, relying on protein sorting mechanisms. Here, we show that protein targeting can additionally be determined by mRNA location and translation rate, through modulating protein binding to specific interacting partners. Peripheral localization of the NET1 mRNA and fast translation lead to higher cytosolic retention of the NET1 protein, through promoting its binding to the membrane-associated scaffold protein CASK. By contrast, perinuclear mRNA location and/or slower translation rate favor nuclear targeting, through promoting binding to importins. This mRNA location-dependent mechanism is modulated by physiological stimuli and profoundly impacts NET1 function in cell motility. These results reveal that the location of protein synthesis and the rate of translation elongation act in coordination as a "partner-selection" mechanism that robustly influences protein distribution and function. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.24.538066v1?rss=1 Authors: Zou, C.-X., Ma, Z.-H., Pan, Z.-Q., Xu, D.-D., Suo, F., Jiang, Z., Du, L.-L. Abstract: Selective macroautophagy of the endoplasmic reticulum (ER) and the nucleus, referred to as ER-phagy and nucleophagy, respectively, are processes whose mechanisms remain incompletely understood. Through an imaging-based screen, we find that fission yeast Yep1, the ortholog of human REEP1-4, is essential for ER-phagy and nucleophagy. Yep1 is required for the autophagosomal enclosure of cargos during ER-phagy and nucleophagy. In its absence, membrane structures derived from the nucleus and cortical ER accumulate in the cytoplasm. Its ER-phagy role depends on its abilities to self-interact and shape membranes, and requires its C-terminal amphipathic helices. In addition, we show that human REEP1-4 and budding yeast Atg40 can substitute for the ER-phagy function of Yep1, and Atg40 is a divergent homolog of Yep1 and REEP1-4. Our study unveils an unexpected mechanism governing the autophagosomal enclosure of ER-phagy/nucleophagy cargos, and sheds new light on the functions and evolution of REEP family proteins. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.26.538393v1?rss=1 Authors: Zhang, W., Zhao, X., Qi, X., Kimber, S. J., Hooper, N., Wang, T. Abstract: Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy (CADASIL) is the most common genetic small vessel disease caused by variants in the NOTCH3 gene. Patients with CADASIL experience recurrent strokes, developing into cognitive defect and vascular dementia. CADASIL is a late-onset vascular condition, but migraine and brain MRI lesions appear in CADASIL patients as early as their teens and twenties, suggesting an abnormal neurovascular interaction at the neurovascular unit (NVU) where microvessels meet the brain parenchyma. To understand the molecular mechanisms of CADASIL, we established induced pluripotent stem cell (iPSC) models from CADASIL patients and differentiated the iPSCs into the major NVU cell types including brain microvascular endothelial-like cells (BMECs), vascular mural cells (MCs), astrocytes and cortical projection neurons. We then built an in vitro NVU model by co-culturing different neurovascular cell types in Transwells and evaluated the blood brain barrier (BBB) function by measuring transendothelial electrical resistance (TEER). Results showed that, while the wild-type MCs, astrocytes and neurons could all independently and significantly enhance TEER values of the iPSC-BMECs, such capability of MCs from iPSCs of CADASIL patients was significantly impeded. Additionally, the barrier function of the BMECs from CADASIL iPSCs was significantly impaired, accompanied with disorganised tight junctions in iPSC-BMECs, which could not be effectively rescued by the wild-type MCs, astrocytes and neurons. Our findings provide new insight into early disease pathologies on the neurovascular interaction and BBB function at the molecular and cellular levels for CADASIL, which helps inform future therapeutic development. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.25.538283v1?rss=1 Authors: Wong, S.-S., Wainman, A., Saurya, S., Raff, J. W. Abstract: Mitotic centrosomes assemble when centrioles recruit large amounts of pericentriolar material (PCM) around themselves in preparation for cell division. How the mitotic PCM grows to the correct size is unclear. In Drosophila syncytial embryos, thousands of mitotic centrosomes assemble in a common cytoplasm as the embryo proceeds through 13 rounds of near-synchronous nuclear division. During nuclear cycles (NCs) 11-13 these divisions gradually slow, and we find that mitotic centrosomes respond by reciprocally slowing their growth rate and increasing their growth period so that they grow to a consistent size at each cycle. This size homeostasis is enforced, at least in part, by the Cdk/Cyclin cell cycle oscillator (CCO). Moderate levels of CCO activity appear to initially promote centrosome growth by stimulating Polo/PLK1 recruitment to centrosomes, while higher levels of activity subsequently inhibit centrosome growth by phosphorylating centrosome proteins to decrease their centrosomal recruitment and/or maintenance as the embryos enter mitosis. Thus, the CCO initially promotes, and subsequently restricts, mitotic centrosome growth to help ensure that centrosomes grow to a consistent size. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.23.537998v1?rss=1 Authors: Zelmer, A. R., Starczak, Y., Solomon, L. B., Richter, K., Yang, D., Atkins, G. J. Abstract: The intracellular infection of osteocytes represents a clinically important aspect of osteomyelitis. However, few human osteocyte in vitro models exist and the differentiation of immature osteoblasts to an osteocyte stage typically takes at least 4-weeks of culture, making the study of this process challenging and time consuming. The osteosarcoma cell line Saos-2 has proved to be a useful model of human osteoblast differentiation through to a mature osteocyte-like stage. Culture under osteogenic conditions in a standard 5% CO2 and normoxic (21% O2) atmosphere results in reproducible mineralisation and acquisition of mature osteocyte markers over the expected 28-35 day culture period. In order to expedite experimental assays, we tested whether reducing available oxygen to mimic concentrations experienced by osteocytes in vivo would increase the rate of differentiation of Saos-2 cells. Cells cultured in a 5% CO2, 1% O2 atmosphere exhibited accelerated deposition of mineral, reaching near saturation by 14 days as demonstrated with the Alizarin Red and Von Kossa staining. The gene expression of the major hypoxia-induced transcription factor HIF1 and the key osteogenic transcription factor RUNX2 were both elevated under 1% O2. Early (COLA1, MEPE) and mature (PHEX, DMP1 and SOST) osteocyte markers were also upregulated earlier under hypoxic compared to normoxic growth conditions. Thus, culture under low oxygen accelerates key markers of osteocyte differentiation, resulting in a useful human osteocyte-like in vitro cell model within 14 days. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.23.537908v1?rss=1 Authors: Dora, M., Obara, C. J., Abel, T., Lippincott-Schwartz, J., Holcman, D. Abstract: The endoplasmic reticulum (ER) is a structurally complex, membrane-enclosed compartment that stretches from the nuclear envelope to the extreme periphery of eukaryotic cells. The organelle is crucial for numerous distinct cellular processes, but how these processes are spatially regulated within the structure is unclear. Traditional imaging-based approaches to understanding protein dynamics within the organelle are limited by the convoluted structure and rapid movement of molecular components. Here, we introduce a combinatorial imaging and machine learning-assisted image analysis approach to track the motion of photoactivated proteins within the ER of live cells. We find that simultaneous knowledge of the underlying ER structure is required to accurately analyze fluorescently-tagged protein redistribution, and after appropriate structural calibration we see all proteins assayed show signatures of Brownian diffusion-dominated motion over micron spatial scales. Remarkably, we find that in some cells the ER structure can be explored in a highly asymmetric manner, likely as a result of uneven connectivity within the organelle. This remains true independently of the size, topology, or folding state of the fluorescently-tagged molecules, suggesting a potential role for ER connectivity in driving spatially regulated biology in eukaryotes. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.25.538277v1?rss=1 Authors: Cmentowski, V., D'Amico, E., Ciossani, G., Wohlgemuth, S., Owa, M., Dynlacht, B. D., Musacchio, A. Abstract: Chromosome biorientation on the mitotic spindle is prerequisite to errorless genome inheritance. CENP-E (kinesin 7) and Dynein-Dynactin (DD), microtubule motors with opposite polarity, promote biorientation from the kinetochore corona, a polymeric structure whose assembly requires MPS1 kinase. The corona building block consists of ROD, Zwilch, ZW10, and the DD adaptor Spindly (RZZS). How CENP-E and DD are scaffolded and mutually coordinated in the corona remains unclear. Here, we report near-complete depletion of RZZS and DD from kinetochores after depletion of CENP-E and the outer kinetochore protein KNL1. With inhibited MPS1, CENP-E, which we show binds directly to RZZS, is required to retain kinetochore RZZS. An RZZS phosphomimetic mutant bypasses this requirement. With active MPS1, CENP-E is dispensable for corona expansion, but strictly required for physiological kinetochore accumulation of DD. Thus, we identify the corona as an integrated scaffold where CENP-E kinesin controls DD kinetochore loading for coordinated bidirectional transport of chromosome cargo. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.22.537784v1?rss=1 Authors: Majumder, A., Joshi, R., Mukherjee, S., Suryawanshi, T., Shukla, S. Abstract: Human mesenchymal stem cells (hMSCs) are multipotent cells that can differentiate into adipocytes, chondrocytes and osteoblasts. Due to their differentiation potential, hMSCs are among the most frequently used cells for therapeutic applications in tissue engineering and regenerative medicine. However, the number of cells obtained through isolation alone is insufficient for hMSC-based therapies and basic research, necessitating their in-vitro expansion. Conventionally, this is often carried out on rigid surfaces such as tissue culture petriplates (TCPs). However, during in-vitro expansion, hMSCs lose their proliferative ability and multilineage differentiation potential, making them unsuitable for clinical use. Although multiple approaches have been tried to maintain hMSC stemness over prolonged expansion, finding a suitable culture system to achieve this remains an unmet need. Recently, few research groups including ours have shown that hMSCs maintain their stemness over long passages when cultured on soft substrate. In addition, it has been shown that hMSCs cultured on soft substrates have more condensed chromatin and lower levels of histone acetylation compared to those cultured on stiff substrates. It has also been shown that condensing/decondensing chromatin by deacetylation/acetylation can delay/hasten replicative senescence in hMSCs during long-term expansion on TCPs. However, how chromatin condensation/decondensation influences nuclear morphology and DNA damage - which are strongly related to the onset of senescence and cancer - is still not known. To answer this question, here we cultured hMSCs for long duration (P4-P11) in presence of epigenetic modifiers histone acetyltransferase inhibitor (HATi) which promotes chromatin condensation by preventing histone acetylation and histone deacetylase inhibitor (HDACi) which promotes chromatin decondensation and investigated their effect on various nuclear markers related to senescence and cancer. We have found that consistent acetylation causes severe nuclear abnormalities whereas chromatin condensation by deacetylation helps in safeguarding nucleus from damages caused by in-vitro expansion. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.23.537965v1?rss=1 Authors: Ramezani, M., Wagenknecht-Wiesner, A., Wang, T., Holowka, D. A., Eliezer, D., Baird, B. Abstract: Alpha synuclein (a-syn) is an intrinsically disordered protein prevalent in neurons, and aggregated forms are associated with synucleinopathies including Parkinson' disease (PD). Despite the biomedical importance and extensive studies, the physiological role of a-syn and its participation in etiology of PD remain uncertain. We showed previously in model RBL cells that a-syn colocalizes with mitochondrial membranes, depending on formation of N-terminal helices and increasing with mitochondrial stress. We have now characterized this colocalization and functional correlates in RBL, HEK293, and N2a cells. We find that expression of a-syn enhances stimulated mitochondrial uptake of Ca2+ from the ER, depending on formation of its N-terminal helices but not on its disordered C-terminal tail. Our results are consistent with a-syn acting as a tether between mitochondria and ER, and we show increased contacts between these two organelles using structured illumination microscopy. We tested mitochondrial stress caused by toxins related to PD, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP/MPP+) and carbonyl cyanide m-chlorophenyl hydrazone (CCCP), and found that a-syn prevents recovery of stimulated mitochondrial Ca2+ uptake. The C-terminal tail, and not N-terminal helices, is involved in this inhibitory activity, which is abrogated when phosphorylation site serine-129 is mutated (S129A). Correspondingly, we find that MPTP/MPP+ and CCCP stress is accompanied by both phosphorylation (pS129) and aggregation of a-syn. Overall, our results indicate that a-syn can participate as a tethering protein to modulate Ca2+ flux between ER and mitochondria, with potential physiological significance. A-syn can also prevent cellular recovery from toxin-induced mitochondrial dysfunction, which may represent a pathological role of a-syn in the etiology of PD. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.22.537920v1?rss=1 Authors: Dresselhaus, E. C., Harris, K. P., Koles, K., Pescosolido, M. F., Ermanoska, B., Rozencwaig, M., Soslowsky, R. C., Stewart, B. A., Rodal, A. A. Abstract: Exosomes are membrane-bound vesicles released by many cells including neurons, carrying cargoes involved in signaling and disease. It has been unclear whether exosomes promote intercellular signaling in vivo or serve primarily to dispose of unwanted cargo. This is because manipulations of exosome cargo expression or traffic often result in their depletion from the donor cell, making it difficult to distinguish whether these cargoes act cell-autonomously or through transcellular transfer. Exosomes arise when multivesicular endosomes fuse with the plasma membrane, releasing their intralumenal vesicles outside the cell. We show that loss of multivesicular endosome-generating ESCRT (endosomal sorting complex required for transport) machinery disrupts release of exosome cargoes from Drosophila motor neurons, without depleting them from the donor presynaptic terminal. Cargoes and autophagic vacuoles accumulate in presynaptic terminals, suggesting that compensatory autophagy follows endosome dysfunction. Surprisingly, exosome cargoes Synaptotagmin-4 (Syt4) and Evenness Interrupted (Evi) retain many of their signaling activities upon ESCRT depletion, despite being trapped in presynaptic terminals. Thus, these cargoes may not require intercellular transfer, and instead are likely to function cell autonomously in the motor neuron. Our results indicate that synaptic exosome release depends on ESCRT, and serves primarily as a proteostatic mechanism for at least some cargoes. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.23.538008v1?rss=1 Authors: Wang, T. S., Coppens, I., Brady, N. R., Hamacher-Brady, A. Abstract: Damaged mitochondria can be subject to lysosomal degradation via mitophagy. However, whole-organelle degradation exhibits relatively slow kinetics and thus its impact may be limited in response to acute, fast-acting cellular stress. We previously reported that in Parkin-deficient cells endolysosomes directly target mitochondria when subjected to bioenergetic stress. Here, using high-resolution live cell imaging we reveal a striking level of dynamic targeting of Rab5+ early endosomes to stressed mitochondria, culminating in a switch-like accumulation in the entire mitochondrial population, independently of canonical autophagy. This process of rapid, largescale Rab5+ vesicle trafficking to mitochondria coincides with, and is mediated by, XIAP E3 ligase activated mitochondrial ubiquitylation and results in ultrastructural changes to, and degradation of, intra-mitochondrial components. Mitochondria-targeting vesicles include early endosomal subpopulations marked by Rab5 effector APPL1 and ubiquitin-binding endocytic adaptors OPTN, TAX1BP1 and Tollip, and Rab7-positive late endosomes/lysosomes. In Parkin expressing cells, XIAP- and Parkin-dependent mitochondrial targeting and resulting processing modes are competitively regulated. Together, our data suggest that XIAP-mediated targeting of endolysosomes to mitochondria functions as a stress-responsive, sub-organelle level mitochondrial processing mode that is distinct from, and competitive to, Parkin-mediated mitophagy. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.24.538041v1?rss=1 Authors: Franzkoch, R., Anand, A., Breitsprecher, L., Psathaki, O. E., Barisch, C. Abstract: The infection course of Mycobacterium tuberculosis is highly dynamic and comprises sequential stages that require damaging and crossing of several membranes to enable the translocation of the bacteria into the cytosol or their escape from the host. Many important breakthroughs such as the restriction of vacuolar and cytosolic mycobacteria by the autophagy pathway and the recruitment of sophisticated host repair machineries to the Mycobacterium-containing vacuole have been gained in the Dictyostelium discoideum/M. marinum system. Despite the availability of well-established light and advanced electron microscopy techniques in this system, a correlative approach that integrates both methodologies with almost native ultrastructural preservation is still lacking at the moment. This is most likely due to the low ability of D. discoideum to adhere to surfaces, which results in cell loss even after fixation. To address this problem, we improved the adhesion of cells and developed a straightforward and convenient workflow for 3D-correlative light and electron microscopy. This approach includes high-pressure freezing, which is an excellent technique for preserving membranes. Thus, our method allows to monitor the ultrastructural aspects of vacuole escape which is of central importance for the survival and dissemination of bacterial pathogens. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.23.538011v1?rss=1 Authors: Tengganu, I. F., Arias Padilla, L. F., Munera Lopez, J., Liu, J., Brown, P. T., Hu, K. Abstract: Apicomplexans parasitize a wide range of hosts. To infect, the parasite needs to travel through different types of tissues and invade into various types of cells. In tissues and three-dimensional (3-D) matrix, the apicomplexan parasite Toxoplasma gondii moves along a helical path. While many genes, including actin and myosins, have been shown to be important for parasite motility, it remains unknown what allows the parasite to travel over a long distance along a helical path. The cortical microtubules, which are ultra-stable, sprially arranged, and form extensive lateral interaction with the parasite cortex, have been considered to be a candidate structure for guiding the long-distance movement of the parasite. In wild-type parasites, the cortical microtubules in mature parasites are impervious to destabilization by cold-treatment or depolymerizing drugs, which makes it difficult to assess their function. Previously, we generated a mutant (dubbed "TKO" for succinctness) that lacks three microtubule-associated proteins. The loss of these three proteins destabilizes the cortical microtubules in mature parasites. Here we quantify the proportion of parasites with various levels of defects in the microtubule array under different conditions. We found that ~ 80% of the non-dividing TKO parasites have severely curtailed cortical microtubules. The extent of depolymerization is further exacerbated upon the initiation of daughter construction or cold treatment. The cold-induced depolymerization is reversible, with noticeable restoration of the cortical microtubules within 20 min of temperature shift to 37 degree. While microtubule polymerization is essential for generating viable daughter cells, the destabilization of the cortical microtubules in the mature parasite does not affect parasite replication. In a 3-D Matrigel matrix, the TKO mutant parasites can travel directionally over long distances. However, their trajectories are significantly more linear than those of wild-type parasites. In tissue culture, the TKO parasite displays a defect in infection and cytolytic efficiency. Interestingly, the speed and behavior of the parasite's entry into and egress from the host cell are similar to that of the wild-type parasite. These results indicate that the cortical microtubules contribute to the helicity but not the persistence of parasite long-distance movement. Furthermore, host cell entry is less sensitive to structural changes in the parasite than overall infection efficiency, which also include extracellular migration and orient the parasite for proper contact with the host cell. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.21.537899v1?rss=1 Authors: Khotina, V. A., Kalmykov, V. A., Zhuravlev, A. D., Sinyov, V. V., Sobenin, I., Orekhov, A. N., Sukhorukov, V. N. Abstract: BackgroundIt was assumed that the cause of chronic inflammation in atherosclerosis is a disturbance of the innate immunity response, caused, among other factors, by mitochondrial dysfunctions. It was also suggested that mitochondrial dysfunction may be caused by heteroplasmic mutations in mitochondrial DNA. The aim of this study was to evaluate the effect of the mitochondrial nonsense mutation m.15059G greater than A on cellular functions in atherosclerosis: lipoidosis, pro-inflammatory response, and mitophagy. MethodsThe human monocytic cell line THP-1 and cytoplasmic hybrid cell line TC-HSMAM1 were used. An original approach based on the CRISPR/Cas9 system was developed and used for the elimination of mtDNA copies carrying the m.15059G greater than A mutation in the MT-CYB gene. Using this approach, the Cas9-TC-HSMAM1 cells with an eliminated m.15059G greater than A mutation was obtained. The gene expression levels of genes encoding enzymes related to cholesterol metabolism were analyzed by quantitative RT-PCR. The evaluation of pro-inflammatory cytokine secretion was assessed using ELISA. Mitophagy in cells was detected using confocal microscopy. ResultsIn contrast to intact TC-HSMAM1 cybrids, in Cas9-TC-HSMAM1 cells, incubation with atherogenic LDL led to a decrease in the expression of the gene encoding fatty acid synthase (FASN). It was found that TC-HSMAM1 cybrids are characterized by defective mitophagy and are also unable to reduce the production of pro-inflammatory cytokines (to form immune tolerance) in response to repeated LPS stimulation. Elimination of mtDNA carrying the m.15059G greater than A mutation led to the restoration of immune tolerance and activation of mitophagy in the studied cells. ConclusionsThe m.15059G greater than A mutation was found to be associated with defective mitophagy, immune tolerance, and impaired metabolism of intracellular lipids due to upregulation of the FASN. Thus, this mutation may play an important role in atherogenesis due to its contribution to the chronification of inflammation, which aggravates the progression of atherosclerosis. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.21.537535v1?rss=1 Authors: Sweat, M. E., Cao, Y., Zhang, X., Burnicka-Turek, O., Perez-Cervantes, C., Akerberg, B. N., Ma, Q., Wakimoto, H., Gorham, J. M., Song, M. K., Trembley, M. A., Wang, P., Lu, F., Gianeselli, M., Prondzynski, M., Bortolin, R. H., Seidman, J. G., Seidman, C., Moskowitz, I. P., Pu, W. T. Abstract: Understanding how the atrial and ventricular chambers of the heart maintain their distinct identity is a prerequisite for treating chamber-specific diseases. Here, we selectively inactivated the transcription factor Tbx5 in the atrial working myocardium of the neonatal mouse heart to show that it is required to maintain atrial identity. Atrial Tbx5 inactivation downregulated highly chamber specific genes such as Myl7 and Nppa, and conversely, increased the expression of ventricular identity genes including Myl2. Using combined single nucleus transcriptome and open chromatin profiling, we assessed genomic accessibility changes underlying the altered atrial identity expression program, identifying 1846 genomic loci with greater accessibility in control atrial cardiomyocytes compared to KO aCMs. 69% of the control-enriched ATAC regions were bound by TBX5, demonstrating a role for TBX5 in maintaining atrial genomic accessibility. These regions were associated with genes that had higher expression in control aCMs compared to KO aCMs, suggesting they act as TBX5-dependent enhancers. We tested this hypothesis by analyzing enhancer chromatin looping using HiChIP and found 510 chromatin loops that were sensitive to TBX5 dosage. Of the loops enriched in control aCMs, 73.7% contained anchors in control-enriched ATAC regions. Together, these data demonstrate a genomic role for TBX5 in maintaining the atrial gene expression program by binding to atrial enhancers and preserving tissue specific chromatin architecture of atrial enhancers. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.21.537900v1?rss=1 Authors: Wen, B., Zheng, H.-X., Deng, D.-X., Zhang, Z.-D., Heng, J.-H., Liao, L.-D., Xu, L.-Y., Li, E.-M. Abstract: The translesion DNA synthesis (TLS) pathway mediated by proliferating cell nuclear antigen (PCNA) monoubiquitination is an essential mechanism by which cancer cells bypass DNA damage caused by DNA replication stress to maintain genomic stability and cell survival. Chromatin assembly factor 1 subunit A (CHAF1A) traditionally promotes histone assembly during DNA replication. Here, we revealed that CHAF1A is a novel regulator of the TLS pathway. High expression of CHAF1A is significantly associated with poor prognosis in cancer patients. CHAF1A promotes fork restart under DNA replication stress and maintains genome integrity. CHAF1A enhances the interaction between PCNA and E3 ubiquitin protein ligase RAD18 and promotes PCNA monoubiquitination, thereby promoting the recruitment of Y-family DNA polymerase Pol {eta} and enhancing cancer cell resistance to stimuli that trigger replication fork blockade. Mechanistically, CHAF1A-mediated PCNA monoubiquitination is independent of CHAF1A-PCNA interaction. CHAF1A interacts with both RAD18 and replication protein A2 (RPA2), mediating RAD18 binding on chromatin in response to DNA replication stress. Taken together, these findings improve our understanding of the mechanisms that regulate the TLS pathway and provide insights into the relationship between CHAF1A and the malignant progression of cancers. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.21.537652v1?rss=1 Authors: Diep, D. T. V., Collado, J., Hugenroth, M., Wälte, M., Schmidt, O., Fernandez-Busnadiego, R., Bohnert, M. Abstract: The lipid droplet (LD) organization proteins Ldo16 and Ldo45 affect multiple aspects of LD biology in yeast. They are linked to the LD biogenesis machinery seipin, and their loss causes defects in LD positioning, protein targeting, and breakdown. However, their molecular roles remained enigmatic. Here we report that Ldo16/45 form a tether-complex with Vac8 for creation of vacuole lipid droplet (vCLIP) contact sites, which can form in the absence of seipin. The phosphatidylinositol transfer protein Pdr16 is a further vCLIP-resident recruited by Ldo45. While only an LD-subpopulation is engaged in vCLIPs at glucose-replete conditions, nutrient stress results in vCLIP expansion, and vCLIP defects impair lipophagy upon prolonged starvation. In summary, Ldo16/45 are multifunctional proteins that orchestrate formation of a metabolically-regulated contact site. Our studies suggest an unexpected link between LD biogenesis and breakdown, and open the door to a deeper understanding of how lipid homeostasis is maintained during metabolic challenges. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.21.537823v1?rss=1 Authors: Duran, M., Ariceta, G., Semidey, M. E., Castells-Esteve, C., Lu, B., Meseguer, A., Cantero, G. Abstract: Mutations in the Cl-/H+ antiporter ClC-5 cause Dent Disease 1 (DD1), a rare primary tubulopathy that eventually progresses to renal failure. In fact, even with normal kidney function, DD1 patients present renal tubulointerstitial fibrosis. However, the link between ClC-5 loss-of-function and renal fibrosis remains unclear. Here, we have shown that DD1 mice models lacking ClC-5 present higher renal collagen deposition and fibrosis. Accordingly, deletion of ClC-5 in human renal proximal tubule epithelial cells (CLCN5 KD) recapitulates this effect. We have demonstrated that CLCN5 KD causes an increase of collagen I (Col I) and IV (Col IV) intracellular levels by promoting their transcription through {beta}-catenin pathway and impairing their lysosomal-mediated degradation. In addition, CLCN5 KD cells release more Col I and IV at the extracellular space that form fibres with altered properties and resistance to removal compared to control cells. Altogether, we describe a new regulatory mechanism for collagens production and release by ClC-5, which is altered in DD1 and provides a better understanding of disease progression to renal fibrosis. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.21.537899v1?rss=1 Authors: Khotina, V. A., Kalmykov, V. A., Zhuravlev, A. D., Sinyov, V. V., Sobenin, I., Orekhov, A. N., Sukhorukov, V. N. Abstract: BackgroundIt was assumed that the cause of chronic inflammation in atherosclerosis is a disturbance of the innate immunity response, caused, among other factors, by mitochondrial dysfunctions. It was also suggested that mitochondrial dysfunction may be caused by heteroplasmic mutations in mitochondrial DNA. The aim of this study was to evaluate the effect of the mitochondrial nonsense mutation m.15059G greater than A on cellular functions in atherosclerosis: lipoidosis, pro-inflammatory response, and mitophagy. MethodsThe human monocytic cell line THP-1 and cytoplasmic hybrid cell line TC-HSMAM1 were used. An original approach based on the CRISPR/Cas9 system was developed and used for the elimination of mtDNA copies carrying the m.15059G greater than A mutation in the MT-CYB gene. Using this approach, the Cas9-TC-HSMAM1 cells with an eliminated m.15059G greater than A mutation was obtained. The gene expression levels of genes encoding enzymes related to cholesterol metabolism were analyzed by quantitative RT-PCR. The evaluation of pro-inflammatory cytokine secretion was assessed using ELISA. Mitophagy in cells was detected using confocal microscopy. ResultsIn contrast to intact TC-HSMAM1 cybrids, in Cas9-TC-HSMAM1 cells, incubation with atherogenic LDL led to a decrease in the expression of the gene encoding fatty acid synthase (FASN). It was found that TC-HSMAM1 cybrids are characterized by defective mitophagy and are also unable to reduce the production of pro-inflammatory cytokines (to form immune tolerance) in response to repeated LPS stimulation. Elimination of mtDNA carrying the m.15059G greater than A mutation led to the restoration of immune tolerance and activation of mitophagy in the studied cells. ConclusionsThe m.15059G greater than A mutation was found to be associated with defective mitophagy, immune tolerance, and impaired metabolism of intracellular lipids due to upregulation of the FASN. Thus, this mutation may play an important role in atherogenesis due to its contribution to the chronification of inflammation, which aggravates the progression of atherosclerosis. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.21.537899v1?rss=1 Authors: Khotina, V. A., Kalmykov, V. A., Zhuravlev, A. D., Sinyov, V. V., Sobenin, I., Orekhov, A. N., Sukhorukov, V. N. Abstract: Objective: It was assumed that the cause of chronic inflammation in atherosclerosis is a disturbance of the innate immunity response, caused, among other factors, by mitochondrial dysfunctions. It was also suggested that mitochondrial dysfunction may be caused by heteroplasmic mutations in mitochondrial DNA. The aim of this study was to evaluate the effect of the mitochondrial nonsense mutation m.15059G greater than A on cellular functions in atherosclerosis: lipoidosis, pro-inflammatory response, and mitophagy. Approach and Results: The human monocytic cell line THP-1 and cytoplasmic hybrid cell line TC-HSMAM1 were used. An original approach based on the CRISPR/Cas9 system was developed and used for the elimination of mtDNA copies carrying the m.15059G greater than A mutation in the MT-CYB gene. Using this approach, the Cas9-TC-HSMAM1 cells with an eliminated m.15059G greater than A mutation was obtained. The gene expression levels of genes encoding enzymes related to cholesterol metabolism were analyzed by quantitative RT-PCR. The evaluation of pro-inflammatory cytokine secretion was assessed using ELISA. Mitophagy in cells was detected using confocal microscopy. In contrast to intact TC-HSMAM1 cybrids, in Cas9-TC-HSMAM1 cells, incubation with atherogenic LDL led to a decrease in the expression of the gene encoding fatty acid synthase (FASN). It was found that TC-HSMAM1 cybrids are characterized by defective mitophagy and are also unable to reduce the production of pro-inflammatory cytokines (to form immune tolerance) in response to repeated LPS stimulation. Elimination of mtDNA carrying the m.15059G greater than A mutation led to the restoration of immune tolerance and activation of mitophagy in the studied cells. Conclusion: The m.15059G greater than A mutation was found to be associated with defective mitophagy, immune tolerance, and impaired metabolism of intracellular lipids due to upregulation of the FASN. Thus, this mutation may play an important role in atherogenesis due to its contribution to the chronification of inflammation, which aggravates the progression of atherosclerosis. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.21.537806v1?rss=1 Authors: Kumarswamy, R., Ravi, A. B. Abstract: Abdominal Aortic Aneurysms (AAAs) are asymptomatic vascular diseases with life threatening outcomes. Smooth-muscle cell (SMC) dysfunction plays an important role in AAA development. The contributions of non-coding genome, specifically the role of long non-coding RNAs (lncRNAs) in SMC dysfunction are relatively unexplored. We investigated the role of lncRNA TUG1 in the pathology of AAA. TUG1 was identified through lncRNA profiling in Angiotensin-II (Ang-II) treated SMCs. TUG1 was upregulated in Ang-II treated SMCs in vitro and its expression increased with progression of aneurysm in mouse model of Ang-II induced AAA. Ang-II induced TUG1 was blunted by inhibition of Notch signaling and TUG1 is demonstrated to be a transcriptional target of Notch. AAA tissues exhibited inversely correlated expression of TUG1 and SMC contractile markers. TUG1 knock-down via siRNA/shRNA increased SMC differentiation. ChIP, DNA-RNA IP, and RNA-IP experiments demonstrated that TUG1 interacts with transcriptional repressor KLF4 and aides in its recruitment to Myocardin promoter, thereby repressing SMC differentiation. In summary, we show a novel role for lncRNA TUG1 in Ang-II induced AAA wherein it modulates SMC differentiation via KLF4-Myocardin axis. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.21.537815v1?rss=1 Authors: Paterou, A., Tyc, J., Sunter, J., Vaughan, S., Gull, K., Dean, S. Abstract: African trypanosomes are medically important parasites that cause Sleeping sickness in humans and nagana in animals. In addition to their pathogenic role, they have emerged as valuable model organisms for studying fundamental biological processes. Protein tagging is a powerful tool for investigating protein localization and function. In a previous study, we developed two plasmids for rapid and reproducible protein tagging in trypanosomes, which enabled the localisation of all proteins in the trypanosome cell. However, the limited selection of fluorescent protein tags and selectable markers restricted the flexibility of this approach. Here, we present an expanded set of greater than 100 vectors that utilizes universal primer annealing sequences, enabling protein tagging with a range of fluorescent and biochemical tags using five different selection markers. We evaluated the suitability of various fluorescent proteins for live cell imaging and determined their brightness and stability under different fixation conditions. Finally, we determined the optimal fluorescent protein for a set of specific experimental conditions demonstrating the utility of this toolkit. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.21.537794v1?rss=1 Authors: Miyata, Y., Nishida, E. Abstract: A protein kinase DYRK1A encoded in human chromosome-21 is the major contributor for multiple symptoms observed in Down syndrome patients. In addition, DYRK1A dysfunction has been associated with various neuronal disorders, including autism spectrum disorder and Alzheimer disease. Here we identified FAM53C as a novel suppressive binding partner of DYRK1A. FAM53C bound to the catalytic kinase domain of DYRK1A, whereas DCAF7/WDR68, the major known DYRK1A-binding protein, binds to its N-terminal domain. The binding of FAM53C inhibited the protein kinase activity of DYRK1A to itself and an exogenous substrate MAPT/Tau. FAM53C did not bind directly to DCAF7/WDR68, whereas DYRK1A tethered FAM53C and DCAF7/WDR68 by binding concurrently to both of them. DYRK1A possesses a nuclear localization signal and accumulates in the nucleus when overexpressed in cells. FAM53C induced cytoplasmic re-localization of DYRK1A and DCAF7/WDR68. FAM53C is thus a binding suppressor of DYRK1A, anchoring it in an inactive state in the cytoplasm. The results explain for the first time why endogenous DYRK1A is distributed in the cytoplasm in the normal brain tissues. FAM53C-dependent regulation of DYRK1A may play a significant role in gene expression modification caused by DYRK1A. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.19.537613v1?rss=1 Authors: Gupta, A., Ruturaj,, Mishra, M., Saha, S., Maji, S., Rodriguez-Boulan, E., Schreiner, R. Abstract: We suggest a model of apico-basolateral sorting in polarized epithelia using homologous Cu-ATPases as membrane cargoes. In polarized epithelia, upon copper treatment, homologous copper-ATPases ATP7A and ATP7B traffic from trans-Golgi network (TGN) to basolateral and apical membranes respectively. We characterized sorting pathways of Cu-ATPases between TGN and plasma-membrane and identified the machinery involved. ATP7A and ATP7B reside on distinct domains of TGN and in high copper, ATP7A traffics directly to basolateral membrane, whereas ATP7B traverses common-recycling, apical-sorting and apical-recycling endosomes en-route to apical membrane. Mass-spectrometry identified regulatory partners of ATP7A and ATP7B that include Adaptor Protein-1 complex. Upon knocking-out pan-AP-1, sorting of both copper-ATPases are disrupted. ATP7A loses polarity and localizes on both apical and basolateral surfaces in high copper. Contrastingly, ATP7B loses TGN-retention but retains apical polarity that becomes copper-independent. Using isoform-specific knockouts, we found that AP-1A provides directionality and TGN-retention for both Cu-ATPases, whereas, AP-1B governs polarized trafficking of ATP7B solely. Trafficking phenotypes of Wilson disease-causing ATP7B mutants that disrupts putative ATP7B-AP1 interaction further substantiates the role of AP-1 in apical sorting of ATP7B. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.20.537668v1?rss=1 Authors: Tatickova, M., Trebichalska, Z., Kyjovska, D., Otevrel, P., Kloudova, S., Holubcova, Z. Abstract: Egg quality is a limiting factor of female fertility and assisted reproductive technology (ART) success. Oocytes recovered from hyperstimulated ovaries often display morphological anomalies suspected to compromise their fertilization and developmental potential. Knowledge of (ab)normal oocytes intracellular organization is vital to establish reliable criteria for morphological evaluation of oocytes intended for in vitro fertilization (IVF). Here, we investigated the fine morphology of 22 dysmorphic IVF oocytes exhibiting different types of cytoplasmic irregularities, namely (1) refractile bodies, (2) centrally-located cytoplasmic granularity (CLCG), (3) smooth endoplasmic reticulum (SER) disc, and (4) vacuoles. Transmission electron microscopy (TEM) revealed the structural basis of these aberrations and indicated that the underlying cause of two of the studied morphotypes was inordinate organelle clustering. To address the mechanism required for accurate organelle positioning, we used cytoskeleton-targeting chemical compounds and performed a series of inhibition experiments involving a total of 133 human oocytes maturing in vitro. Fluorescence and electron microscopy showed that disruption of actin, not microtubules, led to the aggregation of subcellular structures resembling the morphological pattern seen in abnormal oocytes. These results imply that actin serves as a regulator of organelle distribution during human oocyte maturation. The ultrastructural analogy between dysmorphic eggs retrieved in IVF cycles and oocytes, in which actin network integrity was perturbed, suggests that dysfunction of the actin cytoskeleton might be implicated in generating common cytoplasmic aberrations. Knowledge of human oocytes inner workings and the origin of morphological abnormalities is a step forward to more objective egg quality assessment in clinical practice. SUMMARY SENTENCEUltrastructural analysis of eggs exhibiting cytoplasmic abnormalities combined with inhibition experiments indicates that dysfunction of the actin network might be involved in the development of oocyte dysmorphisms. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.18.537332v1?rss=1 Authors: Massey-Harroche, D., Conte, V., Gouirand, N., Sebbagh, M., Le Bivic, A., Bazellieres, E. Abstract: Several cellular processes during morphogenesis, tissue healing or cancer progression involve epithelial to mesenchymal plasticity that leads to collective motion (plasticity?). Even though a rich variety of EMP programs exist, a major hallmark unifying them is the initial breaking of symmetry that modifies the epithelial phenotype and axis of polarity. During this process, the actin cytoskeleton and cellular junctions are extensively remodelled correlating with the build-up of mechanical forces. As the collective migration proceeds, mechanical forces generated by the actin cytoskeleton align with the direction of migration ensuring an organized and efficient collective cell behaviour, but how forces are regulated during the breaking of symmetry at the onset of EMP remains an unaddressed question. It is known that the polarity complex CRB3/PALS1/PATJ, and in particular, CRB3 regulates the organization of the actin cytoskeleton associated to the apical domain thus pointing at a potential role of CRB3 in controlling mechanical forces. Whether and how CRB3 influences epithelial biomechanics during the epithelial-mesenchymal plasticity remains, however, largely unexplored. Here, we systematically combine mechanical and molecular analyses to show that CRB3 regulates the biomechanical properties of collective epithelial cells during the initial breaking of symmetry of the EMP. CRB3 interacts with ARP2/3 and controls the remodelling of actin throughout the monolayer via the modulation of the Rho-/Rac-GTPase balance. Taken together, our results identified CRB3, a polarity protein, as a regulator of epithelial monolayer mechanics during EMP Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.19.537556v1?rss=1 Authors: Chen, P., Mishra, S., Levy, D. L. Abstract: What drives nuclear growth? Studying nuclei assembled in Xenopus egg extract and focusing on importin /{beta}-mediated nuclear import, we show that, while nuclear growth depends on nuclear import, nuclear growth and import can be uncoupled. Nuclei containing fragmented DNA grew slowly despite exhibiting normal import rates, suggesting nuclear import itself is insufficient to drive nuclear growth. Nuclei containing more DNA grew larger but imported more slowly. Altering chromatin modifications caused nuclei to grow less while still importing to the same extent or to grow larger without increasing nuclear import. Increasing heterochromatin in vivo in sea urchin embryos increased nuclear growth but not import. These data suggest that nuclear import is not the primary driving force for nuclear growth. Instead, live imaging showed that nuclear growth preferentially occurred at sites of high chromatin density and lamin addition, whereas small nuclei lacking DNA exhibited less lamin incorporation. Our hypothesized model is that lamin incorporation and nuclear growth are driven by chromatin mechanical properties, which depend on and can be tuned by nuclear import. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.20.537628v1?rss=1 Authors: Wang, X., Zhang, W., Zhao, S., Yan, H., Xin, Z., Cui, T., Zang, R., Zhao, L., Wang, H., Zhou, J., Li, X., Yue, W., Xi, J., Zhang, Z., Fang, X., Pei, X. Abstract: Ex vivo RBC production generates unsatisfactory expansion, {beta}-globin expression, and maturation of erythroid cells. The underlying mechanisms behind these limitations and ex vivo terminal erythropoiesis from different origins are largely unexplained. In this study, we mapped an atlas of ex vivo terminally differentiated cells from umbilical cord blood mononuclear cells (UCBMNs) and pluripotent stem cells (PSCs), and observed the differential regulatory dynamics of erythropoiesis from these two origins at a single-cell resolution. We detected the presence of hematopoietic stem progenitor cells (HSPCs), erythroid progenitor (e.g., CFU-E), and non-erythroid cells (e.g., macrophages) in the terminal populations. We observed that UCBMN-derived erythropoiesis is more active than PSC-derived erythropoiesis in terms of the cell cycle, stress erythropoiesis, and autophagy at single cell resolution, which may provide new insights into the limitations in cell expansion, globin expression, and maturation in ex vivo RBC production, respectively. We verified that a stress-erythropoiesis-related gene, TRIB3, increases the expression of globin genes in ex vivo erythropoiesis. As the major unexpected component detected in terminally differentiated cells, CFU-E were further characterized as having high- or low-expansion capacity based on CD99 expression, which generally decreased over erythropoiesis. By inhibiting CD99 gene expression using antagonists, we increased reticulocyte production in the population. Heterogeneous CFU-Es also exist in bone marrow. Moreover, decreased CD99 expression mediates the interactions between macrophages and CFU-E during ex vivo erythropoiesis. Overall, our results provide a reference for facilitating the development of strategies to improve ex vivo RBC regeneration. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.20.537413v1?rss=1 Authors: Haggerty, K. N., Eshelman, S. C., Sexton, L. A., Frimpong, E., Rogers, L. M., Agosto, M. A., Robichaux, M. A. Abstract: Photoreceptor cells in the vertebrate retina have a highly compartmentalized morphology for efficient long-term phototransduction. Rhodopsin, the visual pigment in rod photoreceptors, is densely packaged into the rod outer segment sensory cilium and continuously renewed through essential synthesis and trafficking pathways housed in the rod inner segment. Despite the importance of this region for rod health and maintenance, the subcellular organization of rhodopsin and its trafficking regulators in the mammalian rod inner segment remain undefined. We used super-resolution fluorescence microscopy with optimized retinal immunolabeling techniques to perform a single molecule localization analysis of rhodopsin in the inner segments of mouse rods. We found that a significant fraction of rhodopsin molecules was localized at the plasma membrane in an even distribution along the entire length of the inner segment, where markers of transport vesicles also colocalized. Thus, our results collectively establish a model of rhodopsin trafficking through the inner segment plasma membrane as an essential subcellular pathway in mouse rod photoreceptors. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.20.537623v1?rss=1 Authors: Ortiz, H. R., Flores, P. C., Ramonett, A., Ahmed, T., Ellis, N. A., Langlais, P. R., Karthikeyan, M., Lee, N. Y. Abstract: Dynamic changes in the endoplasmic reticulum (ER) morphology are central to maintaining cellular homeostasis. Microtubules (MT) facilitate the continuous remodeling of the ER network into sheets and tubules by coordinating with many ER-shaping protein complexes, although how this process is controlled by extracellular signals remains unknown. Here we report that TAK1, a kinase responsive to numerous growth factors and cytokines including TGF-beta and TNF-alpha, triggers ER tubulation by activating aTAT1, an MT-acetylating enzyme that enhances ER-sliding. We show that this TAK1/aTAT-dependent ER remodeling promotes cell survival by actively downregulating BOK, an ER membrane-associated proapoptotic effector. While BOK is normally protected from degradation when complexed with IP3R, it is rapidly degraded upon their dissociation during the ER sheets-to-tubules conversion. These findings demonstrate a distinct mechanism of ligand-induced ER remodeling and suggest that the TAK1/aTAT pathway may be a key target in ER stress and dysfunction. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.19.537409v1?rss=1 Authors: Matsumoto, T., Higaki, T., Takatsuka, H., Kutsuna, N., Ogata, Y., Hasezawa, S., Umeda, M., Inada, N. Abstract: ACTIN DEPOLYMERIZING FACTOR (ADF) is a conserved protein that regulates the organization and dynamics of actin microfilaments. Eleven ADFs in the Arabidopsis thaliana genome are grouped into four subclasses, and subclass I ADFs, ADF1-4, are all expressed throughout the plant. Previously, we showed that subclass I ADFs function in the regulation of the response against powdery mildew fungus as well as in the regulation of cell size and endoreduplication. Here, we report a new role of subclass I ADFs in the regulation of nuclear organization and gene expression. Through a microscopic observation of epidermal cells in mature leaves, we found that the size of chromocenters in both adf4 and transgenic lines where expression of subclass I ADFs are downregulated (ADF1-4Ri) was reduced compared with that of wild-type Col-0. A. thaliana possesses eight ACTIN genes, among which ACT2, -7, and -8 are expressed in vegetative organs. The chromocenter size in act7, but not in the act2/8 double mutant, was enlarged compared with that in Col-0. Microarray analysis revealed that 1,818 genes were differentially expressed in adf4 and ADF1-4Ri. In particular, expression of 22 nucleotide-binding leucine-rich repeat (NLR) genes, which are involved in effector-triggered plant immunity, was reduced in adf4 and ADF1-4Ri. qRT-PCR confirmed the altered expressions shown with microarray analysis. Overall, these results suggest that ADF regulates various aspects of plant physiology through its role in regulation of nuclear organization and gene expression. The mechanism how ADF and ACTIN regulate nuclear organization and gene expression is discussed. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.21.537797v1?rss=1 Authors: Alvarez-Guerra, I., Block, E., Broeskamp, F., Gabrijelcic, S., de Ory, A., Habernig, L., Andreasson, C., Levine, T., Höög, J. L., Büttner, S. Abstract: Lipid droplets (LDs) are fat storage organelles critical for energy and lipid metabolism. Upon nutrient exhaustion, cells consume LDs via gradual lipolysis or via lipophagy, the en bloc uptake of LDs into the vacuole. Here, we show that LDs dock to the vacuolar membrane via a contact site that is required for lipophagy in yeast. The LD-localized LDO proteins carry an intrinsically disordered region that associates with vacuolar Vac8 to form vCLIP, the vacuolar-LD contact site. Nutrient limitation drives vCLIP formation, and its inactivation blocks lipophagy. Vac8 is sufficient to recruit LDs to cellular membranes. We establish a functional link between lipophagy and microautophagy of the nucleus, both requiring Vac8 to form respective contact sites upon metabolic stress. In sum, we unravel the molecular architecture of vCLIP, a contact site required for lipophagy, and find that Vac8 provides a platform for multiple and competing contact sites associated with autophagy. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.20.537668v1?rss=1 Authors: Tatickova, M., Trebichalska, Z., Kyjovska, D., Otevrel, P., Kloudova, S., Holubcova, Z. Abstract: Egg quality is a limiting factor of female fertility and assisted reproductive technology (ART) success. Oocytes recovered from hyperstimulated ovaries often display morphological anomalies suspected to compromise their fertilization and developmental potential. Knowledge of (ab)normal oocytes intracellular organization is vital to establish reliable criteria for morphological evaluation of oocytes intended for in vitro fertilization (IVF). Here, we investigated the fine morphology of 22 dysmorphic IVF oocytes exhibiting different types of cytoplasmic irregularities, namely (1) refractile bodies, (2) centrally-located cytoplasmic granularity (CLCG), (3) smooth endoplasmic reticulum (SER) disc, and (4) vacuoles. Transmission electron microscopy (TEM) revealed the structural basis of these aberrations and indicated that the underlying cause of two of the studied morphotypes was inordinate organelle clustering. To address the mechanism required for accurate organelle positioning, we used cytoskeleton-targeting chemical compounds and performed a series of inhibition experiments involving a total of 133 human oocytes maturing in vitro. Fluorescence and electron microscopy showed that disruption of actin, not microtubules, led to the aggregation of subcellular structures resembling the morphological pattern seen in abnormal oocytes. These results imply that actin serves as a regulator of organelle distribution during human oocyte maturation. The ultrastructural analogy between dysmorphic eggs retrieved in IVF cycles and oocytes, in which actin network integrity was perturbed, suggests that dysfunction of the actin cytoskeleton might be implicated in generating common cytoplasmic aberrations. Knowledge of human oocytes inner workings and the origin of morphological abnormalities is a step forward to more objective egg quality assessment in clinical practice. SUMMARY SENTENCEUltrastructural analysis of eggs exhibiting cytoplasmic abnormalities combined with inhibition experiments indicates that dysfunction of the actin network might be involved in the development of oocyte dysmorphisms. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.20.537668v1?rss=1 Authors: Tatickova, M., Trebichalska, Z., Kyjovska, D., Otevrel, P., Kloudova, S., Holubcova, Z. Abstract: Egg quality is a limiting factor of female fertility and assisted reproductive technology (ART) success. Oocytes recovered from hyperstimulated ovaries often display morphological anomalies suspected to compromise their fertilization and developmental potential. Knowledge of (ab)normal oocyte's intracellular organization is vital to establish reliable criteria for morphological evaluation of oocytes intended for in vitro fertilization (IVF). Here, we investigated the fine morphology of 22 dysmorphic IVF oocytes exhibiting different types of cytoplasmic irregularities, namely (1) refractile bodies, (2) centrally-located cytoplasmic granularity (CLCG), (3) smooth endoplasmic reticulum (SER) disc, and (4) vacuoles. Transmission electron microscopy (TEM) revealed the structural basis of these aberrations and indicated that the underlying cause of two of the studied morphotypes was inordinate organelle clustering. To address the mechanism required for accurate organelle positioning, we used cytoskeleton-targeting chemical compounds and performed a series of inhibition experiments involving a total of 133 human oocytes maturing in vitro. Fluorescence and electron microscopy showed that disruption of actin, not microtubules, led to the aggregation of subcellular structures resembling the morphological pattern seen in abnormal oocytes. These results imply that actin serves as a regulator of organelle distribution during human oocyte maturation. The ultrastructural analogy between dysmorphic eggs retrieved in IVF cycles and oocytes, in which actin network integrity was perturbed, suggests that dysfunction of the actin cytoskeleton might be implicated in generating common cytoplasmic aberrations. Knowledge of human oocytes' inner workings and the origin of morphological abnormalities is a step forward to more objective egg quality assessment in clinical practice. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.20.537668v1?rss=1 Authors: Tatickova, M., Trebichalska, Z., Kyjovska, D., Otevrel, P., Kloudova, S., Holubcova, Z. Abstract: Egg quality is a limiting factor of female fertility and assisted reproductive technology (ART) success. Oocytes recovered from hyperstimulated ovaries often display morphological anomalies suspected to compromise their fertilization and developmental potential. Knowledge of (ab)normal oocyte's intracellular organization is vital to establish reliable criteria for morphological evaluation of oocytes intended for in vitro fertilization (IVF). Here, we investigated the fine morphology of 22 dysmorphic IVF oocytes exhibiting different types of cytoplasmic irregularities, namely (1) refractile bodies, (2) centrally-located cytoplasmic granularity (CLCG), (3) smooth endoplasmic reticulum (SER) disc, and (4) vacuoles. Transmission electron microscopy (TEM) revealed the structural basis of these aberrations and indicated that the underlying cause of two of the studied morphotypes was inordinate organelle clustering. To address the mechanism required for accurate organelle positioning, we used cytoskeleton-targeting chemical compounds and performed a series of inhibition experiments involving a total of 133 human oocytes maturing in vitro. Fluorescence and electron microscopy showed that disruption of actin, not microtubules, led to the aggregation of subcellular structures resembling the morphological pattern seen in abnormal oocytes. These results imply that actin serves as a regulator of organelle distribution during human oocyte maturation. The ultrastructural analogy between dysmorphic eggs retrieved in IVF cycles and oocytes, in which actin network integrity was perturbed, suggests that dysfunction of the actin cytoskeleton might be implicated in generating common cytoplasmic aberrations. Knowledge of human oocytes' inner workings and the origin of morphological abnormalities is a step forward to more objective egg quality assessment in clinical practice. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.21.537835v1?rss=1 Authors: Wu, Z., Huang, W., He, X., Dutta, S., Paul, C., Fan, G.-C., Kanisicak, O., Xu, M., Liang, J., Wang, Y. Abstract: Ischemia-reperfusion (I/R) injury is a common occurrence in various surgical procedures used to treat heart diseases. However, the role of insulin-like growth factor 2 receptor (IGF2R) during the process of myocardial I/R remains unclear. Therefore, this study aims to investigate the expression, distribution, and functionality of IGF2R in various I/R-associated models (such as reoxygenation, revascularization, and heart transplant). Loss-of-function studies (including myocardial conditional knockout and CRISPR interference) were performed to clarify the role of IGF2R in I/R injuries. Following hypoxia, IGF2R expression increased, but this effect was reversed upon restoration of oxygen levels. Loss of myocardial IGF2R was found to enhance the cardiac contractile functions, and reduced cell infiltration or cardiac fibrosis of I/R mouse models compared to the genotype control. CRISPR-inhibition of IGF2R decreased cell apoptotic death under hypoxia. RNA sequencing analysis indicated that myocardial IGF2R played a critical role in regulating the inflammatory response, innate immune response, and apoptotic process following I/R. Integrated analysis of the mRNA profiling, pulldown assays, and mass spectrometry identified granulocyte-specific factors as potential targets of myocardial IGF2R in the injured heart. In conclusion, myocardial IGF2R emerges as a promising therapeutic target to ameliorate inflammation or fibrosis following I/R injuries. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.19.537428v1?rss=1 Authors: Pryce Roberts, A., Dec, K., Tyrrell, V., O'Donnell, V. B., Harwood, A. J., Williams, J. Abstract: Sporadic Alzheimer's disease is the leading cause of dementia worldwide and the Apolipoprotein-E4 allele (APOE) is the strongest genetic risk factor but despite its importance, its role in disease pathogenesis is incompletely understood. The APOE gene encodes Apolipoprotein E (ApoE). Astrocytes are the main source of ApoE in the central nervous system (CNS) and are essential for homeostasis in health and disease. In response to CNS insult, a coordinated multicellular inflammatory response is triggered causing reactive astrogliosis with changes in astrocytic gene expression, cellular structure and function. Using a human embryonic stem-cell line with the neutral APOE33 genotype, we used CRISPR Cas-9 gene-editing technology to create isogenic APOE lines with an APOE44 genotype. We developed a modified protocol designed to produce quiescent astrocytes and then stimulated them to induce an astrogliotic A1 phenotype. Several potentially pathological APOE44-related phenotypes were identified in both quiescent cells and reactive A1 astrocytes including significantly decreased phagocytosis and impaired glutamate uptake in APOE44 astrocytes. There were also key differences in the inflammatory profiles of APOE33 and APOE44 astrocytes characterised by significantly decreased secretion of IL6, IL8 and several oxylipins in APOE44 quiescent astrocytes. In A1 astrocytes there was a pro-inflammatory phenotype in APOE44 astrocytes with increases in GRO, ENA78, IL6 and IL8, a decrease in IL10 as well as significant differences in oxylipin expression suggestive of a defect in their immunomodulatory function. As TNF- induced signaling in astrocytes is driven by Nuclear factor kappa B (NF-{kappa}B) we investigated the proteins of this pathway and found significantly higher levels of the p65 and I{kappa}B sub-units in both quiescent and A1 APOE44 astrocytes. This suggests that perturbation of NF-{kappa}B signaling may contribute to the damaging cell phenotypes that we observe and provides a new direction for targeted disease therapeutics. Given the large numbers of existing drugs that act on the NF-{kappa}B pathway, this could be realised in a relatively short timeframe. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.19.537540v1?rss=1 Authors: Khan, S. T., Ahuja, N., Taib, S., Vohra, S., Cleaver, O., Nunes, S. S. Abstract: The pancreatic islet vasculature displays tissue-specific physiological and functional adaptations that support rapid glucose sensing and insulin response by beta-cells. To uncover the transcriptomic basis of this specialization, we performed a meta-analysis of multi-organ single cell RNA sequencing atlases employing a unique strategy to avoid transcriptomic contamination. We identified biologically relevant genes involved in sphingosine-1-phosphate-mediated insulin secretion (PLPP1, RDX, CDC42), islet basement membrane formation (SPARC, COL15A1), endothelial cell (EC) permeability (PLVAP, EHD4), membrane transporters (CD320, SLCO2A1) and developmental transcription factors (NKX2-3, AHR). These were validated in silico in an independent dataset. We further established the first integrated transcriptomic atlas of human pancreatic ECs and described two unique capillary subpopulations: exocrine and endocrine pancreas ECs. We validated the spatial localization of key markers using RNAscope and immunofluorescence staining on mouse pancreatic tissue cross-sections. Our findings provide novel insights into pancreatic EC heterogeneity and islet EC function with potential implications in therapeutic strategies. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.19.537567v1?rss=1 Authors: Zink, J., Froemel, T., Wittig, I., Fleming, I., Benz, P. M. Abstract: The interaction of Eph receptor tyrosine kinases with their transmembrane ligands; the ephrins, is important for the regulation of cell-cell communication. Ephrin-Eph signaling is probably best known for the discrimination of arterial and venous territories by repulsion of venous endothelial cells away from those with an arterial fate. Ultimately, cell repulsion is mediated by initiating the collapse of the actin cytoskeleton in membrane protrusions. Here, we investigated the role of the Ena/VASP family of actin binding proteins in endothelial cell repulsion initiated by ephrin ligands. Human endothelial cells dynamically extended sheet-like lamellipodia over ephrin-B2 coated surfaces. While lamellipodia of control siRNA transfected cells rapidly collapsed, resulting in a pronounced cell repulsion from the ephrin-B2 surfaces, the knockdown of Ena/VASP proteins impaired the cytoskeletal collapse of membrane protrusions and the cells no longer avoided the repulsive surfaces. Mechanistically, ephrin-B2 stimulation elicited the EphB-mediated tyrosine phosphorylation of VASP, which abrogated its interaction with the focal adhesion protein Zyxin. Nck2 was identified as a novel VASP binding protein, which only interacted with the tyrosine phosphorylated VASP protein. Nck links Eph-receptors to the actin cytoskeleton. Therefore, we hypothesize that Nck-Ena/VASP complex formation is required for actin reorganization and/or Eph receptor internalization downstream of ephrin-Eph interaction in endothelial cells, with implications for endothelial navigation and pathfinding. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.18.537280v1?rss=1 Authors: Kita, T., Chiba, K., Wang, J., Nakagawa, A., Niwa, S. Abstract: Kinesin-3 is a family of microtubule-dependent motor proteins that transport various cargos within the cell. However, the mechanism underlying kinesin-3 activations remains largely elusive. In this study, we compared the biochemical properties of two Caenorhabditis elegans kinesin-3 family proteins, KLP-6 and UNC-104. Both KLP-6 and UNC-104 are predominantly monomeric in solution. As previously shown for UNC-104, non-processive KLP-6 monomer is converted to a processive motor when artificially dimerized. We present evidence that releasing the autoinhibition is sufficient to trigger dimerization of monomeric UNC-104 at nanomolar concentrations, which results in processive movement of UNC-104 on microtubules, although it has long been thought that enrichment in the phospholipid microdomain on cargo vesicles is required for the dimerization and processive movement of UNC-104. In contrast, KLP-6 remains to be a non-processive monomer even when its autoinhibition is unlocked, suggesting a requirement of other factors for full activation. By examining the differences between KLP-6 and UNC-104, we identified a coiled-coil domain called CC2 that is required for the dimerization and processive movement of UNC-104. Our results suggest a common activation mechanism for kinesin-3 family members, while also highlighting their diversification. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.19.537428v1?rss=1 Authors: Pryce Roberts, A., Dec, K., Tyrrell, V., O'Donnell, V. B., Harwood, A. J., Williams, J. Abstract: Sporadic Alzheimer's disease is the leading cause of dementia worldwide and the Apolipoprotein-E4 allele (APOE) is the strongest genetic risk factor but despite its importance, its role in disease pathogenesis is incompletely understood. The APOE gene encodes Apolipoprotein E (ApoE). Astrocytes are the main source of ApoE in the central nervous system (CNS) and are essential for homeostasis in health and disease. In response to CNS insult, a coordinated multicellular inflammatory response is triggered causing reactive astrogliosis with changes in astrocytic gene expression, cellular structure and function. Using a human embryonic stem-cell line with the neutral APOE33 genotype, we used CRISPR Cas-9 gene-editing technology to create isogenic APOE lines with an APOE44 genotype. We developed a modified protocol designed to produce quiescent astrocytes and then stimulated them to induce an astrogliotic A1 phenotype. Several potentially pathological APOE44-related phenotypes were identified in both quiescent cells and reactive A1 astrocytes including significantly decreased phagocytosis and impaired glutamate uptake in APOE44 astrocytes. There were also key differences in the inflammatory profiles of APOE33 and APOE44 astrocytes characterised by significantly decreased secretion of IL6, IL8 and several oxylipins in APOE44 quiescent astrocytes. In A1 astrocytes there was a pro-inflammatory phenotype in APOE44 astrocytes with increases in GRO, ENA78, IL6 and IL8, a decrease in IL10 as well as significant differences in oxylipin expression suggestive of a defect in their immunomodulatory function. As TNF- induced signaling in astrocytes is driven by Nuclear factor kappa B (NF-{kappa}B) we investigated the proteins of this pathway and found significantly higher levels of the p65 and I{kappa}B sub-units in both quiescent and A1 APOE44 astrocytes. This suggests that perturbation of NF-{kappa}B signaling may contribute to the damaging cell phenotypes that we observe and provides a new direction for targeted disease therapeutics. Given the large numbers of existing drugs that act on the NF-{kappa}B pathway, this could be realised in a relatively short timeframe. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.18.537325v1?rss=1 Authors: Falzoni, S., Chiozzi, P., Vultaggio-Poma, V., Tarantini, M., Adinolfi, E., Boldrini, P., Giuliani, A. L., Gorecki, D. C., Di Virgilio, F. Abstract: Microparticles (MPs) are ubiquitously secreted by all cells and play a fundamental role in numerous biological processes such as cell-to-cell communication, cell differentiation, inflammation, and cell energy transfer. Ligation of the P2X7 receptor (P2X7R) by extracellular ATP (eATP) is the well-known stimulus for MP release, which affects their contents in a cell-specific fashion. We investigated MP release and functional impact in mouse microglial cell lines characterized for high (N13-P2X7RHigh) or low (N13-P2X7RLow) expression of the P2X7R. Stimulation with extracellular ATP triggered a P2X7R-dependent release of a MP population enriched with naked mitochondria. Released mitochondria were taken up and incorporated into the mitochondrial network of the recipient cells in a P2X7R-dependent fashion. Other constituents of the MP cargo, e.g. NLRP3 and the P2X7R itself, were also delivered to the recipient cells. Transfer of mitochondria, NLRP3 and P2X7R increased the energy level of the recipient cells and conferred a pro-inflammatory phenotype. These data show that P2X7R-dependent exchange of MPs and mitochondria modulates energy metabolism and inflammatory responses, pointing to the P2X7R as a master regulator of intercellular organelle and MP trafficking in mouse microglia. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.17.537276v1?rss=1 Authors: Anand, A., Mazur, A.-C., Rosell-Arevalo, P., Franzkoch, R., Breitsprecher, L., Listian, S. A., Hüttel, S. V., Müller, D., Schäfer, D. G., Vormittag, S., Hilbi, H., Maniak, M., Gutierrez, M., Barisch, C. Abstract: Several intracellular pathogens, such as Mycobacterium tuberculosis, damage endomembranes to access the cytosol and subvert innate immune responses. The host counteracts endomembrane damage by recruiting repair machineries that retain the pathogen inside the vacuole. Here, we show that the endoplasmic reticulum (ER)-Golgi protein oxysterol binding protein (OSBP) and its Dictyostelium discoideum homologue OSBP8 are recruited to the Mycobacterium-containing vacuole (MCV) after ESX-1-dependent membrane damage. Lack of OSBP8 causes a hyperaccumulation of phosphatidylinositol-4-phosphate (PI4P) on the MCV and decreased cell viability. OSBP8-depleted cells had reduced lysosomal and degradative capabilities of their vacuoles that favoured mycobacterial growth. In agreement with a function of OSBP8 in membrane repair, human macrophages infected with M. tuberculosis recruited OSBP in an ESX-1 dependent manner. These findings identified an ER-dependent repair mechanism for restoring MCVs in which OSBP8 functions to equilibrate PI4P levels on damaged membranes. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.16.537100v1?rss=1 Authors: Köckert, M., Okafornta, C. W., Hill, C., Ryndyk, A., Striese, C., Müller-Reichert, T., Paliulis, L., Fabig, G. Abstract: Spermatogenesis leads to the formation of functional sperm cells. Here we have applied high-pressure freezing in combination with transmission electron microscopy (TEM) to study the ultrastructure of sperm development in subadult males of the praying mantid Hierodula membranacea. We show the ultrastructure of different stages of sperm development in this species. In addition, we have applied serial-section electron tomography of the nebenkern to demonstrate in three dimensions (3D) that this organelle is composed of two interwoven segments that are connected by a zipper-like structure at opposing positions. Our approach will enable further ultrastructural analyses of the nebenkern also in other organisms. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.19.537449v1?rss=1 Authors: Bulos, M. L., Grzelak, E. M., Li-Ma, C., Chen, E., Hull, M., Johnson, K. A., Bollong, M. J. Abstract: Yes-associated protein (YAP), the downstream effector of the evolutionarily conserved Hippo pathway, promotes cellular proliferation and coordinates certain regenerative responses in mammals. Small molecule activators of YAP may therefore display therapeutic utility in treating disease states involving insufficient proliferative repair. From a high-throughput chemical screen of the comprehensive drug repurposing library ReFRAME, here we report the identification of SM04690, a clinical stage inhibitor of CLK2, as a potent activator of YAP driven transcriptional activity in cells. CLK2 inhibition promotes alternative splicing of the Hippo pathway protein AMOTL2, producing an exon-skipped gene product that can no longer associate with membrane-bound proteins, resulting in decreased phosphorylation and membrane localization of YAP. This study reveals a novel mechanism by which pharmacological perturbation of alternative splicing inactivates the Hippo pathway and promotes YAP dependent cellular growth. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.19.537575v1?rss=1 Authors: Zink, J., Froemel, T., Boon, R. A., Fleming, I., Benz, P. M. Abstract: Endothelial tip cells are essential for VEGF-induced angiogenesis, but underlying mechanisms are elusive. Endothelial-specific deletion of EVL, a member of the mammalian Ena/VASP protein family, reduced the expression of the tip cell marker protein endothelial cell specific molecule-1 (Esm1) and compromised the radial sprouting of the vascular plexus in the postnatal mouse retina. The latter effects could at least partly be attributed to reduced VEGF receptor 2 (VEGFR2) internalization and signaling but the underlying mechanisms(s) are not fully understood. In the present study, we revealed that the expression of the long non-coding RNA H19 was significantly reduced in endothelial cells from postnatal EVL-/- mice and in siRNA-transfected human endothelial cells under hypoxic conditions. H19 was recently shown to promote VEGF expression and bioavailability via Esm1 and hypoxia inducible factor 1 (HIF-1). Similar to EVL-/- mice, the radial outgrowth of the vascular plexus was significantly delayed in the postnatal retina of H19-/- mice. In summary, our data suggests that loss of EVL not only impairs VEGFR2 internalization and downstream signaling, but also impairs VEGF expression and bioavailability in the hypoxic retina via downregulation of lncRNA H19. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.18.537262v1?rss=1 Authors: Iwamoto, Y., Ye, A. A., Shirazinejad, C., Hurley, J. H., Drubin, D. G. Abstract: Lentiviruses express non-enzymatic accessory proteins whose function is to subvert cellular machinery in the infected host. The HIV-1 accessory protein Nef hijacks clathrin adaptors to degrade or mislocalize host proteins involved in antiviral defenses. Here, we investigate the interaction between Nef and clathrin-mediated endocytosis (CME), a major pathway for membrane protein internalization in mammalian cells, using quantitative live-cell microscopy in genome-edited Jurkat cells. Nef is recruited to CME sites on the plasma membrane, and this recruitment correlates with an increase in the recruitment and lifetime of CME coat protein AP-2 and late-arriving CME protein dynamin2. Furthermore, we find that CME sites that recruit Nef are more likely to recruit dynamin2, suggesting that Nef recruitment to CME sites promotes CME site maturation to ensure high efficiency in host protein downregulation. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.18.537383v1?rss=1 Authors: Wang, Y.-J., Seibert, H., Ahn, L. Y., Schaffer, A. E., Mu, T.-W. Abstract: Recent advances in genetic diagnosis identified variants in genes encoding GABAA receptors as causative for genetic epilepsy. Here, we selected eight disease-associated variants in the alpha1 subunit of GABAA receptors causing mild to severe clinical phenotypes and showed that they are loss of function, mainly by reducing the folding and surface trafficking of the alpha1 protein. Furthermore, we sought client protein-specific pharmacological chaperones to restore the function of pathogenic receptors. Applications of positive allosteric modulators, including Hispidulin and TP003, increase the functional surface expression of the alpha1 variants. Mechanism of action study demonstrated that they enhance the folding and assembly and reduce the degradation of GABAA variants without activating the unfolded protein response in HEK293T cells and human iPSC-derived neurons. Since these compounds cross the blood-brain barrier, such a pharmacological chaperoning strategy holds great promise to treat genetic epilepsy in a GABAA receptor-specific manner. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.18.537310v1?rss=1 Authors: Stamatiou, K., Huguet, F., Spanos, C., Rappsilber, J., Vagnarelli, P. Abstract: Background: The proliferation antigen Ki-67 has been widely used in clinical settings for cancer staging for many years but investigations on its biological functions have lagged. Recently, Ki-67 was shown to regulate both the composition of the chromosome periphery and chromosome behaviour in mitosis as well as to play a role in heterochromatin organisation and gene transcription. However, a role for Ki-67 in regulating cell cycle progression has never been reported. The progress towards understanding Ki-67 function have been limited by the tools available to deplete the protein coupled to its abundance and fluctuation during the cell cycle. Results: Here we have used an auxin-inducible degron tag (AID) to achieve a rapid and homogeneous degradation of Ki-67 in HCT116 cells. This system, coupled with APEX2 proteomics and phospho-proteomics approaches, allowed us to show for the first time that Ki-67 plays a role during DNA replication. In its absence, DNA replication is severely delayed, the replication machinery is unloaded, causing DNA damage that is not sensed by the canonical pathways and dependant on HUWE1 ligase. This leads to replication and sister chromatids cohesion defects, but it also triggers an interferon response mediated by the cGAS/STING pathway in all the cell lines tested. Conclusions: We have unveiled a new function of Ki-67 in DNA replication and genome maintenance that is independent of its previously known role in mitosis and gene regulation. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.19.537536v1?rss=1 Authors: Opalko, H. E., Geng, S., Hall, A. R., Vavylonis, D., Moseley, J. B. Abstract: Pattern forming networks have diverse roles in cell biology. Rod-shaped fission yeast cells use pattern formation to control the localization of mitotic signaling proteins and the cytokinetic ring. During interphase, the kinase Cdr2 forms membrane-bound multiprotein complexes termed nodes, which are positioned in the cell middle due in part to the node inhibitor Pom1 enriched at cell tips. Node positioning is important for timely cell cycle progression and positioning of the cytokinetic ring. Here, we combined experimental and modeling approaches to investigate pattern formation by the Pom1-Cdr2 system. We found that Cdr2 nodes accumulate near the nucleus, and Cdr2 undergoes nucleocytoplasmic shuttling when cortical anchoring is reduced. We generated particle-based simulations based on tip inhibition, nuclear positioning, and cortical anchoring. We tested model predictions by investigating Pom1-Cdr2 localization patterns after perturbing each positioning mechanism, including in both anucleate and multinucleated cells. Experiments show that tip inhibition and cortical anchoring alone are sufficient for the assembly and positioning of nodes in the absence of the nucleus, but that the nucleus and Pom1 facilitate the formation of unexpected node patterns in multinucleated cells. These findings have implications for spatial control of cytokinesis by nodes and for spatial patterning in other biological systems. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.18.537395v1?rss=1 Authors: Castello-Serrano, I., Heberle, F. A., Diaz-Rohrer, B., Ippolito, R., Shurer, C. R., Lujan, P., Campelo, F., Levental, K. R., Levental, I. Abstract: The organelles of eukaryotic cells maintain distinct protein and lipid compositions required for their specific functions. The mechanisms by which many of these components are sorted to their specific locations remain unknown. While some motifs mediating subcellular protein localization have been identified, many membrane proteins and most membrane lipids lack known sorting determinants. A putative mechanism for sorting of membrane components is based on membrane domains known as lipid rafts, which are laterally segregated nanoscopic assemblies of specific lipids and proteins. To assess the role of such domains in the secretory pathway, we applied a robust tool for synchronized secretory protein traffic (RUSH, Retention Using Selective Hooks) to protein constructs with defined affinity for raft phases. These constructs consist solely of single-pass transmembrane domains (TMDs) and, lacking other sorting determinants, constitute probes for membrane domain-mediated trafficking. We find that while raft affinity can be sufficient for steady-state PM localization, it is not sufficient for rapid exit from the endoplasmic reticulum (ER), which is instead mediated by a short cytosolic peptide motif. In contrast, we find that Golgi exit kinetics are highly dependent on raft affinity, with raft preferring probes exiting Golgi ~2.5-fold faster than probes with minimal raft affinity. We rationalize these observations with a kinetic model of secretory trafficking, wherein Golgi export can be facilitated by protein association with raft domains. These observations support a role for raft-like membrane domains in the secretory pathway and establish an experimental paradigm for dissecting its underlying machinery. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.18.537291v1?rss=1 Authors: Sahu, B. S., Mukherjee, M., Mukherjee, C., Ghosh, V., Jain, A., sadhukhan, s., Dagar, s. Abstract: Dense core vesicles (DCVs) and synaptic vesicles (SVs) are specialised secretory vesicles (SSVs) in neurons/neuroendocrine cells harbouring cargo whose abnormal release is associated with pathophysiology. Endoplasmic Reticulum (ER) stress and inter-organellar communication are also associated with disease biology. In pursuit of investigating the cell physiological consequences arising from the crosstalk of a stressed ER and DCVs, ER stress was modelled in PC12 neuroendocrine cells using Thapsigargin (Tg). DCV exocytosis was severely compromised in ER-stressed PC12 cells, reversed by Docosahexaenoic acid (DHA). Experiments with Tunicamycin(Tm), an independent ER stressor, yielded similar results. Concurrently, ER stress caused impaired DCV exocytosis also in INS-1 cells. Molecular analysis revealed blunted SNAP25 expression, potentially attributed to augmented levels of ATF4 (a well-known CREB inhibitor) and its transcriptional regulator CREB (also known to regulate key granulogenic players Chromogranin A, Secretogranin II). Our studies revealed severe defects in DCV exocytosis in ER-stressed cells for the first time, mediated by reduced levels of key 'exocytotic' and 'granulogenic' switches regulated via the CREB/ATF4/eIF2 axis. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.19.537530v1?rss=1 Authors: Khan, M. M., Zukowska, J., Jung, J., Galea, G., Tuechler, N., Halavatyi, A., Tischer, C., Haberkant, P., Stein, F., Jung, F., Landry, J., Khan, A. M., Oorschot, V., Becher, I., Neumann, B., Muley, T., Winter, H., Duerr, J., Mall, M., Savitski, M., Pepperkok, R. Abstract: Excessive deposition of fibrillar collagen in the interstitial extracellular matrix (ECM) of human lung tissue causes fibrosis, which can ultimately lead to organ failure. Despite our understanding of the molecular mechanisms underlying the disease, a cure for pulmonary fibrosis has not yet been found. In this study, we screened an FDA-approved drug library containing 712 drugs and found that Dextromethorphan (DXM), a cough expectorant, significantly reduces the amount of excess fibrillar collagen deposited in the ECM in in-vitro cultured primary human lung fibroblasts (NHLF) and ex-vivo cultured human precision-cut lung slice (hPCLS) models of lung fibrosis. Reduced extracellular fibrillar collagen levels in the ECM upon DXM treatment are due to a reversible trafficking inhibition of collagen type I (COL1) in the endoplasmic reticulum (ER) in TANGO1 and HSP47 positive structures. Mass spectrometric analysis shows that DXM causes hyper-hydroxylation of proline and lysine residues on Collagen (COL1, COL3, COL4, COL5, COL7, COL12) and Latent-transforming growth factor beta-binding protein (LTBP1 and LTBP2) peptides coinciding with their secretion block. In addition, thermal proteome profiling of cells treated with DXM shows increased thermal stability of prolyl- hydroxylases such as P3H2, P3H3, P3H4, P4HA1 and P4HA2, suggesting a change in activity. Transcriptome analysis of pro-fibrotic stimulated NHLFs and hPCLS upon DXM treatment showed activation of an anti-fibrotic program via regulation of pathways such as those involved in the MMP-ADAMTS axis, WNT, and fibroblast-to-myofibroblast differentiation. Taken together, the data obtained from both in-vitro and ex-vivo models of fibrogenesis show that Dextromethorphan has potent anti-fibrotic activity by efficient inhibition of COL1 membrane trafficking in the ER. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.19.537577v1?rss=1 Authors: Laban, H., Froemel, T., Fleming, I., Benz, P. M. Abstract: Macrophage polarization plays an important role in tissue regeneration. Numerous factors and signaling molecules affect polarization processes. Here we investigated the consequences of the genetic deletion of vasodilator-stimulated phosphoprotein (VASP), which increases macrophage M1 polarization through augmented signal transducer and activator of transcription 1 (STAT1) signaling, and AMP-activated protein kinase (AMPK), which attenuates inflammation by inhibiting STAT1 expression and signaling. While a basal activity of AMPK (phosphorylation on Thr172) was detected in macrophages from wild-type mice, AMPK phosphorylation was significantly reduced in VASP-deficient M1 macrophages in vitro and the expression of the pro-inflammatory cytokines TNF and IL-1{beta} was increased in these cells. Consistent with the role of AMPK in macrophage phagocytosis, VASP-/- macrophage phagocytosis was also significantly impaired. Interestingly, impaired phagocytosis could be rescued by exogenous activation of AMPK. Mechanistically, we found that VASP binds directly to protein phosphatase 1 regulatory subunit 6 (PP1-R6) and we hypothesize that VASP-binding to PP1-R6/PP1 limits the PP1-dependent de-phosphorylation of AMPK in wild-type cells. Conversely, AMPK dephosphorylation by the PP1-R6/PP1 complex is enhanced in the absence of VASP. In summary, we have identified a link between VASP and AMP-activated protein kinase (AMPK) activity, which may contribute to the pro-inflammatory phenotype of VASP-deficient macrophages. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.18.537143v1?rss=1 Authors: Manolis, D., Hasan, S., Ettelaie, C., Maraveyas, A., O'Brien, D. P., Kessler, B. M., Kramer, H. B., Nikitenko, L. L. Abstract: Background: G protein-coupled receptor (GPCR) calcitonin receptor-like receptor (CLR) signalling is implicated in skin-related and cardiovascular diseases, migraine and cancer. However, beyond its agonists and receptor activity-modifying proteins (RAMPs), proteins which bind to CLR and define its properties in primary human cells remain insufficiently understood. Aim: We aimed to profile the CLR interactome in primary human dermal lymphatic endothelial cells (HDLEC), where this GPCR is expressed. Materials and methods: Immunoprecipitation (IP) of core- and terminally-glycosylated CLR from primary in vitro cultured HDLEC was conducted using rabbit polyclonal anti-human CLR serum (with pre-immune serum serving as a control) and confirmed by immunoblotting. Total HDLEC and co-immunoprecipitated CLR proteomes were analysed by label-free quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS). Quantitative in-situ proximity ligation assay (PLA) using ZEISS LSM 710 confocal microscope and ZEN Blue 3.0 and Image J software was performed to confirm LC-MS/MS findings. All experiments were repeated at least three times (biological replicates). For statistical analysis of PLA data, distribution was analysed using Shapiro-Wilk normality test followed by an unpaired t-test or Mann-Whitney test with a p-value of less than or equal to 0.05 interpreted as significant. For MS data of CLR IP samples, statistical analysis was performed using t-test with a permutation-based false discovery rate (FDR)-adjusted p-value of less than or equal to 0.006 interpreted as significant. Results: A total of 4,902 proteins were identified and quantified by LC-MS/MS in primary HDLEC and 46 were co-immunoprecipitated with CLR (p less than 0.006). Direct interaction with the GPCR was confirmed for five of these by PLA (p less than 0.01). Conclusions: This is the first study of its kind to identify novel binding partners of CLR expressed in primary human cells. Our integrative quantitative approach, combining immunoprecipitation of core- and terminally-glycosylated CLR, LC-MS/MS, and PLA, could be applied in a similar fashion to study its interactome in a variety of human cells and tissues, and its contribution to a range of diseases, where the role of this GPCR is implicated. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.19.536389v1?rss=1 Authors: Garcia, M.-I., Dinsart, G., Leprovots, M., Lefort, A., Libert, F., Quesnel, Y., Veiten, A., Vassart, G., Huysseune, S., Parmentier, M. Abstract: The gastrointestinal epithelium constitutes a chemosensory system for microbiota-derived metabolites such as Short Chain Fatty Acids (SCFA). In this study, we investigated spatial distribution of Olfr78, one of the SCFA receptors, in the mouse intestine and studied the transcriptome of colon enteroendocrine cells expressing Olfr78. The receptor is principally detected in the enterochromaffin and L subtypes in the proximal and distal colon, respectively. Using the Olfr78-GFP and VilCre/Olfr78flox transgenic mouse lines, we reveal that loss of epithelial Olfr78 results in impaired enterochromaffin cell differentiation, blocking cells in an undefined secretory lineage state. This is accompanied by dysbiosis, characterized by an increased Firmicutes/Bacteroidetes ratio, as well as a less efficient antioxidant system in colon crypts. Using organoid cultures, we further show that maintenance of enterochromaffin cells involves activation of the Olfr78 receptor via the SCFA ligand acetate. Altogether, this work provides evidence that Olfr78 contributes to colon homeostasis by regulating enterochromaffin cell differentiation. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.17.537198v1?rss=1 Authors: Yeh, A. R., Hoeprich, G. J., Goode, B. L., Martin, A. C. Abstract: Actin networks undergo rearrangements that influence cell and tissue shape. Actin network assembly and organization is regulated in space and time by a host of actin binding proteins. The Drosophila Synaptotagmin-like protein, Bitesize (Btsz), is known to organize actin at epithelial cell apical junctions in a manner that depends on its interaction with the actin-binding protein, Moesin. Here, we showed that Btsz functions in actin reorganization at earlier, syncytial stages of Drosophila embryo development. Btsz was required for the formation of stable metaphase pseudo-cleavage furrows that prevented spindle collisions and nuclear fallout prior to cellularization. While previous studies focused on Btsz isoforms containing the Moesin Binding Domain (MBD), we found that isoforms lacking the MBD also function in actin remodeling. Consistent with this, we found that the C-terminal half of BtszB cooperatively binds to and bundles F-actin, suggesting a direct mechanism for Synaptotagmin-like proteins regulating actin organization during animal development. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.16.537086v1?rss=1 Authors: Zhang, J., Qiu, R., Bieger, B. D., Oakley, E., Oakley, B. R., Egan, M. J., Xiang, X. Abstract: Functions of protein SUMOylation remain incompletely understood in different cell types. The budding yeast SUMOylation machinery interacts with LIS1, a protein critical for dynein activation, but dynein-pathway components were not identified as SUMO-targets in the filamentous fungus Aspergillus nidulans. Via A. nidulans forward genetics, here we identified ubaBQ247*, a loss-of-function mutation in a SUMO-activation enzyme UbaB. Colonies of the ubaBQ247*, {triangleup}ubaB and {triangleup}sumO mutants looked similar and less healthy than the wild-type colony. In these mutants, about 10% of nuclei are connected by abnormal chromatin bridges, indicating the importance of SUMOylation in the completion of chromosome segregation. Nuclei connected by chromatin bridges are mostly in interphase, suggesting that these bridges do not prevent cell-cycle progression. UbaB-GFP localizes to interphase nuclei just like the previously studied SumO-GFP, but the nuclear signals disappear during mitosis when the nuclear pores are partially open, and the signals reappear after mitosis. The nuclear localization is consistent with many SUMO-targets being nuclear proteins, for example, topoisomerase II whose SUMOylation defect gives rise to chromatin bridges in mammalian cells. Unlike in mammalian cells, however, loss of SUMOylation in A. nidulans does not apparently affect the metaphase-to-anaphase transition, further highlighting differences in the requirements of SUMOylation in different cell types. Finally, loss of UbaB or SumO does not affect dynein- and LIS1-mediated early-endosome transport, indicating that SUMOylation is unnecessary for dynein or LIS1 function in A. nidulans. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.17.537129v1?rss=1 Authors: Hardt, R., Dehghani, A., Schoor, C., Goedderz, M., Cengiz Winter, N., Ahmadi, S., Sharma, R., Schork, K., Eisenacher, M., Gieselmann, V., Winter, D. Abstract: Oligodendrocytes are generated via a two-step mechanism from pluripotent neural stem cells (NSCs): after differentiation of NSCs to oligodendrocyte precursor/NG2 cells (OPCs), they further develop into mature oligodendrocytes. The first step of this differentiation process is only incompletely understood. In this study, we utilized the neurosphere assay to investigate NSC to OPC differentiation in a time course-dependent manner by mass spectrometry-based (phospho-) proteomics. We identify double cortin like kinase 1 (Dclk1) as one of the most prominently regulated proteins in both datasets, and show that it undergoes a gradual transition between its short/long isoform during NSC to OPC differentiation. This is regulated by phosphorylation of its SP-rich region, resulting in inhibition of proteolytic Dclk1 long cleavage, and therefore Dclk1 short generation. Through interactome analyses of different Dclk1 isoforms by proximity biotinylation, we characterize their individual putative interaction partners and substrates. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.16.537099v1?rss=1 Authors: Pei, X., Zheng, F., Li, Y., Lin, Z., Zhang, Y., Han, X., Feng, Y., Li, F., Yang, J., Li, T., Tian, Z., Cao, K., Ren, D., Li, C. Abstract: Idiopathic pulmonary fibrosis (IPF) is marked with the replacement of normal alveolar tissue by thicker and harder fibrous material, damaged exchange ability. Currently, nintedanib and pirfenidone, are the only FDA-approved drugs with limited efficacy for IPF, which indicated an urgent need to explore new therapies. Disulfiram (DSF), an acetaldehyde dehydrogenase inhibitor, used as anti-alcohol treatment. Despite reported with anti-hepatic fibrosis effect of DSF, the underlying mechanism remains unclear. In our study, DSF exhibited regulative impact on abnormal proliferation, EMT and ECM production in cell models of IPF including primary DHLF-IPF cells and TGF-{beta}1-stimulated A549 cells. The absence of COX-2 was restored by DSF treatment, together with elevated prostaglandin biosynthesis both in vitro and in vivo models of IPF. Furthermore, the anti-fibrotic effect of DSF was impeded with COX-2 knockdown or pharmacological inhibition in TGF-{beta}1-stimulated A549 cells, however, exogenous PGE2 reclaimed with anti-EMT function. In established animal model of IPF, DSF ameliorated declined lung function and histopathological changes, and restrained the lung hydroxyproline content. Together, these findings suggest that the anti-fibrotic effect of DSF was achieved through re-activation of COX-2 mediated PGE2 biosynthesis. The above results suggest that DSF can be applied therapeutically in fibrotic conditions. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.17.537087v1?rss=1 Authors: Birnbaum, R., Biswas, J., Singer, R., Sharp, D. Abstract: Cell motility requires strict spatiotemporal control of protein expression. During cell migration, mRNA localization and local translation in subcellular areas like the leading edge and protrusions are particularly advantageous for regulating the reorganization of the cytoskeleton. Fidgetin-Like 2 (FL2), a microtubule severing enzyme (MSE) that restricts migration and outgrowth, localizes to the leading edge of protrusions where it severs dynamic microtubules. FL2 is primarily expressed during development but in adulthood, is spatially upregulated at the leading edge minutes after injury. Here, we show mRNA localization and local translation in protrusions of polarized cells are responsible for FL2 leading edge expression after injury. The data suggests that the RNA binding protein IMP1 is involved in the translational regulation and stabilization of FL2 mRNA, in competition with the miRNA let-7. These data exemplify the role of local translation in microtubule network reorganization during migration and elucidate an unexplored MSE protein localization mechanism. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.17.537214v1?rss=1 Authors: Sylvester-Armstrong, K. R., Reeder, C. F., Powell, A., Becker, M. W., Hagan, D. W., Chen, J., Mathews, C. E., Wasserfall, C. H., Atkinson, M. A., Egerman, R., Phelps, E. A. Abstract: Pancreatic beta cells are among the slowest replicating cells in the human body. Human beta cells usually do not increase in number with exceptions being during the neonatal period, in cases of obesity, and during pregnancy. This project explored maternal serum for stimulatory potential on human beta cell proliferation and insulin output. Gravid, full-term women who were scheduled to undergo cesarean delivery were recruited for this study. A human beta cell line was cultured in media supplemented with serum from pregnant and non-pregnant donors and assessed for differences in proliferation and insulin secretion. A subset of pregnant donor sera induced significant increases in beta cell proliferation and insulin secretion. Pooled serum from pregnant donors also increased proliferation in primary human beta cells but not primary human hepatocytes indicating a cell-type specific effect. This study suggests stimulatory factors in human serum during pregnancy could provide a novel approach for human beta cell expansion. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.17.535593v1?rss=1 Authors: Aureille, J., Barnett, S., Arnal, I., Lafanechere, L., Low, B. C., Kanchanawong, P., Mogilner, A., Bershadsky, A. Abstract: Microtubules regulate cell polarity and migration by local activation of focal adhesion turnover, but the mechanism of this process is insufficiently understood. Molecular complexes containing KANK family proteins connect microtubules with the major component of focal adhesions, talin. Local optogenetic activation of KANK1-mediated links which promoted microtubule targeting to individual focal adhesion resulting in its centripetal sliding and rapid disassembly. The sliding is preceded by a local increase of traction force due to accumulation of myosin-II and actin in the proximity of the focal adhesion. Knockdown of Rho activator GEF-H1 prevented development of traction force and abolished sliding and disassembly of focal adhesion upon KANK activation. Other players participating in microtubule-driven KANK-dependent focal adhesion disassembly include kinases ROCK and PAK, as well as microtubules/focal adhesions associated proteins Kinesin-1, APC and TAT. Finally, we propose a physical model of a microtubule-driven focal adhesion disruption involving local GEF-H1/RhoA/ROCK dependent activation of contractility which is consistent with experimental data. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.17.537183v1?rss=1 Authors: Stephan, T., Stoldt, S., Barbot, M., Carney, T. D., Lange, F., Bates, M., Bou Dib, P., Shcherbata, H., Meinecke, M., Riedel, D., Dennerlein, S., Rehling, P., Jakobs, S. Abstract: Mitochondria are essential eukaryotic double-membrane organelles. The convoluted mitochondrial inner membrane forms highly organized invaginations, termed cristae, which are crucial for energy metabolism. Cristae formation requires MICOS, a conserved hetero-oligomeric inner membrane complex. The MICOS core subunit MIC10 is a small transmembrane protein that oligomerizes through highly conserved glycine-rich motifs to control cristae formation. Sequence alignments show that D. melanogaster exhibits three MIC10-like proteins with different tissue-specific expression patterns. Here, we show that the ubiquitously expressed Dmel_CG41128/MINOS1b/DmMIC10b is the major MIC10 orthologue in flies. Loss of DmMIC10b disturbs cristae architecture of mitochondria and reduces the life-span and fertility of flies. Moreover, using fluorescence nanoscopy and electron tomography, we demonstrate that despite its high similarity to the MIC10 proteins from yeast and humans, DmMIC10b exhibits the unique ability to polymerize into elongated filaments upon overexpression. DmMIC10b filaments form bundles which accumulate in the intermembrane space and alter the shape of mitochondrial cristae membranes. We show that the formation of the filaments relies on conserved glycine and cysteine residues and is suppressed by co-expression of other MICOS proteins. Thereby, our findings provide new insights into the regulation of MICOS in flies. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.17.537227v1?rss=1 Authors: Jimenez, A. J., Perez, F. Abstract: Cellular processes are regulated by the formation of specific membrane domains with different lipid and protein compositions. Small GTPases play a role in symmetry breaking and compartmentalization, with early endosomes presenting Rab5-enriched domains that regulate vesicle tethering and fusion. Rabaptin5, which binds activated Rab5 and forms dimers, is essential for endosome fusion and promotes Rab5 recruitment to membranes. Liquid-liquid phase separation (LLPS) is a mechanism for the biogenesis and maintenance of membrane-less organelles and intracellular organization. We show that several partners of Rab proteins, such as Rabaptin5, have phase-separation properties. In particular, Rabaptin5 form condensates close to membranes, while promoting the enrichment of several Rabs involved in early steps of endocytosis. We propose that phase separation of Rab partners ensures efficient recruitment of their respective Rabs and domain formation and maintenance. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.17.537109v1?rss=1 Authors: Meza Guzman, L. G., Hyland, C. D., Bidgood, G. M., Leong, E., Shen, Z., Goh, W., Kratina, T., Rautela, J., Vince, J., Nicholson, S., Huntington, N. Abstract: The clinical development of Natural Killer (NK) cell-mediated immunotherapy marks a milestone in the development of new cancer therapies and has gained traction due to the intrinsic ability of the NK cell to target and kill tumour cells. To fully harness the tumour killing ability of NK cells, we need to improve NK cell persistence and overcome suppression of NK cell activation in the tumour microenvironment. The trans-membrane, protein tyrosine phosphatase CD45, regulates NK cell homeostasis, with genetic loss of CD45 in mice resulting in increased numbers of mature NK cells [1-3]. This suggests that CD45-deficient NK cells might display enhanced persistence following adoptive transfer. However, here we demonstrated that adoptive transfer of CD45-deficiency did not enhance NK cell persistence in mice, and instead, the homeostatic disturbance of NK cells in CD45-deficient mice stemmed from a developmental defect in the common lymphoid progenitor population. The enhanced maturation within the CD45-deficient NK cell compartment was intrinsic to the NK cell lineage, and independent of the developmental defect. CD45 is not a conventional immune checkpoint candidate, as systemic loss is detrimental to T and B cell development [4-6], compromising the adaptive immune system. Nonetheless, this study suggests that inhibition of CD45 in progenitor or stem cell populations may improve the yield of in vitro generated NK cells for adoptive therapy. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.15.537011v1?rss=1 Authors: Mesquita, F. S., Abrami, L., Bracq, L., Panyain, N., Mercier, V., Kunz, B., Chuat, A., Carlevaro-Fita, J., Trono, D., van der Goot, F. G. Abstract: SARS-CoV-2 infection requires Spike protein mediating fusion between the viral and cellular membranes. The fusogenic activity of Spike requires its post-translational lipid modification by host S-acyltransferases, predominantly ZDHHC20. Previous observations indicate that SARS-CoV-2 infection augments the S-acylation of Spike when compared to transfection. Here, we find that SARS-CoV-2 infection triggers a change in the transcriptional start site of the zddhc20 gene, both in cells and in an in vivo infection model, resulting in a 67-amino-acid-long N-terminally extended protein with 37-times higher Spike acylating activity, leading to enhanced viral infectivity. Furthermore, we observed the same induced transcriptional change in response to other challenges, such as chemically induced colitis, indicating that SARS-CoV-2 hijacks an existing cell damage response pathway to generate more infectious viruses. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.15.536998v1?rss=1 Authors: Assou, S., Ahmed, E., Morichon, L., Nasri, A., Foisset, F., Bourdais, C., Gros, N., Wong, S., Petit, A. F., Vachier, I., Muriaux, D., Bourdin, A., De Vos, J. Abstract: Airway-liquid interface cultures of primary epithelial cells and of induced pluripotent stem cell-derived airway epithelial cells (ALI and iALI, respectively) are physiologically relevant models for respiratory virus infection studies because they can mimic the in vivo human bronchial epithelium. Here, we investigated gene expression profiles in human airway cultures (ALI and iALI models) infected or not with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) using publicly available and our own bulk and single-cell transcriptome datasets. SARS-CoV-2) infection significantly increased the expression of interferon-stimulated genes (IFI44, IFIT1, IFIT3, IFI35, IRF9, MX1, OAS1, OAS3 and ISG15) and inflammatory genes (NFKBIA, CSF1, FOSL1, IL32 and CXCL10) at day 4 post-infection, indicating activation of the interferon and immune responses to the virus. Extracellular matrix genes (ITGB6, ITGB1 and GJA1) also were altered in infected cells. Single-cell RNA sequencing data revealed that SARS-CoV-2 infection damaged the respiratory epithelium, particularly mature ciliated cells. The expression of genes encoding intercellular communication and adhesion proteins also was deregulated, suggesting a mechanism to promote shedding of infected epithelial cells. These data demonstrate that ALI/iALI models help to understand the airway epithelium response to SARS-CoV-2 infection and are a key tool for developing COVID-19 treatments. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.13.536796v1?rss=1 Authors: Lin, L. L., Torres, M., Pederson, B., Wang, H. H., Wei, X., Li, Z. J., Liu, X., Mao, H., Hanzel, M., Govek, E. E., Lu, Y., Wang, H., Zhao, Z., Hatten, M. E., Sun, S. E., Qi, L. Abstract: Despite recent advances in our understanding of the physiological importance of SEL1L-HRD1 endoplasmic reticulum (ER)-associated protein degradation (ERAD) using cell type-specific knockout (KO) mouse models, its relevance and importance in ataxia pathogenesis remain unknown. Here we show that loss of SEL1L-HRD1 ERAD complex interaction or function in Purkinje cells leads to cerebellar ataxia. Both homozygous knock-in (KI) mice carrying SEL1L variant p.Ser658Pro (S658P) and mice with Purkinje cell-specific deletion of SEL1L exhibit early-onset cerebellar ataxia, although disease severity and progression differ between the models. Structure-function analyses reveal that SEL1L S658P variant impairs, not abolishes, ERAD function by attenuating the interaction between SEL1L and HRD1. Proteomic screen of potential endogenous substrates leads to the identification of Astrotactin 1 and 2, two integral membrane proteins involved in neuronal function and development, whose maturation and biogenesis in the ER depend on SEL1L-HRD1 ERAD activity. These data demonstrate the pathophysiological importance of SEL1L-HRD1 interaction and function in the pathogenesis of cerebellar ataxia. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.12.536627v1?rss=1 Authors: Gilda, J. E., Nahar, A., Kasiviswanathan, D., Tropp, N., Gilinski, T., Lahav, T., Mandel-Gutfreund, Y., Park, S., Cohen, S. Abstract: Proteasome activity is crucial for cellular integrity, but how tissues adjust proteasome content in response to catabolic stimuli is uncertain. Here, we demonstrate that transcriptional coordination by multiple transcription factors is required to increase proteasome content and activate proteolysis in catabolic states. Using denervated mouse muscle as a model system for accelerated proteolysis in vivo, we reveal that a two-phase transcriptional program activates genes encoding proteasome subunits and assembly chaperones to boost an increase in proteasome content. Initially, gene induction is necessary to maintain basal proteasome levels, and in a more delayed phase (7-10 d after denervation) it stimulates proteasome assembly to meet cellular demand for excessive proteolysis. Intriguingly, the transcription factors PAX4 and -PALNRF-1 control the expression of proteasome among other genes in a combinatorial manner, driving cellular adaptation to muscle denervation. Consequently, PAX4 and -PALNRF-1 represent new therapeutic targets to inhibit proteolysis in catabolic diseases (e.g. type-2 diabetes, cancer). Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.13.536716v1?rss=1 Authors: Darmasaputra, G., Chuva de Sousa Lopes, S. M., Clevers, H. C., Galli, M. Abstract: Many plant and animal cells transition from canonical to non-canonical cell cycles during development, resulting in the formation of polyploid cells. Two types of non-canonical cell cycles exist: endoreplication, where cells increase their DNA content without entering M phase, and endomitosis, where cells enter M phase but exit prematurely. Although endoreplication has been extensively studied in plants and insects, much less is known on the regulation of endomitosis, which is the most common mode of polyploidization in mammals. In this study, we use fetal-derived human hepatocyte organoids (Hep-Org), to investigate how human hepatocytes initiate and execute endomitosis. We find that cells in endomitosis M phase have normal mitotic timings, but lose membrane anchorage to the midbody during cytokinesis, resulting in regression of the cytokinetic furrow and formation of binucleate cells. Using immunofluorescence, we find that three cortical anchoring proteins, RacGAP1, anillin, and citron kinase (CIT-K), lose their association with the cell cortex during cytokinetic regression. Moreover, reduction of WNT activity by withdrawal of CHIR99021, a GSK3 inhibitor, from the culturing medium increases the percentage of binucleated cells in Hep-Orgs. This effect is lost in organoids with mutations in the atypical E2F proteins, E2F7 and E2F8, which have been implicated in binucleation of rodent hepatocytes. Together, our results identify how human hepatocytes inhibit cell division in endomitosis, and highlight an evolutionary recurrent mechanism to initiate non-canonical cell cycles in mammals. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.13.536686v1?rss=1 Authors: Veerabhadraswamy, P., Belekar, P., Kothegala, L., Gandasi, N. R. Abstract: Type-2 diabetes (T2D) is characterized by high blood glucose due to compromised insulin secretion from pancreatic beta-cells. Beta-cells primarily comprise insulin-secreting large-dense-core-vesicles/insulin-secretory-granules (ISGs) and also multivesicular-bodies (MVBs). MVBs are vesicles of endosomal origin containing intraluminal vesicles, which upon fusion with the plasma membrane, secrete exosomes. These play a significant role in the physiology and pathology of T2D via intercellular communication. The role of MVBs and their influence on ISGs of beta-cells or their characterization is yet to be uncovered. In our study, we characterized the role of MVBs by comparing them to largely well-characterized ISGs in beta-cells. We compared the density, localization, and exocytosis of MVBs with ISGs in beta-cells. For this, we developed a novel probe where we exploit the efficiency of tetraspanins CD63 and CD151 to label the MVBs in beta-cells. We showed that the beta-cells have a significantly higher density of ISGs than MVBs. MVBs and ISGs are spatially localized apart within beta-cells. The proteins that localize with MVBs are different from the ones that localize with ISGs. Exocytosis of ISGs occurs at the periphery of the beta-cells and takes significantly lesser time when compared to exosome release, which is non-peripheral and takes a longer duration. Further, we also observed a significant reduction in the density of ISGs and MVBs in T2D patients' islets compared to healthy controls. Studying the effect of MVBs on insulin secretion in physiological and T2D conditions has huge potential. This study provides a strong basis to open new avenues for such future studies. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.13.536432v1?rss=1 Authors: Vinnai, B. A., Arianti, R., Györy, F., Fesüs, L., Kristof, E. Abstract: Brown adipose tissue (BAT) dissipates energy in the form of heat majorly via the mitochondrial uncoupling protein 1 (UCP1). The activation of BAT, which is enriched in the neck area and contains brown and beige adipocytes in humans, was considered as a potential therapeutic target to treat obesity. Therefore, finding novel agents that can stimulate the differentiation and recruitment of brown or beige thermogenic adipocytes are important subjects for investigation. The current study investigated how the availability of extracellular thiamine (vitamin B1), an essential cofactor of mitochondrial enzyme complexes that catalyze key steps in the catabolism of nutrients, affects the expression of thermogenic marker genes and proteins and subsequent functional parameters during ex vivo adipocyte differentiation. Therefore, we differentiated primary human adipogenic progenitors that were cultivated from subcutaneous (SC) or deep neck (DN) adipose tissues in the presence of gradually increasing thiamine concentrations during their 14 day long differentiation program. Higher thiamine levels resulted in increased expression of thiamine transporter 1 and 2 both at mRNA and protein levels in human neck area-derived adipocytes. Gradually increasing concentrations of thiamine led to increased basal, cAMP-stimulated, and proton-leak respiration along with elevated mitochondrial biogenesis of the differentiated adipocytes. The extracellular thiamine availability during adipogenesis determined the expression levels of UCP1, PGC1a, CKMT2, and other browning-related genes and proteins in primary SC and DN-derived adipocytes in a concentration-dependent manner. Providing abundant amounts of thiamine further increased the thermogenic competency of the adipocytes. Our study raises the possibility of a novel strategy with long-term thiamine supplementation, which can enhance the thermogenic competency of differentiating neck area-derived adipocytes for preventing or combating obesity. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.13.536664v1?rss=1 Authors: Yi, X., Chen, P., Kang, C., Li, D., Yang, Y., Cai, H., Li, B., Wu, C. Abstract: Cell migration towards stiff substrates has been coined as durotaxis and implicated in development, wound healing and cancer, where complex interplays between immune and non-immune cells are present. Compared to the emerging mechanisms underlying the strongly adhesive mesenchymal durotaxis, little is known about whether immune cells - migrating in amoeboid mode - could follow mechanical cues. Here we develop an imaging-based confined migration device that provides stiffness gradient for cell migration. By tracking live cell trajectory and analyzing the directionality T cells and neutrophils, we observe that amoeboid cells can durotax. We further delineate the underlying mechanism to involve non-muscle myosin IIA (NMIIA) polarization towards the soft-matrix-side but may not require differential actin flow up- or down-stiffness gradient. Using the protista Dictyostelium, we further demonstrate the evolutionary conservation of amoeboid durotaxis. Finally, these experimental phenomena are theoretically captured by an active gel model capable of mechanosensing. Collectively, these results may shed new lights on immune surveillance and recently identified confined migration of cancer cells, within the tumor microenvironment or the inflamed fibrotic tissues. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.13.536717v1?rss=1 Authors: Kang, S. W. S., Cunningham, R. P., Miller, C. B., Cultraro, C. M., Hernandez, J., Jenkins, L. M., Lobanov, A., Cam, M., Porat-Shliom, N. Abstract: In the liver, mitochondria are exposed to different concentrations of nutrients due to their spatial positioning across the periportal (PP) and pericentral (PC) axis. How these mitochondria sense and integrate these signals to respond and maintain homeostasis is not known. Here, we combined intravital microscopy, spatial proteomics, and functional assessment to investigate mitochondrial heterogeneity in the context of liver zonation. We found that PP and PC mitochondria are morphologically and functionally distinct; beta-oxidation and mitophagy were elevated in PP regions, while lipid synthesis was predominant in the PC mitochondria. In addition, comparative phosphoproteomics revealed that mitophagy and lipid synthesis are regulated by phosphorylation in a zonated manner. Furthermore, we demonstrated that acute pharmacological modulation of nutrient sensing through AMPK and mTOR shifted mitochondrial phenotypes in the PP and PC regions of the intact liver. This study highlights the role of protein phosphorylation in mitochondrial structure, function, and overall homeostasis in hepatic metabolic zonation. The findings have important implications for liver physiology and disease. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.12.536672v1?rss=1 Authors: Fujiwara, Y., Saito, K., Sun, F., Inoue, E., Schimenti, J., Okada, Y., Handel, M. A. Abstract: An unbiased screen for discovering novel genes for fertility identified the spcar3, spermatocyte arrest 3, mutant phenotype. The spcar3 mutation identified a new allele of the Setx gene, encoding senataxin, a DNA/RNA helicase that regulates transcription termination by resolving DNA/RNA hybrid R-loop structures. Although mutations in the human SETX gene cause neural disorders, Setxspcar3 mutant mice do not show any apparent neural phenotype, but instead exhibit male infertility and female subfertility. Histology of the Setxspcar3 mutant testes revealed absence of spermatids and mature spermatozoa in the seminiferous tubules. Cytological analysis of chromosome spread preparations of the Setxspcar3 mutant spermatocytes revealed normal synapsis, but aberrant DNA damage in the autosomes, and defective formation of the sex body. Furthermore, Setxspcar3 testicular cells exhibited abnormal accumulation of R-loops compared to wild type testicular cells. Transient expression assays identified regions of the senataxin protein required for sub-nuclear localization. Together, these results not only confirm that senataxin is required for normal meiosis and spermatogenesis but also provide a new resource for determination of its role in maintaining R-loop formation and genome integrity. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.13.536018v1?rss=1 Authors: Stalder, D., Yakunin, I., Gershlick, D. C. Abstract: ACBD3 is a protein known to localise to the Golgi apparatus, and recruits various proteins to the Golgi, including PI4KIIIbeta, but the mechanism of its recruitment has remained unclear. This study demonstrates there are two mechanisms for ACBD3 recruitment to the Golgi. First, we identified that an MWT374-376 motif in the unique region upstream of the GOLD domain in ACBD3 is essential for Golgi localisation. Second, we use unbiased proteomics to demonstrate that ACBD3 interacts with SCFD1, a Sec1/Munc-18 (SM) protein, and a SNARE protein, SEC22B. CRISPR-KO of SCFD1 causes ACBD3 to become cytosolic. We also found that ACBD3 is redundantly recruited to the Golgi apparatus by two golgins, golgin-45 and giantin, which bind to ACBD3 through interaction with the MWT374-376 motif. Taken together, our results demonstrate that ACBD3 is recruited to the Golgi in a two-step sequential process, with the SCFD1-mediated interaction occurring upstream of the interaction with the golgins. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.12.536533v1?rss=1 Authors: Domenig, S. A., Lenardic, A., Zvick, J., Tarnowska-Sengul, M., Bundschuh, N., Bacchin, G., Ghosh, A., Bar-Nur, O. Abstract: Satellite cells, the stem cells of skeletal muscle tissue, hold a prodigious regeneration capacity. However, low satellite cell yield from autologous or donor-derived muscles precludes adoption of satellite cell transplantation for the treatment of muscle diseases including Duchenne muscular dystrophy (DMD). To address this limitation, here we investigated whether sufficient quantity of satellite cells can be produced in allogeneic or xenogeneic animal hosts. First, we report on exclusive satellite cell production in intraspecies mouse chimeras by injection of CRISPR/Cas9-corrected DMD-induced pluripotent stem cells (iPSCs) into blastocysts carrying an ablation system of host Pax7+ satellite cells. Additionally, injection of genetically-corrected DMD-iPSCs into rat blastocysts produced interspecies rat-mouse chimeras harboring mouse muscle stem cells that efficiently restored dystrophin expression in DMD mice. This study thus provides a proof-of-principle for the generation of therapeutically-competent stem cells between divergent species, raising the possibility of procuring human stem cells in large animals for regenerative medicine purposes. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.12.536397v1?rss=1 Authors: Huang, Y., Dong, X., Sun, S. Y., Lim, T.-K., Lin, Q., He, C. Y. Abstract: Arl13b and Arl3 are ciliary GTPases implicated in human Joubert Syndrome, affecting ciliary membrane and axoneme organization. Although the mechanism of Arl13b as a guanine nucleotide exchange factor (GEF) of Arl3 and the function of Arl13b and Arl3 in ciliary membrane protein transport are well established, their role in axoneme biogenesis is unclear. In Trypanosoma brucei, TbArl13 acts as a GEF for two distinct TbArl3 proteins, TbArl3A and TbArl3C. Here, we identified the T. brucei homolog of ODA16, a cargo adapter facilitating intraflagellar transport (IFT) of motile ciliary components, as an effector of both TbArl3A and TbArl3C. Depletion of TbArl3 GTPases stabilized TbODA16 interaction with IFT, while active TbArl3 variants displaced TbODA16 from IFT, demonstrating a mechanism of TbArl3 in motile ciliary cargo transport. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.12.536536v1?rss=1 Authors: Di Biagi, L., Malacaria, E., Aiello, F. A., Valenzisi, P., Marozzi, G., Franchitto, A., Pichierri, P. Abstract: Replication gaps can arise as a consequence of perturbed DNA replication and their accumulation might undermine the stability of the genome. Loss of RAD52, a protein involved in the regulation of fork reversal, promotes accumulation of parental ssDNA gaps during replication perturbation. Here, we demonstrate that this is due to the engagement of Polalpha downstream of the extensive degradation of perturbed replication forks after their reversal, and is not dependent on PrimPol. Polalpha is hyper-recruited at parental ssDNA in the absence of RAD52, and this recruitment is dependent on fork reversal enzymes and RAD51. Of note, we report that the interaction between Polalpha and RAD51 is stimulated by RAD52 inhibition, and Polalpha-dependent gap accumulation requires nucleation of RAD51 suggesting that it occurs downstream strand invasion. Altogether, our data indicate that RAD51-Polalpha-dependent repriming is essential to promote fork restart and limit DNA damage accumulation when RAD52 function is disabled. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.12.536514v1?rss=1 Authors: Awatade, N. T., Reid, A. T., Nichol, K. S., Budden, K. F., Veerati, P. C., Pathinayake, P. S., Grainge, C. L., Hansbro, P. M., Wark, P. A. Abstract: Introduction: Primary air liquid interface (ALI) cultures of bronchial epithelial cells are used extensively to model airway responses. A recent advance is the development of conditional reprogramming that enhances proliferative capability. Several different media and protocols are utilized, yet even subtle differences may influence cellular responses. We compared the morphology and functional responses, including innate immune responses to rhinovirus infection in conditionally reprogrammed primary bronchial epithelial cells (pBECs) differentiated using two commonly used culture media. Methods: pBECs from healthy participants (n = 5) were CR using gamma-irradiated 3T3 fibroblasts and Rho Kinase inhibitor. CRpBECs were differentiated at ALI in either PneumaCultTM (PN-ALI) or Bronchial Epithelial Growth Medium (BEGM)-based differentiation media (BEBM:DMEM, 50:50, LonzaTM) - (AB-ALI) for 28 days. Transepithelial electrical resistance (TEER), immunofluorescence, histology, cilia activity, ion channel function, and expression of cell markers were analyzed. Viral load was assessed by RT-qPCR and anti-viral factors quantified by Legendplex following Rhinovirus-A1b (RVA1b) infection. Results: CRpBECs differentiated in PneumaCultTM were smaller and had a lower TEER and cilia beat frequency (CBF) compared to BEGM media. PneumaCultTM media cultures exhibited significantly increased FOXJ1 expression, more ciliated cells with a larger active area, increased intracellular mucins, and increased calcium-activated chloride channel current. However, there were no significant changes in viral RNA or host antiviral responses. Conclusion: There are distinct structural and functional differences in CRpBECs cultured in the two commonly used ALI differentiation media. Such factors need to be taken into consideration when designing and comparing CRpBECs ALI experiments. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.10.536321v1?rss=1 Authors: Hu, Y., Cheng, B., Zhang, J., Shen, Q., Sun, Z., Luo, Y. Abstract: Ferroptosis contribute to temporomandibular joint osteoarthritis (TMJOA) lesion development is still poorly understood. In this study, we used different TMJOA animal models to detect whether ferroptosis is related to onset of TMJOA which modelling by monosodium iodoacetate (MIA), IL-1{beta}, occlusion disorder (OD) and unilateral anterior crossbite (UAC). Immunohistochemical staining and Western blot analysis were used to detect ferroptosis proteins and cartilage degradation related protein expression. Our results revealed that lower level of ferroptosis-related proteins GPX4 in cartilage layer, but the level of ACSL4 and P53 increase in that of condyle. Injection of ferroptosis inhibitor liproxstatin-1 (Lip-1) effectively decrease ACSL4, P53 and TRF expression. In vitro, IL-1{beta} induced the reduction of cartilage extracellular matrix expression in mandibular condylar chondrocytes (MCCs). Lip-1 maintain the morphology and function of mitochondria, and inhibited the aggravation of lipid peroxidation and reactive oxygen species (ROS) production which induced by IL-1{beta}. These results suggested that chondrocytes ferroptosis play an important role in the development and progression of TMJOA. Inhibition of condylar chondrocyte ferroptosis could be a promising therapeutic strategy for TMJOA. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.11.536468v1?rss=1 Authors: Giri, S., Suo, C., Pardi, R., Fishbein, G. A., Rezvani, K., Chen, Y., Wang, X. Abstract: BACKGROUND: Neointimal hyperplasia (NH) is a common pathological response to vascular injury and mediated primarily by vascular smooth muscle cell (VSMC) migration and proliferation. The COP9 signalosome (CSN) is formed by 8 canonical subunits (CSN1 through CSN8) with its deneddylation activity residing in CSN5. Each or some of CSN subunits may have deneddylation-independent function. Despite strong evidence linking the CSN to cell cycle regulation in cancer cells, the role of the CSN in vascular biology remains obscure. METHODS: Neointimal CSN5 expression in the lung tissue of pulmonary hypertension (PAH) patients was assessed with immunohistochemistry. Adult mice with smooth muscle cell-restricted CSN5 knockout (CSN5-SMKO) or CSN8 hypomorphism (CSN8-hypo) and cultured mouse VSMCs were studied to determine the role and governing mechanisms of the CSN in NH. NH was induced by ligation of the left common carotid artery (LCCA) and PDGF-BB stimulation was used to mimic the vascular injury in cell cultures. RESULTS: Remarkably higher CSN5 levels were detected in the neointimal VSMCs of the pulmonary arteries of human PAH. LCCA ligation induced NH and significantly increased the mRNA and protein levels of CSN subunits in the LCCA wall of adult wild type mice. CSN5-SMKO impaired Cullin deneddylation and the nuclear export of p27 in vessel walls and markedly inhibited VSMC proliferation in mice. On the contrary, CSN8-hypo significantly exacerbated NH and VSMC proliferation in vivo and in cellulo. Cytoplasmic CSN5 mini-complexes and the nuclear export of p27 were significantly increased in CSN8-hypo mouse vessels and cultured CSN8-hypo VSMCs. Nuclear export inhibition with leptomycin attenuated the PDGF-BB-induced increases in VSMC proliferation in both CSN8-hypo and control VSMCs. Further, genetically disabling CSN5 nuclear export but not disabling CSN5 deneddylase activity suppressed the hyperproliferation and restored p27 nuclear localization in CSN8 hypomorphic VSMCs. Interestingly, CSN deneddylase inhibition by CSN5i-3 did not alter the hyperproliferation of cultured CSN8-hypo VSMCs but suppressed wild type VSMC proliferation in cellulo and in vivo and blocked neointimal formation in wild type mice. CONCLUSION: The CSN promotes VSMC proliferation and NH in injured vessels through deneddylation activity and CSN5-mediated nuclear export. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.11.536411v1?rss=1 Authors: Arslan, F. N., Hannezo, E., Merrin, J., Loose, M., Heisenberg, C.-P. Abstract: Metazoan development relies on the formation and remodeling of cell-cell contacts. Dynamic reorganization of adhesion receptors and the actomyosin cell cortex in space and time play a central role in cell-cell contact formation and maturation. Yet, how this process is mechanistically achieved remains unclear. Here, by building a biomimetic assay composed of progenitor cells adhering to supported lipid bilayers functionalized with E-cadherin ectodomains, we show that cortical Actin flows, driven by the depletion of Myosin-2 at the cell contact center, mediate the dynamic reorganization of adhesion receptors and cell cortex at the contact. E-cadherin-dependent downregulation of the small GTPase RhoA at the forming contact leads to both a depletion of Myosin-2 and a decrease of F-actin at the contact center. This depletion of Myosin-2 causes centrifugal F-actin flows, leading to further accumulation of F-actin at the contact rim and the progressive redistribution of E-cadherin from the contact center to the rim. Eventually, this combination of actomyosin downregulation and flows at the contact determine the characteristic molecular organization, with E-cadherin and F-actin accumulating at the contact rim, where they are needed to mechanically link the contractile cortices of the adhering cells. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.10.536202v1?rss=1 Authors: Gonzalez-Novo, R., Zamora Carreras, H., De Lope-Planelles, A., Lopez-Menendez, H., Roda-Navarro, p., Monroy, F., Wang, L., Toseland, C. P., Redondo-Munoz, J. Abstract: The interplay between cells and their surrounding microenvironment drives multiple cellular functions, including migration, proliferation, and cell fate transitions. The nucleus is a mechanosensitive organelle that adapts external mechanical and biochemical signals provided by the environment into nuclear changes with functional consequences for cell biology. However, the morphological and functional changes of the nucleus induced by 3D extracellular signals remain unclear. Here, we demonstrated that cells derived from 3D conditions show an aberrant nuclear morphology and mislocalization of lamin B1 from the nuclear periphery. We found that actin polymerization and protein kinase C (PKC) activity mediate the abnormal distribution of lamin B1 in 3D conditions-derived cells. Further experiments indicated that these cells show altered chromatin compaction, gene transcription and cellular functions such as cell viability and migration. By combining biomechanical techniques, such as force compression and single-nucleus analysis by atomic force microscopy, optical tweezers, and super-resolution microscopy, we have determined that the nucleus from 3D conditions-derived cells show a different mechanical behaviour and biophysical signature than the nucleus from control cells. Together, our work substantiates novel insights into how the extracellular environment alters the cell biology by promoting consistent changes in the chromatin, morphology, lamin B1 distribution, and the mechanical response of the nucleus. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.09.536174v1?rss=1 Authors: Gregory, E. F., Kalra, S., Brock, T., Bonne, G., Luxton, G. G., Hopkins, C., Starr, D. A. Abstract: Striated muscle laminopathies caused by missense mutations in the nuclear lamin gene LMNA are characterized by cardiac dysfunction and often skeletal muscle defects. Attempts to predict which LMNA variants are pathogenic and to understand their physiological effects lags behind variant discovery. We created Caenorhabditis elegans models for striated muscle laminopathies by introducing pathogenic human LMNA variants and variants of unknown significance at conserved residues within the lmn-1 gene. Severe missense variants reduced fertility and/or motility in C. elegans. Nuclear morphology defects were evident in the hypodermal nuclei of many lamin variant strains, indicating a loss of nuclear envelope integrity. Phenotypic severity varied within the two classes of missense mutations involved in striated muscle disease, but overall, variants associated with both skeletal and cardiac muscle defects in humans lead to more severe phenotypes in our model than variants predicted to disrupt cardiac function alone. We also identified a separation of function allele, lmn-1(R204W), that exhibited normal viability and swimming behavior but had a severe nuclear migration defect. Thus, we established C. elegans avatars for striated muscle laminopathies and identified LMNA variants that offer insight into lamin mechanisms during normal development. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.09.536157v1?rss=1 Authors: Aktar, K., Davies, T., Leontiou, I., Clark, I., Spanos, C., Wallace, E., Tuck, L., Jeyaprakash, A. A., Hardwick, K. Abstract: Cryptococcus neoformans is an opportunistic, human fungal pathogen which undergoes fascinating switches in cell cycle control and ploidy when it encounters stressful environments such as the human lung. Here we carry out a mechanistic analysis of the spindle assembly checkpoint (SAC) which regulates the metaphase to anaphase transition, focusing on Mps1 kinase and the downstream checkpoint components Mad1 and Mad2. We demonstrate that Cryptococcus mad1- or mad2- strains are unable to respond to microtubule perturbations, continuing to re-bud and divide, and die rapidly as a consequence. Fluorescent tagging of Chromosome 3, using a lacO array and mNeonGreen-lacI fusion protein, demonstrates that mad mutants are unable to maintain sister-chromatid cohesion in the absence of microtubule polymers. Thus, the classic checkpoint functions of the SAC are conserved in Cryptococcus. In interphase, GFP-Mad1 is enriched at the nuclear periphery, and it is recruited to unattached kinetochores in mitosis. Purification of GFP-Mad1 followed by mass spectrometric analysis of associated proteins show that that it forms a complex with Mad2 and that it interacts with other checkpoint signalling components (Bub1) and effectors (Cdc20 and APC/C sub-units) in mitosis. We also demonstrate that overexpression of Mps1 kinase is sufficient to arrest Cryptococcus cells in mitosis, and show that this arrest is dependent on both Mad1 and Mad2. We find that a C-terminal fragment of Mad1 is an effective in vitro substrate for Mps1 kinase and map several Mad1 phosphorylation sites. Some sites are highly conserved within the C-terminal Mad1 structure and we demonstrate that mutation of threonine 667 (T667A) leads to loss of checkpoint signalling and abrogation of the GAL-MPS1 arrest. Thus Mps1-dependent phosphorylation of C-terminal Mad1 residues is a critical step in Cryptococcus spindle checkpoint signalling. Finally, we analyse the phenotype of mad and mps1 mutants during titan cell generation: quantitating viability of titan cells and their daughters generated during the ensuing reductive division. The mad1, mad2 and mps1 mutants show significantly reduced viability: many titans are dead and others produce slow growing colonies. We propose that these Cryptococcus neoformans checkpoint proteins have important roles in ensuring high fidelity chromosome segregation during stressful conditions, such that those heightened during its polyploid infection cycle. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.08.535951v1?rss=1 Authors: Ling, Y. H., Chen, Y. Y., Leung, K. N., Chan, K. M., Liu, W. K. Abstract: CCHCR1 (coiled-coil alpha-helical rod protein 1) is a candidate gene of psoriasis and was recently identified as a novel component in P-bodies, the site for regulating mRNA turnover, and a protein in centrosomes. Little is known about its transcriptional regulation or its functions in cell cycle progression. In this study, CCHCR1 was found in a close (287-bp) head-to-head orientation with its neighboring gene, TCF19. The transcription of the CCHCR1/TCF19 gene pair was controlled by a shared bidirectional promoter and was induced at the G1/S transition of the cell cycle. The 287-bp intergenic sequence was sufficient for the G1/S expression of both genes, but the expression of CCHCR1 was further promoted by the presence of exon 1 of TCF19 and CCHCR1. The expression of the CCHCR1/TCF19 gene pair was dependent on the E2F1 transcription factor. E2F1 binding sites were predicted in the CCHCR1/TCF19 bidirectional promoter by in silico analysis. Overexpression of E2F1 induced the expression of CCHCR1 and TCF19. In addition, E2F1 knockdown inhibited both CCHCR1 and TCF19 expression. Knockdown of CCHCR1 or TCF19 reduced cell count but only depletion of CCHCR1 significantly induced p21 expression, implying that CCHCR1 and TCF19 may both regulate cell growth but in divergent pathways. Taken together, we revealed a bidirectional regulation of the CCHCR1/TCF19 gene pair in the G1/S transition and provide a new perspective to understand the role of CCHCR1 as a candidate gene of psoriasis. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.07.535979v1?rss=1 Authors: Chowdhury, D., Dhabal, S., Bhatt, M., Maity, D., Chakraborty, S., Priyadarshi, S., Haldar, S. Abstract: Methotrexate is a well-known antineoplastic drug used to prevent cancer aggravation. Despite being a targeted therapeutic approach, its administration comes with the risk of cancer recurrence, plausibly through its proven off-target effect on focal adhesions. Since FA dynamics is dependent on force transmission through its constituent proteins, including talin, methotrexate might affect the mechanical activity of these proteins. Here we have combined single-molecule studies, computational dynamics, cell-based assays, and genomic analysis to unveil the focal adhesion-regulating role of methotrexate central to its effect on talin dynamics and downstream pathways. Interestingly, our single-molecule force spectroscopic study shows that methotrexate modulates the bimodal force distribution of talin in a concentration-dependent manner. Steered molecular dynamics reveal that methotrexate-talin interactions alter talin mechanical stability exposing their vinculin binding sites. Finally, we found that methotrexate-regulated talin-dynamics remodel cancer cell mechanical phenotypes like cell polarity, adhesion, and migration by regulating talin-vinculin association-mediated YAP signaling. These results further correlate with genomic analysis of methotrexate-treated patients, demonstrating its clinical importance. Taken together, these findings disseminate the effects of methotrexate-modulated mechanosensitivity of adhesion proteins on cellular events. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.03.535492v1?rss=1 Authors: Manandhar, B., Pandzic, E., Deshpande, N., Chen, S.-Y., Wasinger, V., Kockx, M., Glaros, E. N., Ong, K. L., Thomas, S. R., Wilkins, M., Whan, R., Cochran, B. J., Rye, K.-A. Abstract: Objective: High cholesterol levels in pancreatic {beta}-cells cause oxidative stress and decrease insulin secretion. {beta}-cells can internalize apolipoprotein (apo) A-I, which increases insulin secretion. This study asks whether internalization of apoA-I improves {beta}-cell insulin secretion by reducing oxidative stress. Approach: Ins-1E cells were cholesterol-loaded by incubation with cholesterol-methyl-{beta}-cyclodextrin. Insulin secretion in the presence of 2.8 or 25 mM glucose was quantified by radioimmunoassay. Internalization of fluorescently labelled apoA-I by {beta}-cells was monitored by flow cytometry. The effects of apoA-I internalization on {beta}-cell gene expression was evaluated by RNA sequencing. ApoA-I binding partners on the {beta}-cell surface were identified by mass spectrometry. Mitochondrial oxidative stress was quantified in {beta}-cells and isolated islets with MitoSOX and confocal microscopy. Results: An F1-ATPase {beta}-subunit on the {beta}-cell surface was identified as the main apoA-I binding partner. {beta}-cell internalization of apoA-I was time-, concentration-, temperature-, cholesterol- and F1-ATPase {beta}-subunit-dependent. {beta}-cells with internalized apoA-I (apoA-I+ cells) had higher cholesterol and cell surface F1-ATPase {beta}-subunit levels than {beta}-cells without internalized apoA-I (apoA-I- cells). The internalized apoA-I co-localized with mitochondria and was associated with reduced oxidative stress and increased insulin secretion. The ATPase inhibitory factor 1, IF1, attenuated apoA-I internalization and increased oxidative stress in Ins-1E {beta}-cells and isolated mouse islets. Differentially expressed genes in apoA-I+ and apoA-I- Ins-1E cells were related to protein synthesis, the unfolded protein response, insulin secretion and mitochondrial function. Conclusions: These results establish that {beta}-cells are functionally heterogeneous and apoA-I restores insulin secretion in ?-cells with elevated cholesterol levels by improving mitochondrial redox balance. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.06.535932v1?rss=1 Authors: Lewis, T. R., Phan, S., Castillo, C. M., Kim, K.-Y., Coppenrath, K., Thomas, W., Hao, Y., Skiba, N. P., Horb, M. E., Ellisman, M. H., Arshavsky, V. Y. Abstract: The first steps of vision take place within a stack of tightly packed disc-shaped membranes, or "discs", located in the outer segment compartment of photoreceptor cells. In rod photoreceptors, discs are enclosed inside the outer segment and contain deep indentations in their rims called "incisures". The presence of incisures has been documented in a variety of species, yet their role remains elusive. In this study, we combined traditional electron microscopy with three-dimensional electron tomography to demonstrate that incisures are formed only after discs become completely enclosed. We also observed that, at the earliest stage of their formation, discs are not round as typically depicted but rather are highly irregular in shape and resemble expanding lamellipodia. Using genetically manipulated mice and frogs and measuring outer segment protein abundances by quantitative mass spectrometry, we further found that incisure size is determined by the molar ratio between peripherin-2, a disc rim protein critical for the process of disc enclosure, and rhodopsin, the major structural component of disc membranes. While a high perpherin-2 to rhodopsin ratio causes an increase in incisure size and structural complexity, a low ratio precludes incisure formation. Based on these data, we propose a model whereby normal rods express a modest excess of peripherin-2 over the amount required for complete disc enclosure in order to ensure that this important step of disc formation is accomplished. Once the disc is enclosed, the excess peripherin-2 incorporates into the rim to form an incisure. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.07.536031v1?rss=1 Authors: Balasooriya, G. I., Spector, D. L. Abstract: Spatiotemporal gene regulation is fundamental to the biology of diploid cells. Therefore, effective communication between two alleles and their geometry in the nucleus is important. However, the mechanism that fine-tunes the expression from each of the two alleles of an autosome is enigmatic. Establishing an allele-specific gene expression visualization system in living cells, we show that alleles of biallelically expressed Cth and Ttc4 genes are paired prior to acquiring monoallelic expression. We found that active alleles of monoallelic genes are preferentially localized at Sun1-enriched domains at the nuclear periphery. These peripherally localized active DNA loci are enriched with adenine-thymidine-rich tandem repeats that interact with Hnrnpd and reside in a Hi-C-defined A compartment within the B compartment. Our results demonstrate the biological significance of T1A3 tandem repeat sequences in genome organization and how the regulation of gene expression, at the level of individual alleles, relates to their spatial arrangement. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.06.535955v1?rss=1 Authors: ji, h., Yue, L., Sun, H., Chen, R., Zhou, R., Xiao, A., Yang, Y., Wang, R., You, C., Liu, Y. Abstract: Intracranial aneurysm (IA) is pouch-like pathological dilations of cerebral arteries, which often affects middle-aged people and culminates in life-threatening hemorrhagic stroke. A deeper knowledge of the cellular and gene expression perturbations in human IA tissue deepens our understanding of disease mechanisms and facilitates developing pharmacological targets for unruptured IA. In this study, 21,332 qualified cells were obtained from cell-sparse ruptured and unruptured human IA tissues and a detailed cellular profile was determined, including conventional endothelial cells, smooth muscle cells (SMC), fibroblasts and the newly identified pericytes. Notably, striking proportion of immune cells were identified in IA tissue, with the number of monocyte/macrophages and neutrophils being remarkably higher in ruptured IA. By leveraging external datasets and machine learning algorithms, a subset of macrophages characterized by high expression of CCL3 and CXCL3, and transcriptional activation of NF-{kappa}B and HIVEP2 was identified as the cell most associated with IA rupture. Further, the interactome of CCL3/CXCL3 macrophages disclosed their role in regulating vascular cell survival and orchestrating inflammation. In summary, this study illustrated the profile and interactions of vascular and immune cells in human IA tissue and the opportunities for targeting local chronic inflammation. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.07.536038v1?rss=1 Authors: Dougherty, L. L., Avasthi, P. Abstract: At the core of cilia are microtubules which are important for establishing length and assisting ciliary assembly and disassembly; however, another role for microtubule regulation on ciliogenesis lies outside of the cilium. The microtubule cytoskeleton is a highly dynamic structure which reorganizes rapidly to assist in cellular processes. Cytoplasmic microtubule dynamics have previously been thought to be necessary to free up tubulin and proteins in the ciliary precursor pool for ciliogenesis. However, we previously found that low concentrations of taxol can stabilize cytoplasmic microtubules during deciliation while allowing normal cilium regrowth. Here we look at the relationship between ciliogenesis and cytoplasmic microtubule dynamics in Chlamydomonas reinhardtii using chemical and mechanical perturbations. We find that not only can stabilized cytoplasmic microtubules allow for normal ciliary assembly, but high calcium concentrations and low pH-induced deciliation cause microtubules to depolymerize separately from ciliary shedding. In addition, we find that through mechanical shearing, cilia regenerate more quickly despite intact cytoplasmic microtubules. Our data suggests that cytoplasmic microtubules are not a sink for a limiting pool of cytoplasmic tubulin, reorganization that occurs following deciliation is a consequence rather than a requirement for ciliogenesis, and intact microtubules in the cytoplasm and the proximal cilium support more efficient ciliary assembly. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.06.535882v1?rss=1 Authors: Ciotta, G., Singh, S., Gupta, A., Torres, D. C., Fu, J., Choudhury, R., Chu, W. K., Choudhary, C., Gahurova, L., Al-Fatlawi, A., Schroeder, M., Aasland, R., Poetsch, A., Anastassiadis, K., Stewart, A. F. Abstract: SETD1A is the histone 3 lysine 4 (H3K4) methyltransferase central to the mammalian version of the highly conserved eight subunit Set1 complex (Set1C) that apparently conveys H3K4 trimethylation (H3K4me3) onto all active Pol II promoters. Accordingly, mouse embryonic stem cells (ESCs) die when SETD1A is removed. We report that death is accompanied by loss of expression of DNA repair genes and accumulating DNA damage. BOD1L and BOD1 are homologs of the yeast Set1C subunit, Shg1, and subunits of the mammalian SETD1A and B complexes. We show that the Shg1 homology region binds to a highly conserved central a-helix in SETD1A and B. Like mutagenesis of Shg1 in yeast, conditional mutagenesis of Bod1l in ESCs promoted increased H3K4 di- and tri-methylation but also, like loss of SETD1A, loss of expression of DNA repair genes, increased DNA damage and cell death. In contrast to similar losses of gene expression, the converse changes in H3K4 methylation implies that H3K4 methylation is not essential for expression of the DNA repair network genes. Because BOD1L becomes highly phosphorylated after DNA damage and acts to protect damaged replication forks, the SETD1A complex and BOD1L in particular are key nodes for the DNA damage repair network. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.06.535851v1?rss=1 Authors: Stankovic, D., Tain, L., Uhlirova, M. Abstract: Co-transcriptional processing of nascent pre-mRNAs by the spliceosome is vital to regulating gene expression and maintaining genome integrity. Here, we show that the deficiency of functional U5 snRNPs in Drosophila imaginal cells causes extensive transcriptome remodeling and accumulation of highly mutagenic R-loops, triggering a robust stress response and cell cycle arrest. Despite compromised proliferative capacity, the U5 snRNP deficient cells increased protein translation and cell size, causing intra-organ growth disbalance before being gradually eliminated via apoptosis. We identify the Xrp1-Irbp18 heterodimer as the primary driver of transcriptional and cellular stress program downstream of U5 snRNP malfunction. Knockdown of Xrp1 or Irbp18 in U5 snRNP deficient cells attenuated JNK and p53 activity, restored normal cell cycle progression and growth, and inhibited cell death. Reducing Xrp1-Irbp18, however, did not rescue the splicing defects and the organismal lethality, highlighting the requirement of accurate splicing for cellular and tissue homeostasis. Our work provides novel insights into the crosstalk between splicing and the DNA damage response and defines the Xrp1-Irbp18 heterodimer as a critical sensor of spliceosome malfunction. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.06.535829v1?rss=1 Authors: Hennies, J., Lleti, J. M. S., Pape, C., Bekbayev, S., Gross, V., Kreshuk, A., Schwab, Y. Abstract: Segmentation of large-volume datasets obtained by volume SEM techniques is a challenging task that generally requires a considerable amount of human effort. Despite recent advances in deep learning leading to the successful segmentation of cellular organelles in a variety of datasets, it is still challenging and time-consuming to produce the necessary data for training a convolutional neural network as well as to set up targeted post-processing pipelines to obtain a good quality full-volume semantic instance segmentation. We present CebraEM, a software package that uses a novel workflow for the segmentation of organelles in volume EM datasets, which helps to minimize the annotation time for the generation of training data. It relies on a generic CNN-based membrane prediction, followed by a well-established machine-learning pipeline that includes over-segmentation before random forest classification and graph multi-cut grouping. The workflow was tested for the segmentation of organelles on different datasets originating from various sample preparations and imaging modalities in volume SEM, in each case resulting in state-of-the-art semantic instance segmentations without additional post-processing. Importantly, by considerably simplifying the segmentation problem, CebraEM empowers single users with the ability to efficiently segment hundreds of gigabytes of data. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.06.535716v1?rss=1 Authors: Montgomery, A. C., Mendoza, C. S., Garbouchian, A., Quinones, G. B., Bentley, M. Abstract: Neurons are polarized cells that require accurate membrane trafficking to maintain distinct protein complements at dendritic and axonal membranes. The Kinesin-3 family members KIF13A and KIF13B are thought to mediate dendrite-selective transport, but the mechanism by which they are recruited to polarized vesicles and the differences in the specific trafficking role of each KIF13 have not been defined. We performed live-cell imaging in cultured hippocampal neurons and found that KIF13A is a dedicated dendrite-selective kinesin. KIF13B confers two different transport modes, both dendrite- and axon-selective transport. Both KIF13s are maintained at the trans-Golgi network by interactions with the heterotetrameric adaptor protein complex AP-1. Interference with KIF13 binding to AP-1 resulted in disruptions to both dendrite- and axon- selective trafficking. We conclude that AP-1 is the molecular link between the sorting of polarized cargoes into vesicles and the recruitment of kinesins that confer polarized transport. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.05.535802v1?rss=1 Authors: Guo, Y., Wei, J., Liu, C., Li, X., Yan, W. Abstract: Diabetes mellitus is a group of chronic diseases characterized by high blood glucose levels. Diabetic patients have a higher risk of sustaining osteoporotic fractures than non-diabetic people. The fracture healing is usually impaired in diabetics and our understanding of the detrimental effects of hyperglycemia on fracture healing is still inadequate. Metformin is the first-line medicine for type-2 diabetes (T2D). However, its effects on bone in T2D patients remain to be studied. To assess the impacts of metformin on fracture healing, we compared the healing process of closed wound fixed fracture, non-fixed radial fracture, and femoral drill-hole injury models in T2D mice with and without metformin treatment. Our results demonstrated that metformin rescued the delayed bone healing and remolding in T2D mice in all the three injury models. The proliferation, osteogenesis, chondrogenesis of the bone marrow stromal cells (BMSCs) derived from WT and T2D mice treated with or without metformin were compared in vitro and in vivo by assessing the subcutaneous ossicle formation of the BMSC implants in recipient T2D mice. In vivo treatment with metformin to T2D mice could effectively rescue the impaired differentiation potential and detrimental lineage commitment of BMSCs, caused by the hyperglycemic conditions. Moreover, the Safranin O staining of cartilage formation in the endochondral ossification under hyperglycemic condition significantly increased at day 14 post-fracture in T2D mice receiving metformin treatment. The chondrocyte transcript factor SOX9 and PGC1, important to maintain chondrocyte homeostasis, were both significantly upregulated in callus tissue isolated at the fracture site of metformin-treated MKR mice on day 12 post-fracture. Metformin also rescued the chondrocyte disc formation of BMSCs isolated from T2D mice. Taken together, our study demonstrated that metformin facilitated bone healing, bone formation and chondrogenesis in T2D mouse models. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.04.535605v1?rss=1 Authors: Tagay, Y., Kheirabadi, S., Ataie, Z., Singh, R., Prince, O., Nguyen, A., Zhovmer, A. S., Ma, X., Sheikhi, A., Tsygankov, D., Tabdanov, E. D. Abstract: Metastasis is a principal cause of death in cancer patients, which remains an unresolved fundamental and clinical problem. Conventionally, metastatic dissemination is linked to the actomyosin-driven cell locomotion. However, locomotion of cancer cells often does not strictly line up with the measured actomyosin forces. Here, we identify a complementary mechanism of metastatic locomotion powered by the dynein-generated forces. These forces that arise within a non-stretchable microtubule network drive persistent contact guidance of migrating cancer cells along the biomimetic collagen fibers. We also show that dynein-powered locomotion becomes indispensable during invasive 3D migration within a tissue-like luminal network between spatially confining hydrogel microspheres. Our results indicate that the complementary contractile system of dynein motors and microtubules is always necessary and in certain instances completely sufficient for dissemination of metastatic breast cancer cells. These findings advance fundamental understanding of cell locomotion mechanisms and expand the spectrum of clinical targets against metastasis. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.05.535690v1?rss=1 Authors: Kirchenwitz, M., Halfen, J., von Peinen, K., Prettin, S., Kollasser, J., Brakebusch, C. H., Rottner, K., Steffen, A., Stradal, T. Abstract: Salmonella enterica serovar Typhimurium manipulates cellular Rho GTPases for host cell invasion by effector protein translocation via the Type III Secretion System (T3SS). The two Guanine nucleotide exchange (GEF) mimicking factors SopE and -E2 and the inositol phosphate phosphatase (PiPase) SopB activate the Rho GTPases Rac1, Cdc42 and RhoA, thereby mediating bacterial invasion. S. Typhimurium lacking these three effector proteins are largely invasion-defective. Type III secretion is crucial for both early and later phases of the intracellular life of S. Typhimurium. Here we investigated whether and how the small GTPase RhoB, known to localize on endomembrane vesicles and at the invasion site of S. Typhimurium, contributes to bacterial invasion and to subsequent steps relevant for S. Typhimurium lifestyle. We show that RhoB is significantly upregulated within hours of Salmonella infection. This effect depends on the presence of the bacterial effector SopB, but does not require its phosphatase activity. Our data reveal that SopB and RhoB bind to each other, and that RhoB localizes on early phagosomes of intracellular S. Typhimurium. Whereas both SopB and RhoB promote intracellular survival of Salmonella, RhoB is specifically required for Salmonella-induced upregulation of autophagy. Finally, in the absence of RhoB, vacuolar escape and cytosolic hyper-replication of S. Typhimurium is diminished. Our findings thus uncover a role for RhoB in Salmonella-induced autophagy, which supports intracellular survival of the bacterium and is promoted through a positive feedback loop by the Salmonella effector SopB. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.04.535566v1?rss=1 Authors: Aguilar-Bravo, B., Arino, S., Blaya, D., Pose, E., Martinez Garcia de la Torre, R. A., Latasa, M. U., Martinez-Sanchez, C., Zanatto, L., Sererols-Vinas, L., Cantallops, P., Affo, S., Coll, M., Thillen, X., Dubuquoy, L., Avila, M. A., Argemi, J. M., Lamas Paz, A., Nevzorova, Y. A., Cubero, J., Bataller, R., Lozano, J. J., Gines, P., Mathurin, P., Sancho-Bru, P. Abstract: Background and Aims: Loss of hepatocyte identity is associated with impaired liver function in alcohol-related hepatitis (AH). In this context, hepatocyte dedifferentiation gives rise to cells with a hepatobiliary (HB) phenotype expressing biliary and hepatocytes markers and showing immature features. However, the mechanisms and the impact of hepatocyte dedifferentiation in liver disease are poorly understood. Methods: HB cells and ductular reaction (DR) cells were quantified and microdissected from liver biopsies from patients with alcohol-related liver disease (ALD). Hepatocyte-specific overexpression or deletion of CXCR4, and CXCR4 pharmacological inhibition were assessed in mouse liver injury. Patient-derived and mouse organoids were generated to assess plasticity. Results: Here we show that HB and DR cells are increased in patients with decompensated cirrhosis and AH, but only HB cells correlate with poor liver function and patients outcome. Transcriptomic profiling of HB cells revealed the expression of biliary-specific genes and a mild reduction of hepatocyte metabolism. Functional analysis identified pathways involved in hepatocyte reprogramming, inflammation, stemness and cancer gene programs. CXCR4 pathway was highly enriched in HB cells, and correlated with disease severity and hepatocyte dedifferentiation. In vitro, CXCR4 was associated with biliary phenotype and loss of hepatocyte features. Liver overexpression of CXCR4 in chronic liver injury decreased hepatocyte specific gene expression profile and promoted liver injury. CXCR4 deletion or its pharmacological inhibition ameliorated hepatocyte dedifferentiation and reduced DR and fibrosis progression. Conclusions: This study shows the association of hepatocyte dedifferentiation with disease progression and poor outcome in AH. Moreover, the transcriptomic profiling of HB cells revealed CXCR4 as a new driver of hepatocyte-to-biliary reprogramming and as a potential therapeutic target to halt hepatocyte dedifferentiation in AH. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.05.535676v1?rss=1 Authors: Brault, J.-B., Liu, Z., Bardin, S., Ladarre, D., Fraisier, V., Tchenio, A., Lenkei, Z., Salamero, J., Delevoye, C., Goud, B., Miserey, S. Abstract: Alzheimer s disease (AD) is the most common form of dementia worldwide. One of AD s main pathological hallmarks is the cerebral plaque deposits of Beta-amyloid (ABeta). ABeta is generated through sequential enzymatic cleavage of the amyloid precursor protein (APP). The Beta-secretase or Beta-site APP-cleaving enzyme 1 (BACE1) initiates this cleavage and is thus key to regulate ABeta formation. Both APP and BACE1 transit through the endolysosomal system but the exact nature of the compartment(s) where APP cleavage occurs as well as the molecular mechanisms that govern their endosomal sorting remain poorly known. Here we show that RAB11 not only regulates BACE1 transport from early/sorting endosomes (EEs/SEs) and drives the exocytosis of BACE1-containing recycling carriers. Moreover, recycling endosome-associated KIF13A, as well as its closely related homolog KIF13B, which are known RAB11 effectors involved in the biogenesis of recycling endosome (RE) from EEs/SEs, also participate in BACE1 endosomal sorting. Importantly, depletion of KIF13A or KIF13B leads to an increase in ABeta generation. Depletion of the BLOC-1 complex, previously described as an essential partner for KIF13A-dependent RE biogenesis, also induces increased amount of ABeta. Altogether, our findings support a model where the EEs/SEs represent a major organelle for ABeta formation and identify the recycling endosome biogenesis machinery as a master coordinator of BACE1 endosomal sorting and transport. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.05.535705v1?rss=1 Authors: Yang, L., Parajuli, N., Wu, P., Liu, J., Wang, X. Abstract: Background: A better understanding of the regulation of proteasome activities can facilitate the search for new therapeutic strategies. A cell culture study shows that cAMP-dependent protein kinase (PKA) activates the 26S proteasome by phosphorylating Ser14 of RPN6 (pS14-RPN6), but this discovery and its physiological significance remain to be established in vivo. Methods: Male and female mice with Ser14 of Rpn6 mutated to Ala (S14A) or Asp (S14D) to respectively block or mimic pS14-Rpn6 were created and used along with cells derived from them. cAMP/PKA were manipulated pharmacologically. Ubiquitin-proteasome system (UPS) functioning was evaluated with the GFPdgn reporter mouse and proteasomal activity assays. Impact of S14A and S14D on proteotoxicity was tested in mice and cardiomyocytes overexpressing the misfolded protein R120G-CryAB (R120G). Results: PKA activation increased pS14-Rpn6 and 26S proteasome activities in wild-type (WT) but not S14A embryonic fibroblasts (MEFs), adult cardiomyocytes (AMCMs), and mouse hearts. Basal 26S proteasome activities were significantly greater in S14D myocardium and AMCMs than in WT counterparts. S14D::GFPdgn mice displayed significantly lower myocardial GFPdgn protein but not mRNA levels than GFPdgn mice. In R120G mice, a classic model of cardiac proteotoxicity, basal myocardial pS14-Rpn6 was significantly lower compared with non-transgenic littermates, which was not always associated with reduction of other phosphorylated PKA substrates. Cultured S14D neonatal cardiomyocytes displayed significantly faster proteasomal degradation of R120G than WT neonatal cardiomyocytes. Compared with R120G mice, S14D/S14D::R120G mice showed significantly greater myocardial proteasome activities, lower levels of total and K48-linked ubiquitin conjugates and of aberrant CryAB protein aggregates, less reactivation of fetal genes and cardiac hypertrophy, and delays in cardiac malfunction. Conclusions: This study establishes in animals that pS14-Rpn6 mediates the activation of 26S proteasomes by PKA and that the reduced pS14-Rpn6 is a key pathogenic factor in cardiac proteinopathy, thereby identifies a new therapeutic target to reduce cardiac proteotoxicity. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.06.535946v1?rss=1 Authors: Bhowmik, A. T., Zhao, S. R., Wu, J. C. Abstract: Electronic cigarettes (e-cigarettes) have become increasingly popular with adolescents in recent years as a result of aggressive marketing schemes, false safety claims, and appealing flavors targeted towards teens from e-cigarette companies. In the past 8 years alone, e-cigarette use amongst youth has increased by 18 times. Although many dangerous effects of smoking e-cigarettes on lungs have come to light, limited and qualitative effort has been made to analyze the impact of smoking e-cigarettes on the human heart directly. In this study, we determined e-liquid cardiotoxicity in both healthy cells and cells with long QT syndrome by treating healthy and diseased human cardiomyocytes with e-liquids with varying nicotine concentrations. These cardiomyocytes were generated from human induced pluripotent stem cells. The cardiomyocytes were divided into 5 groups, a control group and 4 test groups, each treated with e-liquid containing varying amounts of nicotine between 0% and 70%. The cells' biological indicators such as heart rate, pulse pressure, essential protein concentration, and metabolic activity, were measured, characterized using three different functional assays: contractility, Western blot, and viability, and tracked closely over 2 weeks. The results demonstrated that acute exposure to e-liquid led to tachycardia, hypertension, decreased protein levels, and cell death. The rate of cardiotoxicity increases with higher nicotine concentrations. The basal fluid also showed non-negligible toxicity. Under identical conditions, the functionality of the diseased heart cells declined at a faster rate compared to healthy cells. Overall, this work systematically establishes the harmful physiological effects of e-cigarettes on the human heart quantitatively. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.06.535922v1?rss=1 Authors: Tang, V. T., Abbineni, P. S., da Veiga Leprevost, F., Basrur, V., Emmer, B. T., Nesvizhskii, A., Ginsburg, D. Abstract: Most proteins secreted into the extracellular space are first recruited from the endoplasmic reticulum into coat protein complex II (COPII)-coated vesicles or tubules that facilitate their transport to the Golgi apparatus. Although several secreted proteins have been shown to be actively recruited into COPII vesicles/tubules by the cargo receptors LMAN1 and SURF4, the full cargo repertoire of these receptors is unknown. We now report mass spectrometry analysis of conditioned media and cell lysates from HuH7 cells CRISPR targeted to inactivate the LMAN1 or SURF4 gene. We found that LMAN1 has limited clients in HuH7 cells whereas SURF4 traffics a broad range of cargoes. Analysis of putative SURF4 cargoes suggests that cargo recognition is governed by complex mechanisms rather than interaction with a universal binding motif. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.06.535853v1?rss=1 Authors: Danan, C. H., Naughton, K. E., Hayer, K. E., Vellapan, S., McMillan, E. A., Zhou, Y., Matsuda, R., Nettleford, S. K., Katada, K., Parham, L. R., Ma, X., Chowdhury, A., Wilkins, B. J., Shah, P., Weitzman, M. D., Hamilton, K. E. Abstract: The METTL3-METTL14 methyltransferase complex adds N6-methyladenosine (m6A) to mRNA with profound impacts on cell fate. Studies delete METTL3 or METTL14 interchangeably to define the role of m6A in target tissues despite a lack of data confirming that these deletions are equivalent. Intestinal epithelial METTL14 deletion triggers stem cell death in the colon with no overt phenotype in the small intestine. The effect of METTL3 deletion in the same tissues remains unknown. We report that intestinal epithelial METTL3 deletion caused unexpected severe defects in the small intestine, including crypt and villus atrophy associated with cellular senescence and death in the crypt transit amplifying zone. Ribosome profiling and m6A-sequencing demonstrated downregulated translation of hundreds of unique methylated transcripts, including genes essential to growth factor signal transduction, such as Kras. Our study suggests that METTL3 is essential for small intestinal homeostasis via enhanced translation of growth factor signaling in crypt transit amplifying cells. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.05.535746v1?rss=1 Authors: Tschuck, J., Padmanabhan Nair, V., Galhoz, A., Ciceri, G., Rothenaigner, I., Tchieu, J., Tai, H.-M., Stockwell, B. R., Studer, L., Menden, M. P., Vincendeau, M., Hadian, K. Abstract: Development of functional neurons is a complex orchestration of several signaling pathways controlling cell proliferation, differentiation, and homeostasis. However, details about the involved factors are not fully understood. The balance of antioxidants and vitamins is important for neuronal survival, synaptic plasticity, and early neuronal development; thus, we hypothesized that ferroptosis -- a lipid peroxidation dependent cell death modality that is inhibited by antioxidanats -- needs to be suppressed to gain neurons. Our study shows that removal of antioxidants diminishes neuronal development and laminar organization of cortical organoids. Intriguingly, impaired neuronal development in conditions lacking antioxidants can be fully restored when ferroptosis is specifically inhibited by ferrostatin-1, or neuronal differentiation occurs in the presence of sufficient amounts of vitamin A. Mechanistically, vitamin A activates the heterodimeric nuclear receptor complex Retinoic Acid Receptor (RAR)/Retinoid X Receptor (RXR), which upregulates expression of the ferroptosis regulators GPX4, FSP1, GCH1, and ACSL3, amongst others. Therefore, our study reveals that above a certain threshold, vitamin A increases expression of essential cellular gatekeepers of lipid peroxidation. This study uncovers a critical process during early neuronal development, where suppression of ferroptosis by radical-trapping antioxidants or vitamin A is required to obtain maturing neurons and proper laminar organization in cortical organoids. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.04.535641v1?rss=1 Authors: Rebollo, E., Rubi-Sans, G., Cler, M., Valls-Lacalle, L., Nyga, A., Perez-Amodio, S., Mateos-Timoneda, M. A., Engel, E. Abstract: In recent years, the interaction between cells and the extracellular matrix (ECM) has become a new focus in understanding tissue morphogenesis, regeneration, and disease. However, the lack of specific techniques to study the ECM composition in preserved tissue structures remains a major obstacle to explaining ECM changes in response to extrinsic stimuli. To overcome this, we propose a novel strategy that uses multidimensional fluorescence microscopy and computational tools to quantify ECM composition in immunolabeled tissues and/or cell-derived matrices (CDM). This approach includes a detailed protocol for densitometric fluorescence calibration and procedures for image acquisition, processing, and automated quantification. Using this method, we present new data comparing collagen types I, III, and IV, and fibronectin contents in various tissues. These results emphasize the importance of studying ECM composition in situ under both normal homeostatic and disease conditions. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.05.535684v1?rss=1 Authors: Chen, X., Qiao, C., Jiang, T., Liu, J., Meng, Q., Zeng, Y., Chen, H., Zhang, Y., Li, X., Zhang, G., Li, Y., Qiao, H., Wu, J., Tan, S., Li, D., Dai, Q. Abstract: Detection noise significantly degrades the quality of structured illumination microscopy (SIM) images, especially under low-light conditions. Although supervised learning based denoising methods have shown prominent advances in eliminating the noise-induced artifacts, the requirement of a large amount of high-quality training data severely limits their applications. Here we developed a pixel-realignment based self-supervised denoising framework for SIM (PRS-SIM) that trains an SIM image denoiser with only noisy data and substantially removes the reconstruction artifacts. We demonstrated that PRS-SIM generates artifact-free images with 10-fold less fluorescence than ordinary imaging conditions while achieving comparable super-resolution capability to ground truth (GT). Moreover, the proposed method is compatible with multiple SIM modalities such as total internal reflective fluorescence SIM (TIRF-SIM), three-dimensional SIM (3D-SIM), and lattice light-sheet SIM (LLS-SIM). With PRS-SIM, we achieved long-term super-resolution live-cell imaging of various bioprocesses, revealing the clustered distribution of clathrin coated pits and detailed interaction dynamics of multiple organelles and the cytoskeleton. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.03.535494v1?rss=1 Authors: Chance, T., Meledeo, M. A., Cap, A., Darlington, D. N., Bynum, J. A., Wu, X. Abstract: Objective: The causes of endotheliopathy are multifactorial and trauma dependent, and the temporal mechanistic link that it has with acute traumatic coagulopathy (ATC) has yet to be fully determined. Therefore, we sought to define early characteristics and markers of endotheliopathy in two rat models, a time-course of acute lethal hemorrhage shock and polytrauma with hemorrhagic shock. Approach and Results: We sough to answer the following questions: (1) how soon can elevated biomarkers of endotheliopathy be detected in non-survivable (decompensated) hemorrhagic shock; (2) does extended hemorrhage time and accumulated hemorrhage impact biomarker levels; and (3) does the addition of polytrauma contribute to the further elevation of said biomarkers? In this study, we delineated a significant, acute elevation in end plasma levels of syndecan-1, thrombomodulin, and heparan sulfate, whose shedding patterns are a function of time as well as total hemorrhage volume vs. the addition of polytrauma. Additionally, we found that correlation of syndecan-1 and thrombomodulin to lactate levels and prothrombin times at trauma end revealed a potential for these markers to acutely predict downstream consequences of these trauma indications. Conclusion: Our results are of great relevance to the continued effort towards the identification and characterization of vascular dysfunction for early interventions in combat casualty care. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.03.535461v1?rss=1 Authors: Dong, K., He, X., Hu, G., Yao, Y., Zhou, J. Abstract: Objective: Vascular smooth muscle cells (VSMCs) are the primary contractile component of blood vessels and can undergo phenotypic switching from a contractile to a synthetic phenotype in vascular diseases such as coronary artery disease (CAD). This process leads to decreased expression of SMC lineage genes and increased proliferative, migratory and secretory abilities that drive disease progression. Super-enhancers (SE) and occupied transcription factors are believed to drive expression of genes that maintain cell identify and homeostasis. The goal of this study is to identify novel regulator of VSMC homeostasis by screening for SE-regulated transcription factors in arterial tissues. Approach and Results: We characterized human artery SEs by analyzing the enhancer histone mark H3K27ac ChIP-seq data of multiple arterial tissues. We unexpectedly discovered the transcription factor PRDM16, a GWAS identified CAD risk gene with previously well-documented roles in brown adipocytes but with an unknown function in vascular disease progression, is enriched with artery-specific SEs. Further analysis of public bulk RNA-seq and scRNA-seq datasets, as well as qRT-PCR and Western blotting analysis, demonstrated that PRDM16 is preferentially expressed in arterial tissues and in contractile VSMCs but not in visceral SMCs, and down-regulated in phenotypically modulated VSMCs. To explore the function of Prdm16 in vivo, we generated Prdm16 SMC-specific knockout mice and performed histological and bulk RNA-Seq analysis of aortic tissues. SMC-deficiency of Prdm16 does not affect the aortic morphology but significantly alters expression of many CAD risk genes and genes involved in VSMC phenotypic modulation. Specifically, Prdm16 negatively regulates the expression of Tgfb2 that encodes for an upstream ligand of TGF-{beta} signaling pathway, potentially through binding to the promoter region of Tgfb2. These transcriptomic changes likely disrupt VSMC homeostasis and predispose VSMCs to a disease state. Conclusions: Our results suggest that the CAD risk gene PRDM16 is preferentially expressed in VSMCs and is a novel regulator of VSMC homeostasis. Future studies are warranted to investigate its role in VSMCs under pathological conditions such as atherosclerosis. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.04.535541v1?rss=1 Authors: Wells, A., Mendes, C. C., Castellanos, F., Mountain, P., Wright, T., Wainwright, S. M., Stefana, M. I., Harris, A. L., Goberdhan, D. C. I., Wilson, C. Abstract: Secretory cells in glands and the nervous system frequently package and store proteins destined for regulated secretion in dense-core granules (DCGs), which disperse when released from the cell surface. Despite the relevance of this dynamic process to diseases such as diabetes and human neurodegenerative disorders, our mechanistic understanding is relatively limited, because of the lack of good cell models to follow the nanoscale events involved. Here, we employ the prostate-like secondary cells (SCs) of the Drosophila male accessory gland to dissect the cell biology and genetics of DCG biogenesis. These cells contain unusually enlarged DCGs, which are assembled in compartments that also form secreted nanovesicles called exosomes. We demonstrate that known conserved regulators of DCG biogenesis, including the small G-protein Arf1 and the coatomer complex AP-1, play key roles in making SC DCGs. Using real-time imaging, we find that the aggregation events driving DCG biogenesis are accompanied by a change in the membrane associated small Rab GTPases which are major regulators of membrane and protein trafficking in the secretory and endosomal systems. Indeed, a transition from trans-Golgi Rab6 to recycling endosomal protein Rab11, which requires conserved DCG regulators like AP-1, is essential for DCG and exosome biogenesis. Our data allow us to develop a model for DCG biogenesis that brings together several previously disparate observations concerning this process and highlights the importance of communication between the secretory and endosomal systems in controlling regulated secretion. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.03.535481v1?rss=1 Authors: Thoduvayil, S., Weerakkody, J. S., Topper, M., Bera, M., Coleman, J., Li, X., Mariappan, M., Ramakrishnan, S. Abstract: High-throughput quantification of the first- and second-phase insulin secretion dynamics is intractable with current methods. The fact that independent secretion phases play distinct roles in metabolism necessitates partitioning them separately and performing high-throughput compound screening to target them individually. We developed an insulin-nanoluc luciferase reporter system to dissect the molecular and cellular pathways involved in the separate phases of insulin secretion. We validated this method through genetic studies, including knockdown and overexpression, as well as small-molecule screening and their effects on insulin secretion. Furthermore, we demonstrated that the results of this method are well correlated with those of single-vesicle exocytosis experiments conducted on live cells. Thus, we can quantitatively determine the number of vesicles that fuse when a stimulus is applied. We have developed a robust methodology for screening small molecules and cellular pathways that target specific phases of insulin secretion, resulting in a better understanding of insulin secretion, which in turn will result in a more effective insulin therapy through the stimulation of endogenous glucose-stimulated insulin secretion. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.04.535251v1?rss=1 Authors: Dinarello, A., May, M., Amo-Aparicio, J., Azam, T., Gaballa, J. M., Marchetti, C., Tesoriere, A., Ghirardo, R., Redzic, J. S., Webber, W., Atif, S. M., Li, S., Eisenmesser, E. Z., de Graaf, D. M., Dinarello, C. A. Abstract: The IL-1 Family member IL-38 has been characterized as an anti-inflammatory cytokine in human and mouse models of systemic diseases. Here, we examined the role of IL 38 in the small intestine (SI). Immunostaining of SI revealed that IL-38 expression partially confines to intestinal stem cells. Cultures of intestinal organoids reveal IL 38 functions as a growth factor by increasing organoid size via inducing WNT3a. In contrast, organoids from IL 38 deficient mice develop more slowly. This reduction in size is likely due to downregulation of intestinal stemness markers (i.e., Fzd5, Ephb2, Olfm4) expression compared with wild type organoids. IL-38 binding to IL-1R6 is postulated to recruit the co-receptor IL-1R9. Therefore, to analyze the molecular mechanisms of IL-38 signaling, we also examined organoids from IL 1R9 deficient mice. Unexpectedly, these organoids, although significantly smaller than wild type, respond to IL 38, suggesting that IL-1R9 is not involved in IL-38 signaling in the stem cell crypt. Nevertheless, silencing of IL-1R6 disabled the organoid response to the growth property of IL 38, thus suggesting IL-1R6 as the main receptor used by IL-38 in the crypt compartment. In organoids from wild type mice, IL-38 stimulation induced low concentrations of IL-1{beta} which contribute to organoid growth. However, high concentrations of IL 1beta have detrimental effects on the cultures that were prevented by treatment with recombinant IL 38. Overall, our data demonstrate an important regulatory function of IL-38 as a growth factor, and as an anti-inflammatory molecule in the SI, maintaining homeostasis. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.03.535302v1?rss=1 Authors: Griffin, M., Talbott, H., Guardino, N., Guo, J., Spielman, A., Chen, K., Mascharak, S., Parker, J., Henn, D., Liang, N., King, M., Cotterell, A., Bauer-Rowe, K., Abbas, D., Deleon, N. M. D., Fahy, E., Sivaraj, D., Downer, M., Akras, D., Berry, C., Cook, J., Quarto, N., Klein, O. D., Lorenz, P., Gurtner, G., Januszyk, M., Wan, D. C., Longaker, M. T. Abstract: While past studies have suggested that plasticity exists between dermal fibroblasts and adipocytes, it remains unknown whether fat actively contributes to fibrosis in scarring. We show that adipocytes convert to scar-forming fibroblasts in response to Piezo-mediated mechanosensing to drive wound fibrosis. We establish that mechanics alone are sufficient to drive adipocyte-to-fibroblast conversion. By leveraging clonal-lineage-tracing in combination with scRNA-seq, Visium, and CODEX, we define a mechanically naive fibroblast-subpopulation that represents a transcriptionally intermediate state between adipocytes and scar-fibroblasts. Finally, we show that Piezo1 or Piezo2-inhibition yields regenerative healing by preventing adipocytes activation to fibroblasts, in both mouse-wounds and a novel human-xenograft-wound model. Importantly, Piezo1-inhibition induced wound regeneration even in pre-existing established scars, a finding that suggests a role for adipocyte-to-fibroblast transition in wound remodeling, the least-understood phase of wound healing. Adipocyte-to-fibroblast transition may thus represent a therapeutic target for minimizing fibrosis via Piezo-inhibition in organs where fat contributes to fibrosis. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.04.535547v1?rss=1 Authors: Adamo, G., Santonicola, P., Picciotto, S., Gargano, P., Nicosia, A., Longo, V., Aloi, N., Romancino, D. P., Paterna, A., Rao, E., Raccosta, S., Noto, R., Salamone, M., Costa, S., Alfano, C., Zampi, G., Colombo, P., Wei, M., Touzet, N., Manno, M., Di Schiavi, E., Bongiovanni, A. Abstract: Nanoalgosomes are extracellular vesicles (EVs) released by microalgal cells that can mediate intercellular and cross-kingdom communication. In the present study, the optimization of high quality nanoalgosome manufacturing from cultures of the marine microalgae Tetraselmis chuii has been enhanced by quality control procedures, applying robust biophysical and biochemical characterizations. Then, we evaluated the biological properties of nanoalgosomes in pre-clinical models. Our investigation of nanoalgosome biocompatibility included toxicological and genetic analyses, starting from studies on the invertebrate model organism Caenorhabditis elegans and proceeding to hematological and immunological evaluations in mice and human cells. Nanoalgosome biodistribution was evaluated in mice with accurate space-time resolution, and in C. elegans at cellular and subcellular levels. Further examination highlighted the antioxidant and anti-inflammatory bioactivities of nanoalgosomes. This holistic approach to nanoalgosome characterization showcases that nanoalgosomes are innate effectors for novel cosmetic formulations and EV-based therapies. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.30.534178v1?rss=1 Authors: Yu, K., Basu, A., Yau, C., Wolf, D. M., Goodarzi, H., Bandyopadhyay, S., Korkola, J. E., Hirst, G. L., Asare, S., I-SPY 2 TRIAL,, Demichele, A., Hylton, N., Yee, D., Esserman, L., van 't Veer, L., Sirota, M. Abstract: Drug resistance is a major obstacle in cancer treatment and can involve a variety of different factors. Identifying effective therapies for drug resistant tumors is integral for improving patient outcomes. In this study, we applied a computational drug repositioning approach to identify potential agents to sensitize primary drug resistant breast cancers. We extracted drug resistance profiles from the I-SPY 2 TRIAL, a neoadjuvant trial for early stage breast cancer, by comparing gene expression profiles of responder and non-responder patients stratified into treatments within HR/HER2 receptor subtypes, yielding 17 treatment-subtype pairs. We then used a rank-based pattern-matching strategy to identify compounds in the Connectivity Map, a database of cell line derived drug perturbation profiles, that can reverse these signatures in a breast cancer cell line. We hypothesize that reversing these drug resistance signatures will sensitize tumors to treatment and prolong survival. We found that few individual genes are shared among the drug resistance profiles of different agents. At the pathway level, however, we found enrichment of immune pathways in the responders in 8 treatments within the HR+HER2+, HR+HER2-, and HR-HER2- receptor subtypes. We also found enrichment of estrogen response pathways in the non-responders in 10 treatments primarily within the hormone receptor positive subtypes. Although most of our drug predictions are unique to treatment arms and receptor subtypes, our drug repositioning pipeline identified the estrogen receptor antagonist fulvestrant as a compound that can potentially reverse resistance across 13/17 of the treatments and receptor subtypes including HR+ and triple negative. While fulvestrant showed limited efficacy when tested in a panel of 5 paclitaxel-resistant breast cancer cell lines, it did increase drug response in combination with paclitaxel in HCC-1937, a triple negative breast cancer cell line. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.31.534207v1?rss=1 Authors: van Veen, S., Kourti, A., Ausloos, E., Van Asselberghs, J., Van den Haute, C., Baekelandt, V., Eggermont, J., Vangheluwe, P. Abstract: Polyamine homeostasis is disturbed in several human diseases, including cancer, which is hallmarked by increased intracellular polyamine levels and an upregulated polyamine transport system (PTS). So far, the polyamine transporters contributing to the elevated levels of polyamines in cancer cells have not yet been described, despite the fact that polyamine transport inhibitors are considered for cancer therapy. Here, we tested whether upregulation of candidate polyamine transporters of the P5B-transport ATPase family is responsible for the increased PTS in the well-studied breast cancer cell line MCF7 compared to the non-tumorigenic epithelial breast cell line MCF10A. We found that MCF7 cells present elevated expression of a previously uncharacterized P5B-ATPase ATP13A4, which is responsible for the elevated polyamine uptake activity. Furthermore, MCF7 cells are more sensitive to polyamine cytotoxicity, as demonstrated by cell viability, cell death and clonogenic assays. Importantly, overexpression of ATP13A4 WT in MCF10A cells induces a MCF7 polyamine phenotype, with significantly higher uptake of BODIPY-labelled polyamines and increased sensitivity to polyamine toxicity. In conclusion, we establish ATP13A4 as a new polyamine transporter in the human PTS and show that ATP13A4 may play a major role in the increased polyamine uptake of breast cancer cells. ATP13A4 therefore emerges as a candidate therapeutic target for anticancer drugs that block the PTS. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.29.534741v1?rss=1 Authors: Latour, S., Minet, N., Boschat, A.-C., Lane, R., Laughton, D., Beer, P., Asnagli, H., Soudais, C., Bourne, T., Fischer, A., Martin, E. Abstract: The CTP nucleotide is a key precursor of nucleic acids metabolism essential for DNA replication. De novo CTP production relies on CTP synthetases 1 and 2 (CTPS1 and 2) that catalyze the conversion of UTP into CTP. CTP synthetase activity is high in proliferating cells including cancer cells, however, the respective roles of CTPS1 and CTPS2 in cell proliferation are not known. By inactivation of CTPS1 and/or CTPS2 and complementation experiments, we showed that both CTPS1 and CTPS2 are differentially required for cell proliferation. CTPS1 was more efficient in promoting proliferation than CTPS2, in association with a higher intrinsic enzymatic activity that was more resistant to inhibition by 3-Deaza-uridine, an UTP analog. The contribution of CTPS2 to cell proliferation was modest when CTPS1 was expressed, but essential in absence of CTPS1. Public databases analysis of more than 1,000 inactivated cancer cell lines for CTPS1 or CTPS2 confirmed that cell growth is highly dependent of CTPS1 but less of CTPS2. Therefore, our results demonstrate that CTPS1 is the main contributor to cell proliferation. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.02.535304v1?rss=1 Authors: Matsuo, K., Nakajima, Y., Shigeta, M., Kobayashi, D., Sakaki, S., Inoue, S., Takeshita, N., Ueyama, A., Nishikawa, K., Saba, R., Yokoyama, T., Yashiro, K. Abstract: Almost all differentiated mammalian cells have primary cilia on their surface. Ciliary dysfunction causes ciliopathy in humans. Centrosomal protein 290 (CEP290) is a ciliary protein that causes ciliopathies, localizes at the cilial base in ciliated cells, whereas it localizes to the centrosome in non-ciliated proliferating cells. The cilia-dependent function of CEP290 has been extensively studied; however, the cilia-independent function, which is likely responsible for the wider phenotypic spectra of CEP290-related ciliopathies, remains largely unknown. Here, we examined cilia-independent functions of CEP290 in non-ciliated cells. Our study showed that Cep290 function loss suppresses microtubule elongation due to microtubule organizing center malfunction. Surprisingly, CEP290 forms a complex with the adenomatous polyposis coli (APC) protein encoded by the adenomatous polyposis coli gene. The APC-CEP290 complex exists in the centrosome and on microtubule fibers. Notably, the reduced focal adhesion formation is likely responsible for the Cep290 mutant phenotypes, including impaired directed cell migration, shrunken cell shape, and reduced adhesive capacity to the extracellular matrix. The APC-CEP290 complex is consistently important for transporting a focal adhesion molecule, paxillin, to focal adhesions in non-ciliated cells. Thus, our findings provide a novel platform to better understand the ciliopathies. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.02.535296v1?rss=1 Authors: Lee, Y.-T., Savini, M., Chen, T., Yang, J., Zhao, Q., Ding, L., Gao, S. M., Senturk, M., Sowa, J., Wang, J. D., Wang, M. C. Abstract: Healthy mitochondria are critical for reproduction. During aging, both reproductive fitness and mitochondrial homeostasis decline. Mitochondrial metabolism and dynamics are key factors in supporting mitochondrial homeostasis. However, how they are coupled to control reproductive health remains unclear. We report that mitochondrial GTP metabolism acts through mitochondrial dynamics factors to regulate reproductive aging. We discovered that germline-only inactivation of GTP- but not ATP-specific succinyl-CoA synthetase (SCS), promotes reproductive longevity in Caenorhabditis elegans. We further revealed an age-associated increase in mitochondrial clustering surrounding oocyte nuclei, which is attenuated by the GTP-specific SCS inactivation. Germline-only induction of mitochondrial fission factors sufficiently promotes mitochondrial dispersion and reproductive longevity. Moreover, we discovered that bacterial inputs affect mitochondrial GTP and dynamics factors to modulate reproductive aging. These results demonstrate the significance of mitochondrial GTP metabolism in regulating oocyte mitochondrial homeostasis and reproductive longevity and reveal mitochondrial fission induction as an effective strategy to improve reproductive health. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.02.535287v1?rss=1 Authors: Bui, D. L. H., Roach, A., Li, J., Bandekar, S. J., Orput, E., Raghavan, R., Arac, D., Sando, R. Abstract: Adhesion GPCRs (aGPCRs) are a large GPCR class that direct diverse fundamental biological processes. One prominent mechanism for aGPCR agonism involves autoproteolytic cleavage, which generates an activating, membrane-proximal tethered agonist (TA). How universal this mechanism is for all aGPCRs is unclear. Here, we investigate G protein induction principles of aGPCRs using mammalian LPHN3 and CELSR1-3, members of two aGPCR families conserved from invertebrates to vertebrates. LPHNs and CELSRs mediate fundamental aspects of brain development, yet CELSR signaling mechanisms are unknown. We found that CELSR1 and CELSR3 are cleavage-deficient, while CELSR2 is efficiently cleaved. Despite differential autoproteolysis, CELSR1-3 all engage GS, and CELSR1 or CELSR3 TA point mutants retain GS coupling activity. CELSR2 autoproteolysis enhances GS coupling, yet acute TA exposure alone is insufficient. These studies support that aGPCRs signal via multiple paradigms and provide insights into CELSR biological function. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.02.535249v1?rss=1 Authors: Pemberton, J. M., Osterlund, E. J., Schoormann, W., Pogmore, J., Nguyen, D., Leber, B., Andrews, D. Abstract: Anti-apoptotic proteins such as BCL-XL promote cell survival by sequestering pro-apoptotic BCL-2 family members, an activity that frequently contributes to tumorigenesis. Thus, the development of small-molecule inhibitors for anti-apoptotic proteins, termed BH3-mimetics, is revolutionizing how we treat cancer. BH3 mimetics kill cells by displacing sequestered pro-apoptotic proteins to initiate tumor-cell death. Recent evidence has demonstrated that in live cells the BH3-only proteins PUMA and BIM resist displacement by BH3-mimetics, while others like tBID do not. Analysis of the molecular mechanism by which PUMA resists BH3-mimetic mediated displacement from full-length anti-apoptotic proteins (BCL-XL, BCL-2, BCL-W and MCL-1) reveals that both the BH3-motif and a novel binding site within the carboxyl-terminal sequence (CTS) of PUMA contribute to binding. Together these sequences bind to anti-apoptotic proteins, which effectively "double-bolt locks" the proteins to resist BH3-mimetic displacement. The pro-apoptotic protein BIM has also been shown to double-bolt lock to anti-apoptotic proteins however, the novel binding sequence in PUMA is unrelated to that in the CTS of BIM and functions independent of PUMA binding to membranes. Moreover, contrary to previous reports, we find that when exogenously expressed, the CTS of PUMA directs the protein primarily to the endoplasmic reticulum (ER) rather than mitochondria and that residues I175 and P180 within the CTS are required for both ER localization and BH3-mimetic resistance. Understanding how PUMA resists BH3-mimetic displacement will be useful in designing more efficacious small-molecule inhibitors of anti-apoptotic BCL-2 proteins. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.01.535227v1?rss=1 Authors: Yamamoto, M., Miyoshi, M., Morioka, K., Mitani, T., Takaya, T. Abstract: A myogenetic oligodeoxynucleotide, iSN04, is the 18-base single-stranded DNA that acts as an anti-nucleolin aptamer. iSN04 has been reported to restore myogenic differentiation by suppressing inflammatory responses in myoblasts isolated from patients with diabetes or healthy myoblasts exposed to cancer-releasing factors. Thus, iSN04 is expected to be a nucleic acid drug for the muscle wasting associated with chronic diseases. The present study investigated the anti-inflammatory mechanism of iSN04 in the murine myoblast cell line C2C12. Tumor necrosis factor- (TNF-) or Toll-like receptor (TLR) ligands (Pam3CSK4 and FSL-1) induced nuclear translocation and transcriptional activity of nuclear factor-{kappa}B (NF-{kappa}B), resulting in upregulated expression of TNF- and interleukin-6. Pre-treatment with iSN04 significantly suppressed these inflammatory responses by inhibiting the nuclear accumulation of {beta}-catenin induced by TNF- or TLR ligands. These results demonstrate that antagonizing nucleolin with iSN04 downregulates the inflammatory effect mediated by the {beta}-catenin/NF-{kappa}B signaling pathway in myoblasts. In addition, the anti-inflammatory effects of iSN04 were also observed in smooth muscle cells and pre-adipocytes, suggesting that iSN04 may be useful in preventing inflammation induced by metabolic disorders. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.31.535181v1?rss=1 Authors: Dutta, A., Halder, P., Gayen, A., Mukherjee, A., Mukherjee, C., Majumder, S. Abstract: Primary cilia (PCs) that are present in most human cells and perform sensory function or signal transduction are lost in many solid tumors. Previously, we identified VDAC1, best known to regulate mitochondrial bioenergetics, to negatively regulate ciliogenesis. Here, we show that downregulation of VDAC1 in pancreatic cancer-derived Panc1 and glioblastoma-derived U-87 cells significantly increased ciliation. Those PCs were remarkably longer than the control cells. Such increased ciliation inhibited cell cycle, which contributed to reduced proliferation of these cells. VDAC1-depletion also led to longer PCs in quiescent RPE1 cells. Therefore, serum-induced PC disassembly was slower in VDAC1-depleted RPE1 cells. Overall, this study reiterates the importance of VDAC1 in modulating tumorigenesis, due to its novel role in regulating PC length and disassembly. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.01.535208v1?rss=1 Authors: Daly, C., Guseinov, A. A., Hahn, H., Tikhonova, I., Thomsen, A. R. B., Plouffe, B. Abstract: The vasopressin type 2 receptor (V2R) is an essential GPCR in renal regulation of water homeostasis. Upon stimulation, the V2R activates Gs and Gq/11, which is followed by robust recruitment of {beta}-arrestins and receptor internalization into endosomes. Unlike canonical GPCR signaling, the {beta}-arrestin association with the V2R does not terminate Gs activation, and thus, Gs-mediated signaling is sustained while the receptor is internalized. Here, we demonstrate that this V2R ability to co-interact with G protein/{beta}-arrestin and promote endosomal G protein signaling is not restricted to Gs, but also involves Gq/11. Furthermore, our data implies that {beta}-arrestins potentiate Gs/Gq/11activation at endosomes rather than terminating their signaling. Surprisingly, we found that the V2R internalizes and promote endosomal G protein activation independent of {beta}-arrestins to a minor degree. These new observations challenge the current model of endosomal GPCR signaling and suggest that this event can occur in both {beta}-arrestin-dependent and -independent manners. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.30.534992v1?rss=1 Authors: Wald, T., Verma, A., Cooley, V., Marangoni, P., Cazares, O., Sharir, A., Sandoval, E. J., Sung, D., Najibi, H., Drennon, T. Y., Bush, J. O., Joester, D., Klein, O. D. Abstract: Amelogenesis, the formation of dental enamel, is driven by specialized epithelial cells called ameloblasts, which undergo successive stages of differentiation. Ameloblasts secrete enamel matrix proteins (EMPs), proteases, calcium, and phosphate ions in a stage-specific manner to form mature tooth enamel. Developmental defects in tooth enamel are common in humans, and they can greatly impact the well-being of affected individuals. Our understanding of amelogenesis and developmental pathologies is rooted in past studies using epithelial Cre driver and knockout alleles. However, the available mouse models are limited, as most do not allow targeting different ameloblast subpopulations, and constitutive loss of EMPs often results in severe phenotype in the mineral, making it difficult to interpret defect mechanisms. Herein, we report on the de-sign and verification of a toolkit of twelve mouse alleles that include ameloblast-stage specific Cre recombinases, fluorescent reporter alleles, and conditional flox alleles for the major EMPs. We show how these models may be used for applications such as sorting of live stage specific ameloblasts, whole mount imaging, and experiments with incisor explants. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.01.534865v1?rss=1 Authors: He, N., Depta, L., Rossetti, C., Cigler, M., Michon, M., Dan, O. R., Hoock, J. G. F., Barbier, J., Gillet, D., Forrester, A., Winter, G. E., Laraia, L. Abstract: Sterol-binding proteins are important regulators of lipid homeostasis and membrane integrity; however, the discovery of selective small molecule modulators can be challenging due to structural similarities in the sterol binding domains. We report the discovery of highly potent and selective inhibitors of oxysterol binding protein (OSBP), which we term oxybipins. Sterol-containing chemical chimeras aimed at identifying new sterol binding proteins by targeted degradation, led to a significant reduction in Golgi-associated proteins. The degradation was found to occur at lysosomes, concomitant with changes in general protein glycosylation, indicating that the degradation of Golgi proteins was a downstream effect. By establishing a sterol transport protein biophysical assay panel, we discovered that the oxybipins potently inhibited OSBP, resulting in blockage of retrograde trafficking and attenuating Shiga toxin toxicity. As the oxybipins do not target any other sterol transporters tested, we advocate their use as chemical tools to study OSBP function and therapeutic relevance. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.01.535233v1?rss=1 Authors: Zhang, P.-P., Benske, T. M., Paton, J. C., Paton, A. W., Mu, T., Wang, Y.-J. Abstract: The GRIN genes encoding N-methyl-D-aspartate receptor (NMDAR) subunits are remarkably intolerant to variation. Many pathogenic NMDAR variants result in their protein misfolding, inefficient assembly, reduced surface expression, and impaired functionality at the plasma membrane, causing neurological disorders including epilepsy and intellectual disability. Here, we concentrate on the proteostasis maintenance of NMDARs containing epilepsy-associated variations in the GluN2A (or NR2A) subunit, including M705V and A727T. We showed that these two variants are targeted to the proteasome for degradation and have reduced functional surface expression. We demonstrated that the application of BIX, a known small molecule activator of an HSP70 family chaperone BiP (Binding immunoglobulin Protein) in the endoplasmic reticulum (ER), significantly increases total and surface protein levels, and thus the function of the M705V and A727T variants in HEK293T cells. Mechanistic studies revealed that BIX promotes folding, inhibits degradation, and enhances anterograde trafficking of the M705V variant by modest activation of the IRE1 pathway of the unfolded protein response. Our results showed that adapting the ER proteostasis network restores the folding, trafficking, and function of pathogenic NMDAR variants, representing a potential treatment for neurological disorders resulting from NMDAR dysfunction. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.31.535113v1?rss=1 Authors: Litschel, T., Kelley, C. F., Cheng, X., Babl, L., Mizuno, N., Case, L. B., Schwille, P. Abstract: Focal adhesions form liquid-like assemblies around activated integrin receptors at the plasma membrane. Made up of hundreds of proteins, focal adhesions are dynamic structures which can assemble and disassemble quickly, withstand strong actomyosin-applied forces, and form highly stable complexes. How they achieve these flexible characteristics is not well understood. Here, we use recombinant focal adhesion proteins to reconstitute the core structural machinery in vitro, with the goal of understanding the underlying protein dynamics and interactions. We observe liquid-liquid phase separation of the core focal adhesion proteins talin and vinculin for a spectrum of conditions and in combination with several interaction partners. Intriguingly, we show that membrane binding triggers phase separation of these proteins on the membrane, which in turn induces the enrichment of integrin in the clusters. We also introduce a novel experimental setup to probe talin-membrane interactions down to the single protein level. Our results suggest that membrane composition triggers condensate assembly at the membrane, a regulatory mechanism which could widely apply to membrane-localized biomolecular condensates and provide a pathway of how spatial organization of lipids within the membrane can couple into the cytosol. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.30.534746v1?rss=1 Authors: Kettel, P., Marosits, L., Spinetti, E., Rechberger, M., Radler, P., Niedermoser, I., Fischer, I., Versteeg, G. A., Loose, M., Covino, R., Karagoz, G. E. Abstract: Upon accumulation of unfolded proteins at the endoplasmic reticulum (ER), IRE1 activates the unfolded protein response (UPR) to restore protein-folding homeostasis. During ER stress, the ER lumenal domain (LD) of IRE1 drives its clustering on the ER membrane to initiate signaling. How IRE1 LD assembles into high-order oligomers remains largely unknown. By in vitro reconstitution experiments we show that human IRE1 LD forms dynamic biomolecular condensates. IRE1 LD condensates were stabilized when IRE1 LD was tethered to model membranes and upon binding of unfolded polypeptide ligands. Molecular dynamics simulations suggested that weak multivalent interactions are involved in IRE1 LD assemblies. Mutagenesis showed that disordered regions in IRE1 LD control its clustering in vitro and in cells. Importantly, dysregulated clustering led to defects in IRE1 signaling. Our results reveal that membranes and unfolded polypeptides act as scaffolds to assemble dynamic IRE1 condensates into stable, signaling competent clusters. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.31.535139v1?rss=1 Authors: Connor, O. M., Matta, S. K., Friedman, J. R. Abstract: Mitochondria are highly dynamic double membrane-bound organelles that maintain their shape in part through fission and fusion. Mitochondrial fission is performed by the dynamin-related protein Dnm1 (Drp1 in humans), a large GTPase that constricts and divides the mitochondria in a GTP hydrolysis-dependent manner. However, it is unclear whether factors inside mitochondria help coordinate the process and if Dnm1/Drp1 activity alone is sufficient to complete fission of both mitochondrial membranes. Here, we identify an intermembrane space protein required for mitochondrial fission in yeast, which we propose to name Mdi1. Loss of Mdi1 leads to hyper-fused mitochondria networks due to defects in mitochondrial fission, but not lack of Dnm1 recruitment to mitochondria. Mdi1 plays a conserved role in fungal species and its homologs contain a putative amphipathic alpha-helix, mutations in which disrupt mitochondrial morphology. One model to explain these findings is that Mdi1 associates with and distorts the mitochondrial inner membrane to enable Dnm1 to robustly complete fission. Our work reveals that Dnm1 cannot efficiently divide mitochondria without the coordinated function of a protein that resides inside mitochondria. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.31.535026v1?rss=1 Authors: Deolal, P., Ramalingam, K., Das, B., Mishra, K. Abstract: When yeast cells are exposed to nutrient-limiting conditions, they undergo transcriptional and translational reprogramming that results in the remodeling of metabolite utilization and organelle architecture. Organelle membranes and contacts also undergo structural and functional alterations. In the budding yeast Saccharomyces cerevisiae, regulated expression of Uip4 is shown to be a critical effector of nuclear shape and function, particularly during the stationary phase. In this work, we demonstrate that the absence of UIP4 affects the morphology of multiple other organelles including mitochondria, endoplasmic reticulum, vacuole and the distribution of lipid droplets. The results show that modulating carbon source, nitrogen availability and cellular energy state impact Uip4 expression. This expression of Uip4 is controlled by the transcription factor Msn2, downstream of Sch9 signaling pathway. Cells lacking Uip4 have poor survival in the stationary phase of the growth cycle. These cellular changes are concomitant with dysregulation of the global lipidome profile and aberrant organelle interaction. We propose that the dynamic and regulated expression of Uip4 is required to maintain lipid homeostasis and organelle architecture which is ultimately required to survive in nutrient-limiting conditions such as stationary phase. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.30.534617v1?rss=1 Authors: Gonzalez-Jimenez, P., Duarte, S., Martinez, A. E., Navarro-Carrasco, E., Lalioti, V., Pajares, M. A., Perez-Sala, D. Abstract: Cysteine residues can undergo multiple posttranslational modifications with diverse functional consequences, potentially behaving as tunable sensors. The intermediate filament protein vimentin has important implications in pathophysiology, including cancer progression, infection, and fibrosis, and maintains a close interplay with other cytoskeletal structures, such as actin filaments and microtubules. We previously showed that the single vimentin cysteine, C328, is a key target for oxidants and electrophiles. Here, we demonstrate that structurally diverse cysteine-reactive agents, including electrophilic mediators, oxidants and drug-related compounds, disrupt the vimentin network eliciting morphologically distinct reorganizations. As most of these agents display broad reactivity, we pinpointed the importance of C328 by confirming that local perturbations introduced through mutagenesis provoke structure-dependent vimentin rearrangements. Thus, GFP-vimentin wild type (wt) forms squiggles and short filaments in vimentin-deficient cells, the C328F, C328W, and C328H mutants generate diverse filamentous assemblies, and the C328A and C328D constructs fail to elongate yielding dots. Remarkably, vimentin C328H structures resemble the wt, but are strongly resistant to electrophile-elicited disruption. Therefore, the C328H mutant allows elucidating whether cysteine-dependent vimentin reorganization influences other cellular responses to reactive agents. Electrophiles such as 1,4-dinitro-1H-imidazole and 4-hydroxynonenal induce robust actin stress fibers in cells expressing vimentin wt. Strikingly, under these conditions, vimentin C328H expression blunts electrophile-elicited stress fiber formation, apparently acting upstream of RhoA. Analysis of additional vimentin C328 mutants shows that electrophile-sensitive and assembly-defective vimentin variants permit induction of stress fibers by reactive species, whereas electrophile-resistant filamentous vimentin structures prevent it. Together, our results suggest that vimentin acts as a break for actin stress fibers formation, which would be released by C328-aided disruption, thus allowing full actin remodeling in response to oxidants and electrophiles. These observations postulate C328 as a sensor transducing structurally diverse modifications into fine-tuned vimentin network rearrangements, and a gatekeeper for certain electrophiles in the interplay with actin. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.31.535033v1?rss=1 Authors: Aslan, M. K., Fourneaux, C., Yilmaz, A., Stavros, S., Parmentier, R., Paldi, A., Gonin-Giraud, S., deMello, A. J., Gandrillon, O. Abstract: Cell lineage tracking is a long-standing and unresolved problem in biology. Microfluidic technologies have the potential to address this problem, by virtue of their ability to manipulate and process single-cells in a rapid, controllable and efficient manner. Indeed, when coupled with traditional imaging approaches, microfluidic systems allow the experimentalist to follow single-cell divisions over time. Herein, we present a valve-based microfluidic system able to probe the decision-making processes of single-cells, by tracking their lineage over multiple generations. The system operates by trapping single-cells within growth chambers, allowing the trapped cells to grow and divide, isolating sister cells after a user-defined number of divisions and finally extracting them for downstream transcriptome analysis. The platform incorporates multiple cell manipulation operations, image processing-based automation for cell loading and growth monitoring, reagent addition and device washing. To demonstrate the efficacy of the microfluidic workflow, 6C2 (chicken erythroleukemia) and T2EC (primary chicken erythrocytic progenitors) cells are tracked inside the microfluidic device over two generations, with a cell viability rate in excess of 90%. Sister cells are successfully isolated after division and extracted within a 500 nL volume, which is compatible with downstream single-cell RNA sequencing analysis. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.30.534956v1?rss=1 Authors: Oprescu, S. N., Baumann, N., Chen, X., Sun, Q., Zhao, Y., Yue, F., Wang, H., Kuang, S. Abstract: Transcription factors (TFs) play key roles in regulating the differentiation and function of stem cells, including muscle satellite cells (MuSCs), a resident stem cell population responsible for postnatal regeneration of the skeletal muscle. Sox11 belongs to the Sry-related HMG-box (SOX) family of TFs that play diverse roles in stem cell behavior and tissue specification. Analysis of single-cell RNA-sequencing (scRNA-seq) datasets identify a specific enrichment of Sox11 mRNA in differentiating but not quiescent MuSCs. Consistent with the scRNA-seq data, Sox11 levels increase during differentiation of murine primary myoblasts in vitro. scRNA-seq data comparing muscle regeneration in young and old mice further demonstrate that Sox11 expression is reduced in aged MuSCs. Age-related decline of Sox11 expression is associated with reduced chromatin contacts within the topologically associated domains. Unexpectedly, Myod1Cre-driven deletion of Sox11 in embryonic myoblasts has no effects on muscle development and growth, resulting in apparently healthy muscles that regenerate normally. Pax7CreER or Rosa26CreER driven (MuSC-specific or global) deletion of Sox11 in adult mice similarly has no effects on MuSC differentiation or muscle regeneration. These results identify Sox11 as a novel myogenic differentiation marker with reduced expression in quiescent and aged MuSCs, but the specific function of Sox11 in myogenesis remain to be elucidated. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.31.535067v1?rss=1 Authors: Jokl, E., Mullan, A., Simpson, K., Birchall, L., Pearmain, L., Martin, K., Pritchett, J., Shah, R., Hodson, N., Williams, C., Camacho, E., Zeef, L., Donaldson, I., Athwal, V., Hanley, N., Piper Hanley, K. Abstract: Myofibroblasts are responsible for scarring and organ stiffness during fibrosis. The scar propagates mechanical signals inducing a radical transformation in myofibroblast cell state linked to an increasingly pro-fibrotic phenotype. Here, we have discovered mechanical stress from progressive scarring induces nuclear softening and de-repression of heterochromatin. The parallel loss of H3K9Me3 enables a permissive state for distinct chromatin accessibility and profibrotic gene regulation. By integrating chromatin accessibility profiles (ATAC sequencing) we provide insight into the transcription network and open chromatin landscape underlying the switch in profibrotic myofibroblast states, emphasizing mechanoadaptive pathways linked to PAK1 as key drivers. Through genetic manipulation in liver and lung fibrosis, uncoupling PAK1-dependant signaling impaired the mechanoadaptive response in vitro and dramatically improved fibrosis in vivo. Moreover, we provide human validation for mechanisms underpinning PAK1 mediated mechanotransduction in liver and lung fibrosis. Collectively, these observations provide new insight into the nuclear mechanics driving the profibrotic chromatin landscape in fibrosis, highlighting actomyosin-dependent mechanisms linked to chromatin organisation as urgently needed therapeutic targets in fibrosis. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.31.535027v1?rss=1 Authors: Armendariz, I., Lopez de Heredia, U., Soler, M., Puigdemont, A., Ruiz, M. M., Jove, P., Soto, A., Serra, O., Figueras, M. Abstract: The periderm is basic for land plants due to its protective role during radial growth, which is achieved by the polymers deposited in the cell walls. In most trees, like holm oak, the periderm is frequently replaced by subsequent internal periderms yielding a heterogeneous outer bark made of a mixture of periderms and phloem tissues, known as rhytidome. Exceptionally, cork oak forms a persistent or long-lived periderm which results in a homogeneous outer bark of thick phellem cell layers known as cork. Here we use the outer bark of cork oak, holm oak, and their natural hybrids to analyse the chemical composition, the anatomy and the transcriptome, and further understand the mechanisms underlying periderm development. The inclusion of hybrid samples showing rhytidome-type and cork-type barks is valuable to approach to cork and rhytidome development, allowing an accurate identification of candidate genes and processes. The present study underscores that biotic stress and cell death signalling are enhanced in rhytidome-type barks whereas lipid metabolism and cell cycle are enriched in cork-type barks. Development-related DEGs, showing the highest expression, highlight cell division, cell 47 expansion, and cell differentiation as key processes leading to cork or rhytidome-type barks. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.31.535147v1?rss=1 Authors: Hladyshau, S., Stoop, J. P., Kamada, K., Nie, S., Tsygankov, D. V. Abstract: Rho-GTPases are central regulators within a complex signaling network that controls the cytoskeletal organization and cell movement. This network includes multiple GTPases, such as the most studied Rac1, Cdc42, and RhoA, and their numerous effectors that provide mutual regulation and feedback loops. Here we investigate the temporal and spatial relationship between Rac1 and Cdc42 during membrane ruffling using a simulation model which couples GTPase signaling with cell morphodynamics to capture the GTPase behavior observed with FRET-based biosensors. We show that membrane velocity is regulated by the kinetic rate of GTPase activation rather than the concentration of active GTPase. Our model captures both uniform and polarized ruffling. We also show that cell-type specific time delays between Rac1 and Cdc42 activation can be reproduced with a single signaling motif, in which the delay is controlled by feedback from Cdc42 to Rac1. The resolution of our simulation output matches those of the time-lapsed recordings of cell dynamics and GTPase activity. This approach allows us to validate simulation results with quantitative precision using the same pipeline for the analysis of simulated and experimental data. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.31.535108v1?rss=1 Authors: Skoufou-Papoutsaki, N., Adler, S., D'Santos, P., Mannion, L., Mehmed, S., Kemp, R., Smith, A., Perrone, F., Nayak, K., Russell, A., Zilbauer, M., Winton, D. J. Abstract: Organoids are currently one of the most widely used ex vivo models in epithelial biology. Combined with genetic editing strategies, organoids offer a promise of rapid and efficient investigation of gene function in many models of human disease. However, to date, the editing efficiency of organoids with the use of non-viral electroporation methods has been only up to 30%, with implications for the subsequent need for selection including including turnaround time and exhaustion or adaptation of the organoid population. Here, we describe an efficient method of intestinal organoid editing using a Ribonucleoprotein CRISPR-based approach. Editing efficiencies of up to 98% in target genes were robustly achieved across different anatomical gut locations and developmental timepoints from multiple patient samples with no off-target editing. The method allowed us to study the effect of the loss of the tumour suppressor gene, PTEN, in normal human intestinal cells. Analysis of PTEN deficient organoids defined phenotypes that likely relate to its tumour suppressive function in vivo, such as a proliferative advantage and increased organoid budding. Transcriptional profiling revealed differential expression of genes in pathways commonly known to be associated with PTEN loss including mTORC1 activation. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.30.534946v1?rss=1 Authors: Hutchins, J. R. A., Peiffer, I., Urbach, S., Mergny, J.-L., Marin, P., Maiorano, D., MECHALI, M. Abstract: In metazoan cells, replication of genomic DNA initiates from thousands of discrete chromosomal loci known as origins. Proteins such as the Origin Recognition Complex (ORCs) associate with origins, but this does not show clear sequence specificity for DNA binding. Genome-wide origin mapping studies have shown that the region surrounding the replication initiation site contains motifs such as the Origin G-rich Repeated Element (OGRE), proximal to the majority of origins. Here, using an approach coupling DNA affinity purification to quantitative proteomics, we identified proteins that interact specifically with an OGRE. Three of the top-scoring interactors, Dhx36, Pura and Tial1, were selected for further study. We show that Dhx36 and Tial1 localise to the nucleus and their knockdown decreased cells in S-phase resulting in their accumulation in the G1 phase of the cell cycle. Altogether these results indicate that these OGRE-binding factors may play roles in DNA synthesis in mammalian cells. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.30.534941v1?rss=1 Authors: Ingram, S., DeCorte, A., Gentry, A. E., Philpott, M. K., Moldenhauer, T., Stadler, S., Steinberg, C., Millman, J., Ehrhardt, C. J. Abstract: Analysis of DNA mixtures from sexual assault evidence is an ongoing challenge for DNA casework laboratories. There is a significant need for new techniques that can provide information as to the source of DNA, particularly for sexual assault samples that do not involve semen. The goal of this study was to develop a new biological signature system that provides additional probative value to samples comprised of mixtures of epidermal and vaginal cells, as may be observed in cases involving digital penetration. Signatures were based on morphological and autofluorescence properties of individual cells collected through Imaging Flow Cytometry (IFC). Comparisons to reference cell populations from vaginal tissue and epidermal cells collected from hands showed strong multivariate differences across greater than 80 cellular measurements. These differences were used to build a predictive framework for classifying unknown cell populations as originating from epithelial cells associated with digital penetration or epidermal tissue. As part of the classification scheme, posterior probabilities of specific tissue group membership were calculated for each cell, along with multivariate similarity to that tissue type. We tested this approach on cell populations from reference tissue as well as mock casework samples involving digital penetration. Many more cells classifying as non-epidermal tissue were detected in digital penetration samples than control hand swabbings. Minimum interpretation thresholds were developed to minimize false positives; these thresholds were also effective when screening licked hands, indicating the potential utility of this method for a variety of biological mixture types and depositional events relevant to forensic casework. Results showed that samples collected subsequent to digital penetration possessed markedly higher numbers of cells classifying as vaginal tissue as well as higher posterior probabilities for vaginal tissue ( greater than or equal to 0.90) compared to cell populations collected from hands without prior contact with vaginal tissue. Additionally, digital penetration cell populations may be resolved from saliva cell populations and other non-target tissue types. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.31.535068v1?rss=1 Authors: Shi, Q., Zheng, L. Abstract: Long-term space flight will lead to bone loss and osteoblasts dysfunction. The underlying mechanism is still far to reveal. Frizzled-9 (Fzd9) is a Wnt receptor which is essential to osteoblasts differentiation and bone formation. Here we investigate whether Fzd9 plays a role in simulated microgravity (SMG) induced osteoblasts dysfunction. After 1-3 days of SMG, the osteogenic markers were decreased which accompanied the decline of Fzd9 expression. Fzd9 also decreased in the femur of the rats after 3 weeks of hindlimb unloading. Overexpression of Fzd9 will counteract SMG-induced osteoblasts dysfunction. However, Fzd9 overexpression did not affect SMG induced pGSK3 and -catenin expression or sublocalization. Overexpression of Fzd9 regulates the phosphorylation of Akt and ERK, as well as induces F-actin polymerization to form the actin cap, presses the nuclei, and increases the nuclear pore size, which promotes nuclear translocation of YAP. Our study provides mechanistic insights into the role of Fzd9 regulates YAP in SMG-mediated osteoblasts dysfunction and indicates Fzd9 as a potential target to restore osteoblast function in bone diseases and space flight. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.31.535077v1?rss=1 Authors: Santos, C., Rogers, S. L., Carter, A. P. Abstract: Cytoplasmic microtubules are tubular polymers that can harbor small proteins or filaments inside their lumen. The identity of these objects and what causes their accumulation has not been conclusively established. Here, we used cryogenic electron tomography (cryoET) of Drosophila S2 cell protrusions and found filaments inside the microtubule lumen, which resemble those reported recently in human HAP1 cells. The frequency of these filaments increased upon inhibition of the sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) with the small-molecule drug thapsigargin. Subtomogram averaging showed that the luminal filaments adopt a helical structure reminiscent of cofilin-bound actin (cofilactin). Consistent with this, cofilin was activated in cells under the same conditions that increased luminal filament occurrence. Furthermore, RNAi knock-down of cofilin reduced the frequency of luminal filaments with cofilactin morphology. These results suggest that cofilin activation stimulates its accumulation on actin filaments inside the microtubule lumen. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.29.534783v1?rss=1 Authors: Bychkov, I., Topchu, I., Makhov, P., Kudinov, A., Patel, J. D., Boumber, Y. Abstract: Lung cancer is the most frequently diagnosed cancer type and the leading cause of cancer-related deaths worldwide. Non-small cell lung cancer (NSCLC) represents most of the lung cancer. Vascular endothelial growth factor receptor-2 (VEGFR2) is a member of the VEGF family of receptor tyrosine kinase proteins, expressed on both endothelial and tumor cells which is one of the key proteins contributing to cancer development and involved in drug resistance. We previously showed that Musashi-2 (MSI2) RNA-binding protein is associated with NSCLC progression by regulating several signaling pathways relevant to NSCLC. In this study, we performed Reverse Protein Phase Array (RPPA) analysis of murine lung cancer which nominated VEGFR2 protein as strongly positively regulated by MSI2. Next, we validated VEGFR2 protein regulation by MSI2 in several human NSCLC cell line models. Additionally, we found that MSI2 affected AKT signaling via negative PTEN mRNA translation regulation. In silico prediction analysis suggested that both VEGFR2 and PTEN mRNAs have predicted binding sites for MSI2. We next performed RNA immunoprecipitation coupled with quantitative PCR which confirmed that MSI2 directly binds to VEGFR2 and PTEN mRNAs, suggesting direct regulation mechanism. Finally, MSI2 expression positively correlated with VEGFR2 and VEGF-A protein levels in human NSCLC samples. We conclude that MSI2/VEGFR2 axis contributes to NSCLC progression and is worth further investigations and therapeutic targeting. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.31.534530v1?rss=1 Authors: Sun, X.-M., Miles, G. J., Craxton, A., Powley, I. R., Galavotti, S., Chernova, T., Dawson, A., Nakas, A., Willis, A. E., Cain, K., MacFarlane, M. Abstract: Malignant pleural mesothelioma (MPM) is an aggressive malignancy linked to asbestos exposure and highly resistant to chemotherapy, potentially due to upregulated expression of the pro-survival proteins, BCL2/BCL-XL/MCL-1. Using clinically-relevant models of MPM we show that patient-derived primary MPM cell lines and ex-vivo 3D tumour explants are highly resistant to apoptosis induced by the BCL2/BCL-XL inhibitor, ABT-737. Importantly, we discover that 2-deoxyglucose (2DG), a glycolytic inhibitor, can sensitize MPM cells to ABT-737 and show this correlates with loss of the pro-survival protein, MCL-1. siRNA knockdown of MCL-1 (MCL-1 KD) combined with ABT-737 induced BAX/BAK-dependent, but BIM/PUMA-independent apoptosis, mimicking 2DG/ABT-737 treatment. MCL-1 KD/ABT-737 induced mitochondrial cytochrome c release and caspase-independent inhibition of mitochondrial respiration. Moreover, we observed a hitherto unreported caspase-dependent cleavage of glycolytic enzymes and subsequent inhibition of glycolysis. 2DG inhibited ERK/STAT3 activity, decreased MCL-1 mRNA and protein levels, with concurrent activation of AKT, which limited loss of MCL-1 protein. However, co-treatment with a specific AKT inhibitor, AZD5363, and 2DG/ABT-737 potently induced cell death and inhibited clonogenic cell survival, while in MPM 3D tumour explants MCL-1 protein expression decreased significantly following 2DG or 2DG/AZD5363 treatment. Notably, a similar synergy was observed in MPM cell lines and MPM 3D tumour explants using ABT-737 in combination with the recently developed MCL-1 inhibitor, S63845. Importantly, our study provides a mechanistic explanation for the chemoresistance of MPM and highlights how this can be overcome by a combination of metabolic reprogramming and/or simultaneous targeting of MCL-1 and BCL-2/BCL-XL using BH3-mimetics. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.30.534966v1?rss=1 Authors: Wang, C., Ma, H., Baserga, S., Pederson, T., Huang, S. Abstract: The nucleolus is a multi-functional nuclear body. To tease out the roles of nucleolar structure without resorting to multi-action drugs, we knocked down RNA polymerase I subunit RPA194 in HeLa cells by siRNA. Loss of RPA194 resulted in nucleolar structural segregation and effects on both nucleolus-proximal and distal nuclear components. The perinucleolar compartment was disrupted, centromere-nucleolus interactions were significantly reduced, and the intranuclear locations of specific genomic loci were altered. Moreover, Cajal bodies, distal from nucleoli, underwent morphological and compositional changes. To distinguish whether these global reorganizations are the results of nucleolar structural disruption or inhibition of ribosome synthesis, the pre-ribosomal RNA processing factor, UTP4, was also knocked down, which did not lead to nucleolar segregation, nor the intranuclear effects seen with RPA195A knockdown, demonstrating that they do not arise from a cessation of ribosome synthesis. These findings point to a commutative system that links nucleolar structure to the maintenance and spatial organization of certain nuclear bodies and genomic loci. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.31.531359v1?rss=1 Authors: Ramzan, F., Abrar, F., Kumar, A., Liao, L. M. Q., Campbell, Z. E., Gray, R. V., Akanni, O., Guyn, C. M., Martin, D. D. O. Abstract: Multi-system proteinopathy (MSP) is a rare dominantly-inherited disorder that includes a cluster of diseases, including frontotemporal dementia, inclusion body myopathy, and Paget's disease of bone. MSP is caused by mutations in the gene encoding Valosin-containing protein (VCP). Patients with the same mutation, even within the same family, can present with a different combination of any or all of these diseases, along with amyotrophic lateral sclerosis (ALS). The pleiotropic effects may be linked to the greater than 50 VCP co-factors that direct VCP's many roles in the cell. Small VCP-Interacting Protein (SVIP) is a small protein that directs VCP to autophagosomes and lysosomes. We found that SVIP directs VCP localization to autophagosomes in an acylation-dependent manner. We demonstrate that SVIP is myristoylated at glycine 2 and palmitoylated at cysteines 4 and 7. Acylation of SVIP was required to mediate cell death in the presence of the MSP- associated VCP variant (R155H-VCP), whereby blocking SVIP myristoylation rescues cytotoxicity. Therefore, SVIP acylation may present a novel target in MSP. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.31.535084v1?rss=1 Authors: Otto, T. A., Bergsma, T., Dekker, M., Mouton, S., Gallardo, P., Wolters, J. C., Steen, A., Onck, P. R., Veenhoff, L. L. Abstract: Transport through the NPC relies on intrinsically disordered FG-Nups forming a selective barrier. Away from the NPC, FG-Nups readily form condensates and aggregates, and we address how this behavior is surveilled in cells. FG-Nups, including Nsp1, together with nuclear transport receptor Kap95, form a native cytosolic condensate in yeast. In aged cells this condensate disappears as cytosolic Nsp1 levels decline. Biochemical assays and modeling show that Nsp1 is a modulator of FG-Nup liquid-liquid phase separation, promoting a liquid-like state. Nsp1s presence in the cytosol and condensates is critical, as a reduction of cytosolic levels in young cells induces NPC assembly and transport defects and a general decline in protein quality control, all quantitatively mimicking aging phenotypes. Excitingly, these phenotypes can be rescued by cytosolic Nsp1. We conclude that Nsp1 is a phase state regulator that surveils FG-Nups and impacts general protein homeostasis. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.29.534814v1?rss=1 Authors: Chang, W.-J., Harpel, Z., Circelli, J., Chen, R., Chang, I., Rivera, J., Wu, S., Wei, Z. Abstract: Tetrahymena are ciliated protists that have been used to study the effects of toxic chemicals, including anticancer drugs. In this study, we tested the inhibitory effects of six pyrimidine analogs (5-fluorouracil, floxuridine, 5-deoxy-5-fluorouridine, 5-fluorouridine, gemcitabine, cytarabine) on wild-type CU428 and conditional mutant NP1 Tetrahymena thermophila at room temperature and the restrictive temperature (37{degrees}C) where NP1 does not form the oral apparatus. We found that cytarabine was the only tested analog that did not inhibit growth, and phagocytosis was not required for pyrimidine analog entry. IC50 values did not significantly differ between strains for the same analog at either temperature. To investigate the mechanism of inhibition, we used two pyrimidine bases (uracil and thymine) and three nucleosides (uridine, thymidine, 5-methyluridine) to help determine whether the inhibitory effects from analogs were reversible. We found that the inhibitory effects from 5-fluorouracil could be reversed by uracil and thymine, from floxuridine could be reversed by thymidine, and from 5-deoxy-5-fluorouridine could be reversed by uracil. None of the tested nucleobases or nucleosides could reverse the inhibitory effects of gemcitabine or 5-fluorouridine. Our results suggest that the five pyrimidine analogs act on different sites to inhibit T. thermophila growth and that nucleobases and nucleosides are metabolized differently. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.30.533363v1?rss=1 Authors: Iuliani, I., Mbemba, G., Lagomarsino, M. C., Sclavi, B. Abstract: A long-standing hypothesis sees DNA replication control in E. coli as a central cell cycle oscillator at whose core is the DnaA protein. The consensus is that the activity of the DnaA protein, which is dependent on its nucleotide bound state, is an effector of initiation of DNA replication and a sensor of cell size. However, while several processes are known to regulate the change in DnaA activity, the oscillations in DnaA production and DnaA activity have never been observed at the single cell level, and their correlation with cell volume has yet to be established. Here, we measured the volume-specific production rate of a reporter protein under control of the dnaAP2 promoter in single cells. By a careful dissection of the effects of DnaA-ATP- and SeqA-dependent regulation of dnaAP2 promoter activity two distinct cell-cycle oscillators emerge. The first one, driven by both DnaA activity and SeqA repression, is strongly coupled to cell cycle and cell size, and its minima show the same "adder" behaviour as initiation events. The second, a reporter of DnaA activity in the absence of SeqA binding, is still coupled with cell size but not to the time of cell division, and its minima (corresponding to DnaA activity peaks) show a "sizer-like" behavior, hence deviating from actual initiations. These findings indicate that production of DnaA is tightly coupled to cell volume through the timing of gene duplication, positive and negative regulation by DnaA-ATP itself and SeqA repression, and that DnaA activity peaks are a necessary but not sufficient condition to trigger replication initiation, posing firmer quantitative bases for a mechanistic understanding of cell cycle progression in bacteria. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.30.534893v1?rss=1 Authors: Carvalho, A. M., Silva, B., Pereira, F. B., Kim, H.-N., Almeida, M., Sardao, V. A. Abstract: Estrogen deficiency increases bone resorption and is a major contributor to osteoporosis. However, the molecular mechanisms mediating the effects of estrogen on osteoclasts remain unclear. This study aimed at elucidating the early metabolic effects of RANKL, the essential cytokine for osteoclastogenesis, and 17-beta-estradiol (E2) on osteoclast progenitor cells, using RAW 264.7 macrophage cell line and bone marrow-derived macrophages as biological models. RANKL stimulated complex I activity, oxidative phosphorylation (OXPHOS), and mitochondria-derived ATP production, as early as 3 to 6 h. This up-regulation of mitochondrial bioenergetics was associated with an increased capacity to oxidize TCA cycle substrates, fatty acids, and amino-acids. E2 inhibited all effects of RANKL on mitochondria metabolism. In the presence of RANKL, E2 also decreased cell number and stimulated the mitochondrial-mediated apoptotic pathway, detected as early as 3h. Surprisingly, the pro-apoptotic effects of E2 were associated with an accumulation of p392S-p53 in mitochondria. These findings elucidate early effects of RANKL on osteoclast progenitor metabolism and suggest novel p53-mediated mechanisms that contribute to postmenopausal osteoporosis. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.29.534749v1?rss=1 Authors: van Soest, D. M. K., Polderman, P. E., den Toom, W. T., Zwakenberg, S., De Henau, S., Burgering, B. M. T., Dansen, T. B. Abstract: Reactive Oxygen Species (ROS) derived from mitochondrial respiration are frequently cited as a major source of genomic DNA damage and subsequent mutations that contribute to cancer development and aging. However, experimental evidence showing that ROS released by mitochondrial can directly damage nuclear DNA under (patho)physiological conditions has been largely lacking. In this study we modeled the effects of mitochondrial H2O2 release and compared this to H2O2 production at the nucleosomes in an untransformed human cell line. We used a chemogenetic approach to produce localized H2O2 and combined it with a new method we developed to directly quantify the amount of H2O2 produced. This enabled us to precisely investigate to what extent DNA damage occurs downstream of near- and supraphysiological amounts of localized H2O2 generation. Nuclear H2O2 production gives rise to DNA strand breaks, subsequent activation of the DNA damage response, cell cycle arrest and eventually senescence. Release of H2O2 from mitochondria on the other hand shows none of these effects, even at levels that are orders of magnitude higher than what mitochondria normally produce. Artificially high levels of mitochondrial H2O2 release do result in DNA strand breaks, but in parallel invariably cause ferroptosis-mediated cell death, preventing propagation of DNA damage-induced mutations. This study shows that H2O2 released from mitochondria is unlikely to directly damage genomic DNA, limiting its contribution to oncogenic transformation and aging. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.30.534618v1?rss=1 Authors: Govindaraj, K., Kannan, S., Karperien, M., Post, J. N. Abstract: The multi-lineage differentiation capacity of human mesenchymal stem cells (hMSCs) enables its potential for tissue engineering and regenerative medicine. Master transcription factors play a key role during development, differentiation, homeostasis and disease pathology. RUNX2 is the master transcription factor for bone development, and it regulates several important signaling pathways during chondrogenic and osteogenic differentiation of hMSCs. However, modulation of RUNX2 activity during hMSC differentiation into various lineages is not yet fully described. We differentiated hMSCs into chondro-, osteo-, and adipogenic lineages and studied RUNX2 protein dynamics using Transcription Factor - Fluorescence Recovery After Photobleaching (TF-FRAP) at different time points. The TF-FRAP method can capture the dynamic changes of RUNX2 protein mobility at the single cell level resolution, and cluster analysis shows how RUNX2 dynamics change at subpopulation level in proliferating and differentiating hMSCs. Our data show that although whole hMSC population is exposed to differentiation stimuli, some subpopulations in hMSCs do not respond to environmental cues. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.29.534651v1?rss=1 Authors: Ng, J. Q., Jafarov, T. H., Little, C. B., Wang, T., Ali, A., Ma, Y., Radford, G., Vrbanac, L., Ichinose, M., Whittle, S., Hunter, D., Lannagan, T., Suzuki, N., Goyne, J. M., Kobayashi, H., WANG, T. C., Haynes, D., Menicanin, D., Gronthos, S., Worthley, D. L., Woods, S. L., Mukherjee, S. Abstract: Osteoarthritis (OA), which carries an enormous disease burden across the world, is characterised by irreversible degeneration of articular cartilage (AC), and subsequently bone. The cellular cause of OA is unknown. Here, using lineage tracing in mice, we show that the BMP-antagonist Gremlin 1 (Grem1) marks a novel chondrogenic progenitor (CP) cell population in the articular surface that generates joint cartilage and subchondral bone during development and adulthood. Notably, this CP population is depleted in injury-induced OA, and with age. OA is also induced by toxin-mediated ablation of Grem1 CP cells in young mice. Transcriptomic analysis and functional modelling in mice revealed articular surface Grem1-lineage cells are dependent on Foxo1; ablation of Foxo1 in Grem1-lineage cells led to early OA. This analysis identified FGFR3 signalling as a therapeutic target, and injection of its activator, FGF18, caused proliferation of Grem1-lineage CP cells, increased cartilage thickness, and reduced OA pathology. We propose that OA arises from the loss of CP cells at the articular surface secondary to an imbalance in progenitor cell homeostasis and present a new progenitor population as a locus for OA therapy. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.30.531699v1?rss=1 Authors: Hosseini, A., Lindholm, H. T., Chen, R., Mehdipour, P., Marhon, S. A., Ishak, C. A., De Carvalho, D. D. Abstract: Viral mimicry describes the immune response induced by endogenous stimuli such as dsRNA formed by endogenous retroelements. Activation of viral mimicry has the potential to kill cancer cells or augment anti-tumor immune response. Paradoxically, cancer cells frequently present a dysregulated epigenome, leading to increased expression of retroelements. We previously found that ADAR1 p150 upregulation is an adaptation mechanism to tolerate high retroelement-derived dsRNA levels, leading to a druggable dependency. Here, we systematically identified novel mechanisms of viral mimicry adaptation associated with cancer cell dependencies. We correlated the gene knockout sensitivity from the DepMap dataset and interferon stimulated gene (ISG) expression in the Cancer Cell Line Encyclopedia (CCLE) dataset of 1005 human cell lines and identified pathways such as RNA modification and nucleic acid metabolism. Among the top hits was the RNA decay protein XRN1 as an essential gene for the survival of a subset of cancer cell lines. XRN1-sensitive cancer cell lines have a high level of cytosolic dsRNA and high ISG expression. Furthermore, sensitivity to XRN1 knockout was mediated by MAVS and PKR activation, indicating that the cells die due to XRN1-dependent induction of viral mimicry. XRN1-resistant cell lines had low basal dsRNA levels, but became synthetically dependent on XRN1 upon treatment with viral mimicry inducing drugs such as 5-AZA-CdR or palbociclib. Finally, XRN1-dependency is partly independent of ADAR1 activity. These results confirm the potential for our ISG correlation analysis to discover novel regulators of viral mimicry and show that XRN1 activation is an adaptive mechanism to control high dsRNA stress induced by dysregulated retroelements in cancer cells and creates a dependency that can be explored for novel cancer therapies. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.29.534690v1?rss=1 Authors: Islam, A., Chen, X.-C., Weng, C.-W., Chen, C.-Y., Wang, C.-W., Chen, M.-K., Tikhomirov, A. S., Shchekotikhin, A. E., Chueh, P. J. Abstract: The antibiotic heliomycin (resistomycin), which is generated from Streptomyces resistomycificus, has multiple activities, including anticancer effects. Heliomycin was first described in the 1960s, but its clinical applications have been hindered by extremely low solubility. A series of 4-aminomethyl derivatives of heliomycin were synthesized to increase water solubility; studies showed that they had anti-proliferative effects, but the drug targets remained unknown. In this study, we conducted cellular thermal shift assays and molecular docking simulations to identify and validate the intracellular targets of heliomycin and its water-soluble derivative, 4-(dimethylaminomethyl)heliomycin (designated compound 4-dmH), in p53-functional SAS and p53-mutated HSC-3 oral cancer cells. Consistent with our in silico studies, our cellular thermal shift assays (CETSA) revealed that, in addition to SIRT1, the water-soluble 4-dmH preferentially targeted a tumor-associated NADH oxidase called tNOX or ENOX2. The direct binding of 4-dmH to tNOX inhibited the activity of tNOX and enhanced its ubiquitin-proteasomal protein degradation in both SAS and HSC-3 cells. Moreover, the inhibition of tNOX by 4-dmH decreased the oxidation of NADH to NAD+ which diminished NAD+-dependent SIRT1 deacetylase activity, ultimately inducing apoptosis and significant cytotoxicity in both cell types. We also observed that tNOX and SIRT1 were both upregulated in tumor tissues of oral cancer patients compared to adjacent normal tissues, suggesting their clinical relevance. Finally, the better therapeutic efficacy of 4-dmH was confirmed in tumor-bearing mice, which showed greater tNOX and SIRT1 downregulation and tumor volume reduction when treated with 4-dmH compared to heliomycin. Taken together, our in vitro and in vivo findings suggest that the multifaceted properties of water-soluble 4-dmH enable it to offer superior antitumor value compared to parental heliomycin, and indicated that it functions through targeting the tNOX-NAD+-SIRT1 axis to induce apoptosis in oral cancer cells. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.29.534685v1?rss=1 Authors: Alsehli, H. S., Roy, E., Williams, T., Kuziola, A., Guo, Y., Green, J. B., Gentleman, E., Danovi, D. Abstract: In early human development, gastrulation is tightly associated with lineage specification. The interplay between mechanical forces and biochemical signals during these processes is poorly understood. Here, we dissect the effects of biochemical cues and physical confinement on a 3D in vitro model of gastrulation that uses spheroids formed from human induced pluripotent stem cells (hiPSCs). First, we compare self-renewing versus differentiating media conditions in free-floating cultures, and observe the emergence of organised tri-germ layers. In these unconfined cultures, BMP4 exposure induces polarised expression of SOX17 in conjunction with spheroid elongation. We then physically confine spheroids using PEG-peptide hydrogels and observe dramatically reduced SOX17 expression, albeit rescued if gels that soften over time are used instead. Our study combines high-content imaging, synthetic hydrogels and hiPSCs-derived models of early development to define the drivers causing changes in shape and emergence of germ layers. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.29.534670v1?rss=1 Authors: Wang, Y., Ruan, L., Zhu, J., Zhang, X., Chang, A., Tomaszewski, A., Li, R. Abstract: Mitochondria are the cellular energy hub and central target of metabolic regulation. Mitochondria also facilitate proteostasis through pathways such as the mitochondria as guardian in cytosol (MAGIC) whereby cytosolic misfolded proteins are imported into and degraded inside mitochondria. In this study, a genome-wide screen in yeast uncovered that Snf1, the yeast AMP-activated protein kinase (AMPK), inhibits the import of misfolded proteins into mitochondria while promoting mitochondrial biogenesis under glucose starvation. We show that this inhibition requires a downstream transcription factor regulating mitochondrial gene expression and is likely to be conferred through substrate competition and mitochondrial import channel selectivity. We further show that Snf1/AMPK activation protects mitochondrial fitness in yeast and human cells under stress induced by misfolded proteins such as those associated with neurodegenerative diseases. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.28.534608v1?rss=1 Authors: Asrul, A. Abstract: The use of biological agents as a biocontrol against plant pathogens was often ineffective because it worked slowly. The objective of this research was to examine the potential of local isolates of Bacillus spp. as a biocontrol agent in suppressing Fusarium wilt disease (Fusarium oxysporum f. sp. cepae) on wakegi onions. The research was designed using a completely randomized design with the treatment of rhizosphere bacterial isolates. The treatments consisted of control (without isolate application), isolates KP17, KP5, DB9, DB12, DB18, DG4, and DG11 so that the number of treatments was eight. Each treatment was repeated 5 times and each replication consisted of 10 wakegi onion plants. This research was divided into 2 stages, namely laboratory research which included isolation, characterization of colony morphology of rhizosphere bacterial isolates, and in vitro testing of the inhibitory power of biocontrol agents against pathogens. The test in the greenhouse was in the form of a disease case suppression test. The results obtained seven candidate isolates of biocontrol from 46 isolates obtained from the rhizosphere of the wakegi onion. These isolates had similarities with Bacillus spp. based on colony morphology, physiology, and biochemistry characteristics. Among the isolates found, the DB12 isolate had the potential to be developed as a biocontrol agent compared to other isolates. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.27.534456v1?rss=1 Authors: Li, J., Jin, C., Gustafsson, S., Rao, A., Wabitsch, M., Park, C. Y., Quertermous, T., Bielczyk-Maczynska, E., Knowles, J. W. Abstract: Adipogenesis is a process in which fat-specific progenitor cells (preadipocytes) differentiate into adipocytes that carry out the key metabolic functions of the adipose tissue, including glucose uptake, energy storage, and adipokine secretion. Several cell lines are routinely used to study the molecular regulation of adipogenesis, in particular the immortalized mouse 3T3-L1 line and the primary human Simpson-Golabi-Behmel syndrome (SGBS) line. However, the cell-to-cell variability of transcriptional changes prior to and during adipogenesis in these models is not well understood. Here, we present a single-cell RNA-Sequencing (scRNA-Seq) dataset collected before and during adipogenic differentiation of 3T3-L1 and SGBS cells. To minimize the effects of experimental variation, we mixed 3T3-L1 and SGBS cells and used computational analysis to demultiplex transcriptomes of mouse and human cells. In both models, adipogenesis results in the appearance of three cell clusters, corresponding to preadipocytes, early and mature adipocytes. These data provide a groundwork for comparative studies on human and mouse adipogenesis, as well as on cell-to-cell variability in gene expression during this process. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.23.532505v1?rss=1 Authors: Ge, X., Lu, L., Bai, W., Wang, M., Han, C., Du, H., Wang, N., Gao, M., Li, D., Dong, F. Abstract: Exosomes are small vesicles released from cells and present in various mammal biological fluids, such as bovine milk, which worked for skin care for many years besides dairy. In addition, Exosomes were regarded as a vehicle for intercellular communication. Therefore, we aimed to investigate the novel roles of bovine milk-derived exosomes (MK-Exo) on human skin anti-aging. Purified MK-Exo can be directly uptake by the keratinocytes and fibroblast in vitro and upregulate the expression of the natural factors related to skin moisturizing, including Filaggrin (FLG), Aquaporin 3 (AQP3), CD44 in the keratinocytes and hyaluronidase (HAS2) in the fibroblast, and MK-Exo promoted the cell migration of the fibroblast, while rescue its expression of type I collagen (Col I), type III collagen (Col III) after ultraviolet radiation. Furthermore, the phototoxicity test, photoallergy test, repeated skin irritation test, skin allergy test, and patch test confirm the safety of MK-Exo on the skin. Finally, the roles of MK-Exo in preserving moisture and anti-wrinkle were also identified in humans. Then, MK-Exo was smeared on the facial skin of 31 female volunteers twice a day for 28 days, and the functions were evaluated following the safety assessment in vivo. These studies reveal the novel roles of bovine milk-derived exosomes in human skin aging, which opens a new way of skin care. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.27.534417v1?rss=1 Authors: Itkin, T., Houghton, S., Schreiner, R., Lin, Y., Badwe, C., Voisin, V., Murison, A., Seyedhassantehrani, N., Kaufmann, K. B., Garcia-Prat, L., Booth, G. T., Geng, F., Liu, Y., Gomez-Salinero, J. M., Shieh, J.-H., Redmond, D., Xiang, J. Z., Josefowicz, S. Z., Trapnell, C., Spencer, J. A., Zangi, L., Hadland, B., Dick, J. E., Xie, S. Z., Rafii, S. Abstract: Transition between activation and quiescence programs in hematopoietic stem and progenitor cells (HSC/HSPCs) is perceived to be governed intrinsically and by microenvironmental co-adaptation. However, HSC programs dictating both transition and adaptability, remain poorly defined. Single cell multiome analysis divulging differential transcriptional activity between distinct HSPC states, indicated for the exclusive absence of Fli-1 motif from quiescent HSCs. We reveal that Fli-1 activity is essential for HSCs during regenerative hematopoiesis. Fli-1 directs activation programs while manipulating cellular sensory and output machineries, enabling HSPCs co-adoptability with a stimulated vascular niche. During regenerative conditions, Fli-1 presets and enables propagation of niche-derived Notch1 signaling. Constitutively induced Notch1 signaling is sufficient to recuperate functional HSC impairments in the absence of Fli-1. Applying FLI-1 modified-mRNA transduction into lethargic adult human mobilized HSPCs, enables their vigorous niche-mediated expansion along with superior engraftment capacities. Thus, decryption of stem cell activation programs offers valuable insights for immune regenerative medicine. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.28.534499v1?rss=1 Authors: Rehl, K. M., Selvakumar, J., Hoang, D., Arumugam, K., Gorfe, A., Cho, K.-J. Abstract: Ras proteins are membrane-bound GTPases that regulate essential cellular processes at the plasma membrane (PM). Constitutively active mutations of K-Ras, one of the three Ras isoforms in mammalian cells, are frequently found in human cancers. Ferrocene derivatives, which elevate cellular reactive oxygen species (ROS), have shown to block the growth of non-small cell lung cancers (NSCLCs) harboring oncogenic mutant K-Ras. Here, we developed and tested a novel ferrocene derivative on the growth of human pancreatic ductal adenocarcinoma (PDAC) and NSCLC. Our compound inhibited the growth of K-Ras-dependent PDAC and NSCLC and abrogated the PM binding and signaling of K-Ras, but not other Ras isoforms. These effects were reversed upon antioxidant supplementation, suggesting a ROS-mediated mechanism. We further identified K-Ras His95 residue in the G-domain as being involved in the ferrocene-induced K-Ras PM dissociation via oxidative modification. Together, our studies demonstrate that the redox system directly regulates K-Ras PM binding and signaling via oxidative modification at the His95, and proposes a role of oncogenic mutant K-Ras in the recently described antioxidant-induced metastasis in K-Ras-driven lung cancers. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.28.534628v1?rss=1 Authors: Fontcuberta-Rigo, M., Nakamura, M., Puigbo, P. Abstract: The bone extracellular matrix (ECM) contains minerals deposited on highly cross-linked collagen fibrils, and hundreds of non-collagenous proteins. Some of these proteins are determinant to regulate bone formation and regeneration via signaling pathways, and play important regulatory and structural roles. However, the complete list of bone extracellular matrix proteins, their roles, and the extent of individual and cross-species variation are not yet well understood in both humans and model organisms. Here, we introduce the most comprehensive resource of bone bone extracellular matrix (ECM) proteins that can be used in future studies in research fields such as bone regeneration, osteoporosis and mechanobiology. The Phylobone database (available at https://phylobone.com) includes 255 proteins potentially expressed in the bone extracellular matrix (ECM) of humans and 30 species of vertebrates. A bioinformatics pipeline has been utilized to identify evolutionary relationships of bone ECM proteins. This analysis facilitates the identification of potential model organisms to study the molecular mechanisms of bone regeneration. A network analysis shows the high connectivity of bone ECM proteins. A total of 214 functional protein domains have been identified, including collagen and domains involved in bone formation and resorption. Information from public drug repositories is utilized to identify potential repurposing of existing drugs. The Phylobone database provides a platform to study bone regeneration and osteoporosis in the light of (biological) evolution, and will substantially contribute to the identification of molecular mechanisms and drug targets. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.27.534352v1?rss=1 Authors: Cai, P., Casas, C. J., Hua, L. L., Mikawa, T. Abstract: Individual homologous chromosomes are spatially segregated into haploid chromosome sets along the centrosome axis in an antipairing configuration. Disruption of the antipairing pattern occurs in cancer cells. However, little is known about how this spatial organization of chromosomes is established or maintained. Here, we report that there is a zone of diminished interchromosomal linkage and centromere components between haploid sets in primary and established human epithelial cell lines. Using 4-Dimensional live cell imaging analysis of centromere and centrosome tracking, we show ipsilateral restriction of chromosome oscillations along the diminished zone, coincident with the centrosome and apical-basal axis from mitosis onset to G1 interphase. We propose a biophysical model of axis-dependent ipsilateral restriction of chromosome oscillations for haploid set organization. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.27.534302v1?rss=1 Authors: Yordanov, T. E., Martinez, M. A. E., Esposito, T., Tefft, J. B., Labzin, L. I., Stehbens, S. J., Rowan, A., Hogan, B. M., Chen, C. S., Lauko, J., Lagendijk, A. K. Abstract: Cerebral Cavernous Malformations (CCMs) are vascular lesions that predominantly form in blood vessels of the central nervous system (CNS) upon loss of the CCM multimeric protein complex. The endothelial cells (ECs) within CCM lesions are characterised by overactive MEKK3 kinase and KLF2/4 transcription factor signalling, leading to pathological changes such as increased EC spreading and reduced junctional integrity. Concomitant to aberrant EC signalling, non-autonomous signals from the extracellular matrix (ECM) have also been implicated in CCM lesion growth and these factors might explain why CCM lesions mainly develop in the CNS. Here, we adapted a three dimensional (3D) microfluidic system to examine CCM1 deficient human micro-vessels in distinctive ECMs. We validate that EC pathological hallmarks are maintained in this 3D model. We further show that supplementing the ECM with distinct forms of Hyaluronic Acid (HA), a major ECM component of the CNS, alters CCM1 biology, independent of KLF2/4. This study provides a proof-of-principle that ECM embedded 3D microfluidic models are ideally suited to identify how changes in ECM structure and signalling impact vascular malformations. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.28.534295v1?rss=1 Authors: Tung, V. S. K., Mathews, F., Boruk, M., Suppa, G., Foronjy, R., Pato, M., Pato, C., Knowles, J. A., Evgrafov, O. V. Abstract: Study of the neurodevelopmental molecular mechanisms of schizophrenia requires the development of adequate biological models such as patient-derived cells and their derivatives. We previously used cell lines with neural progenitor properties (CNON) derived from superior or middle turbinates of patients with schizophrenia and control groups to study gene expression specific to schizophrenia. In this study, we compared single cell-RNA seq data from two CNON cell lines, one derived from an individual with schizophrenia (SCZ) and the other from a control group, with two biopsy samples from the middle turbinate (MT), also from an individual with SCZ and a control. In addition, we compared our data with previously published data from olfactory neuroepithelium (1). Our data demonstrated that CNON originated from a single cell type which is present both in middle turbinate and olfactory neuroepithelium. CNON express multiple markers of mesenchymal cells. In order to define relatedness of CNON to the developing human brain, we also compared CNON datasets with scRNA-seq data of embryonic brain (2) and found that the expression profile of CNON very closely matched one of the cell types in the embryonic brain. Finally, we evaluated differences between SCZ and control samples to assess usability and potential benefits of using single cell RNA-seq of CNON to study etiology of schizophrenia. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.28.534513v1?rss=1 Authors: pan, x., ye, f., Ning, P., Zhang, Z., Zhang, B., Chen, G., Gao, W., Qiu, C., Wu, Z., Gong, K., Li, J., Xia, J., Du, Y. Abstract: Hydroxycarboxylic acid receptor 2 (HCAR2) belongs to the family of class A G-protein-coupled receptors with key roles in regulating lipolysis and free fatty acid formation in humans. It is deeply involved in many pathophysiological processes and serves as an attractive target for the treatment of neoplastic, autoimmune, neurodegenerative, inflammatory, and metabolic diseases. Here, we report four cryo-EM structures of human HCAR2-Gi1 complexes with or without agonists, including the drugs niacin and acipimox, and the highly subtype-specific agonist MK-6892. Combined with molecular docking and functional analysis, we have revealed the recognition mechanism of HCAR2 for different agonists and summarized the general pharmacophore features of HCAR2 agonists, which are based on three key residues R1113.36, S17945.52, and Y2847.43. Notably, the MK-6892-HCAR2 structure shows an extended binding pocket relative to other agonist-bound HCAR2 complexes. In addition, the key residues that determine the ligand selectivity between the HCAR2 and HCAR3 are also illuminated. Our findings provide structural insights into the ligand recognition, selectivity, activation, and G protein coupling mechanism of HCAR2, which sheds light on the design of new HCAR2-targeting drugs for greater efficacy, higher selectivity, and fewer or no side effects. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.29.534729v1?rss=1 Authors: Brooks, J. W., Tillu, V., Verma, S., Collins, B. M., Parton, R. G., Yap, A. S. Abstract: As physical barriers, epithelia must preserve their integrity when challenged by mechanical stresses. Cell-cell junctions linked to the cortical cytoskeleton play key roles in this process, often with mechanotransduction mechanisms that reinforce tissues. Caveolae are mechanosensitive organelles that buffer tension via disassembly. Loss of caveolae, through caveolin-1 or cavin1 depletion, causes activation of PtdIns(4, 5)P2 signalling, recruitment of FMNL2 formin, and enhanced cortical actin assembly. How this equates to physiological responses in epithelial cells containing endogenous caveolae is unknown. Here we examined the effect of mechanically-inducing acute disassembly of caveolae in epithelia. We show that perturbation of caveolae, through direct mechanical stress, reinforces the actin cortex at adherens junctions. Increasing interactions with membrane lipids by introducing multiple phosphatidylserine-binding undecad cavin1 (UC1) repeat domains into cavin1 rendered caveolae more stable to mechanical stimuli. This molecular stabilization blocked cortical reinforcement in response to mechanical stress. Cortical reinforcement elicited by the mechanically-induced disassembly of caveolae increased epithelial resilience against tensile stresses. These findings identify the actin cortex as a target of caveola mechanotransduction that contributes to epithelial integrity. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.29.534583v1?rss=1 Authors: Montealegre, S., de Calbiac, H., Straube, M., Debruge, H., Chentout, L., Ciura, S., Imbard, A., Le Guillou, E., Marian, A., Goudin, N., Caccavelli, L., Fabrega, S., Hubas, A., van Endert, P., Dupont, N., Diana, J., Kabashi, E., de Lonlay, P. Abstract: Patients with pathogenic variants in the TANGO2 gene suffer from severe and recurrent rhabdomyolysis (RM) episodes precipitated by fasting. Since starvation promotes autophagy induction, we wondered whether TANGO2-related muscle symptoms result from autophagy insufficiency to meet cellular demands in stress conditions. Autophagy functioning was analyzed in vitro, in primary skeletal muscle cells from TANGO2 patients in basal and fasting conditions. In addition, wce developed a tango2 morphant zebrafish model to assess the effect of tango2 knockdown (KD) on locomotor function and autophagy efficiency in vivo. We report that TANGO2 mutations are associated with decreased LC3-II levels upon starvation in primary muscle cells, but not in fibroblasts. In zebrafish larvae, tango2 knockdown induces locomotor defects characterized by reduced evoked movements which are exacerbated by exposure to atorvastatin, a compound known to cause RM. Importantly, RM features of tango2 KD are also associated with autophagy defects in zebrafish. Calpeptin treatment, a known activator of autophagy, is sufficient to rescue the locomotor function and improves autophagy in zebrafish. LC3-II levels of primary muscle cells of TANGO2 patients are also ameliorated by calpeptin treatment. Overall, we demonstrate that TANGO2 plays an important role in autophagy, and that autophagy efficiency is critical to prevent RM, thus giving rise to new therapeutic perspectives in the prevention of these life-threatening episodes in the context of TANGO2 pathology. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.29.534715v1?rss=1 Authors: van Deventer, S. J., Hoogvliet, I. A., van de Voort, M., Arnold, F., van Spriel, A. Abstract: Tetraspanin proteins play an important role in many cellular processes as they are key organizers of different plasma membrane receptors. Most tetraspanins are highly glycosylated, but the function of this post-translational modification remains largely unstudied. In this study we investigated the glycosylation of CD37 and CD53, two tetraspanins important for cellular and humoral immunity. Broad and cell-specific repertoires of N-glycosylated CD37 and CD53 were observed in human B cells. We generated different glycosylation mutants and analyzed their localization, nanoscale organization and protein interactions. Abrogation of glycosylation in CD37 revealed the importance of this modification for CD37 surface expression, whereas neither surface expression nor nanoscale organization of CD53 was affected by its glycosylation. CD37 interaction with its known partner proteins, CD20 and IL-6R, was not affected by glycosylation, other than via its changed subcellular localization. Surprisingly, glycosylation was found to inhibit the interaction between CD53 and its partner proteins CD45 and CD20. Together, our data show that tetraspanin glycosylation affects their function in immune cells, which adds another layer of regulation to tetraspanin-mediated membrane organization. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.28.534519v1?rss=1 Authors: Peysson, A., Zariohi, N., Gendrel, M., Chambert-Loir, A., Frebault, N., Andrini, O., Boulin, T. Abstract: The plasma membrane of excitable cells is highly structured and molecular scaffolds recruit proteins to specific membrane compartments. Here, we show that potassium channels and proteins belonging to the dystrophin-associated protein complex define multiple types of planar-polarized membrane compartments at the surface of C. elegans muscle cells. Surprisingly, conserved planar cell polarity proteins are not required for this process. However, we implicate a Wnt signaling module involving the Wnt ligand EGL-20, the Wnt receptor CAM-1, and the intracellular effector DSH-1/disheveled in the formation of this cell polarity pattern. Moreover, using time-resolved and tissue-specific protein degradation, we demonstrate that muscle cell polarity is a dynamic state, requiring continued presence of DSH-1 throughout post-embryonic life. Our results reveal the intricate, highly reproducible, and entirely unsuspected complexity of the worm's sarcolemma. This novel case of planar cell polarity in a tractable genetic model organism may provide valuable insight into the molecular and cellular mechanisms that regulate cellular organization, allowing specific functions to be compartmentalized within distinct plasma membrane domains. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.27.534387v1?rss=1 Authors: Sasaki, M., Hara, T., Wang, J. X., Zhou, Y., Kennedy, K. V., Umeweni, N. N., Alston, M. A., Spergel, Z. C., Nakagawa, R., McMillan, E. A., Whelan, K. A., Karakasheva, T. A., Hamilton, K. E., Ruffner, M. A., Muir, A. B. Abstract: Background & Aims: Epithelial disruption in eosinophilic esophagitis (EoE) encompasses both impaired differentiation and diminished barrier integrity. We have shown that lysyl oxidase (LOX), a collagen cross-linking enzyme, is upregulated in the esophageal epithelium in EoE. However, the functional roles of LOX in the esophageal epithelium remains unknown. Methods: We investigated roles for LOX in the human esophageal epithelium using 3-dimensional organoid and air-liquid interface cultures stimulated with interleukin (IL)-13 to recapitulate the EoE inflammatory milieu, followed by single-cell RNA sequencing, quantitative reverse transcription-polymerase chain reaction, western blot, histology, and functional analyses of barrier integrity. Results: Single-cell RNA sequencing analysis on patient-derived organoids revealed that LOX was induced by IL-13 in differentiated cells. LOX-overexpressing organoids demonstrated suppressed basal and upregulated differentiation markers. Additionally, LOX overexpression enhanced junctional protein genes and transepithelial electrical resistance. LOX overexpression restored the impaired differentiation and barrier function, including in the setting of IL-13 stimulation. Transcriptome analyses on LOX-overexpressing organoids identified enriched bone morphogenetic protein (BMP) signaling pathway compared to wild type organoids. Particularly, LOX overexpression increased BMP2 and decreased BMP antagonist follistatin. Finally, we found that BMP2 treatment restored the balance of basal and differentiated cells. Conclusions: Our data support a model whereby LOX exhibits non-canonical roles as a signaling molecule important for epithelial homeostasis in the setting of inflammation via activation of BMP pathway in esophagus. The LOX/BMP axis may be integral in esophageal epithelial differentiation and a promising target for future therapies. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.27.534359v1?rss=1 Authors: Solis, G. P., Koval, A., Valnohova, J., Savitsky, M., Katanaev, V. L. Abstract: GNAO1 mutated in pediatric encephalopathies encodes the major neuronal G-protein G alpha o. Of greater than 40 pathogenic mutations, most are single amino acid substitutions spreading across G alpha o sequence. We perform extensive characterization of G alpha o mutants showing abnormal GTP uptake and hydrolysis, and deficiencies to bind G beta gamma and RGS19. Plasma membrane localization of G alpha o is decreased for a subset of mutations that leads to epileptic manifestations. Pathogenic mutants massively gain interaction with Ric8A/B proteins, delocalizing them from cytoplasm to Golgi. Being general G alpha-subunit chaperones and binding multiple other proteins, Ric8A/B likely mediate the disease dominance when engaging in neomorphic interactions with pathogenic G alpha o. As the strength of G alpha o-Ric8B interactions correlates with disease severity, our study further identifies an efficient biomarker and predictor for clinical manifestations in GNAO1 encephalopathies. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.27.534397v1?rss=1 Authors: Smolen, K. A., Papke, C. M., Swingle, M. R., Musiyenko, A., Li, C., Camp, A. D., Honkanen, R. E., Kettenbach, A. N. Abstract: Variants in the phosphoprotein phosphatase-2 regulatory protein-5D gene (PPP2R5D) cause the clinical phenotype of Jordan's Syndrome (PPP2R5D-related disorder), which includes intellectual disability, hypotonia, seizures, macrocephaly, autism spectrum disorder and delayed motor skill development. The disorder originates from de novo single nucleotide mutations, generating missense variants that act in a dominant manner. Pathogenic mutations altering 13 different amino acids have been identified, with the E198K variant accounting for ~40% of reported cases. Here, we use CRISPR-PRIME genomic editing to introduce a transition (c.592G greater than A) in the PPP2R5D allele in a heterozygous manner in HEK293 cells, generating E198K-heterozygous lines to complement existing E420K variant lines. We generate global protein and phosphorylation profiles of wild-type, E198K, and E420K cell lines and find unique and shared changes between variants and wild-type cells in kinase- and phosphatase-controlled signaling cascades. As shared signaling alterations, we observed ribosomal protein S6 (RPS6) hyperphosphorylation, indicative of increased ribosomal protein S6-kinase activity. Rapamycin treatment suppressed RPS6 phosphorylation in both, suggesting activation of mTORC1. Intriguingly, our data suggest AKT-dependent (E420K) and -independent (E198K) activation of mTORC1. Thus, although upstream activation of mTORC1 differs between PPP2R5D-related disorder genotypes, treatment with rapamycin or a p70S6K inhibitor warrants further investigation as potential therapeutic strategies for patients. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.27.533254v1?rss=1 Authors: Zheng, P., Kumadaki, K., Quek, C., Lim, Z. H., Ashenafi, Y., Yip, Z. T., Newby, J., Alverson, A. J., Jedd, G. Abstract: Diatoms are ancestrally photosynthetic microalgae. However, some underwent a major evolutionary transition, losing photosynthesis to become obligate heterotrophs. The molecular and physiological basis for this transition is unclear. Here, we isolate and characterize new strains of non-photosynthetic diatoms from the coastal waters of Singapore. These diatoms occupy diverse ecological niches and display glucose-mediated catabolite repression, a classical feature of bacterial and fungal heterotrophs. Live-cell imaging reveals deposition of secreted extracellular polymeric substance (EPS). Diatoms moving on pre-existing EPS trails (runners) move faster than those laying new trails (blazers). This leads to cell-to-cell coupling where runners can push blazers to make them move faster. Calibrated micropipettes measure substantial single cell pushing forces, which are consistent with high-order myosin motor cooperativity. Collisions that impede forward motion induce reversal, revealing navigation-related force sensing. Together, these data identify aspects of metabolism and motility that are likely to facilitate and underpin the transition to obligate heterotrophy. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.27.534346v1?rss=1 Authors: Burigotto, M., Vigorito, V., Mattivi, A., Gliech, C., Ghetti, S., Bisio, A., Lolli, G., Holland, A. J., Fava, L. L. Abstract: 53BP1 acts at the crossroads between DNA repair and p53-mediated stress response. With its interactor USP28, it is part of the mitotic surveillance pathway (MSP), a sensor that monitors the duration of cell division, promoting p53-dependent cell cycle arrest when a critical time threshold is surpassed. 53BP1 dynamically associates with kinetochores, being recruited during prophase, and then undergoing a time-dependent loss of affinity. However, the relevance of this behaviour remains unclear. Here, we identify CENP-F as an interaction partner and kinetochore receptor for 53BP1. By engineering human cells with a CENP-F point mutation, we demonstrate that preventing 53BP1 kinetochore localization does not reduce MSP proficiency. Strikingly, however, preventing the loss of 53BP1 from the kinetochore by inhibiting Polo-like kinase 1 (PLK1) restrains MSP activity, a phenomenon that is abrogated in the CENP-F mutant condition. Taken together, we demonstrate that kinetochore-loaded 53BP1 represents an MSP functionally inhibited state and that PLK1-dependent re-localization of 53BP1 represents an important layer of MSP regulation. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.26.534266v1?rss=1 Authors: Sarkar, S., Sharma, H., Ladke, J. S., Raran-Kurussi, S., Bhandari, R., Jaiswal, M. Abstract: Inorganic polyphosphate (polyP), a linear polymer of orthophosphate residues, is found in all living organisms. While the biological and molecular functions of polyP have been studied in prokaryotes and unicellular eukaryotes, in metazoans it has often been referred to as a molecular fossil. Although certain functions of polyP, e.g. in blood clotting, have been uncovered in humans, a genetically tractable model system has not been developed to systematically study polyP function in metazoans. To explore Drosophila as a model to study polyP, we first developed methods to quantify polyP in flies and then analysed polyP levels in various tissues during different developmental stages. Interestingly, we found that the levels of polyP increase before pupal formation and significantly drop during metamorphosis. To check the in-situ localization of polyP, we used polyP binding domain of bacterial protein, namely PPBD to detect endogenous polyP in fly tissues. We report the presence of polyP in cytoplasm and enrichment in the nucleolus in most tissue types. Interestingly, during oogenesis, we found polyP is enriched in early stage egg chambers but get depleted in later stages suggesting that polyP may play a crucial role during ovariole development. Finally, we found polyP to be enriched in fly hemocytes and showed that polyP promotes hemolymph clotting suggesting that polyP function between flies and mammals is conserved. These observations strongly suggest that combining powerful Drosophila genetic and biochemical tools can accelerate the discovery of unknown biological functions of polyP in metazoans. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.27.534392v1?rss=1 Authors: Vedula, P., Fina, M. E., Bell, B. A., Nikonov, S. S., Kashina, A., Dong, D. W. Abstract: Lack of non-muscle {beta}-actin gene (Actb) leads to early embryonic lethality in mice, however mice with {beta}- to {gamma}-actin replacement develop normally and show no detectable phenotypes at young age. Here we investigated the effect of this replacement in the retina. During aging, these mice have accelerated degeneration of retinal structure and function, including elongated microvilli and defective mitochondria of retinal pigment epithelium (RPE), abnormally bulging photoreceptor outer segments (OS) accompanied by reduced transducin concentration and light sensitivity, and accumulation of autofluorescent microglia cells in the subretinal space between RPE and OS. These defects are accompanied by changes in the F-actin binding of several key actin interacting partners, including ezrin, myosin, talin, and vinculin known to play central roles in modulating actin cytoskeleton and cell adhesion and mediating the phagocytosis of OS. Our data show that {beta}-actin protein is essential for maintaining normal retinal structure and function. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.26.534279v1?rss=1 Authors: Meineke, B., Heimgärtner, J., Caridha, R., Block, M., Kimler, K. J., Pires, M. F., Landreh, M., Elsässer, S. J. Abstract: Genetic code expansion via stop codon suppression is a powerful strategy to engineer proteins. Suppressor tRNAs are aminoacylated with noncanonical amino acids (ncAAs) by dedicated aminoacyl-tRNA synthetases (aaRS) and direct ncAA incorporation site-specifically during translation. These pairs of tRNA/aaRS must be orthogonal to the host's tRNAs, aaRS and natural amino acids. Pyrrolysyl-tRNA (PylT)/PylRS pairs from methanogenic archaea, as well as engineered tRNA/aaRS pairs derived from bacteria, are used for genetic code expansion in mammalian cells. Amber suppression is routinely achieved by transient introduction of the components leading to short-term and heterogeneous expression. Here, we demonstrate that stable integration of tRNA/aaRS genes allows for efficient, genetically encoded ncAA incorporation in diverse mammalian cell lines. We extend a general plasmid design and PiggyBac (PB) integration strategy developed for the Methanosarcina mazei PylT/PylRS pair to genomic integration of two tRNA/aaRS pairs of bacterial origin. We further explore suppression of ochre and opal stop codons and parallel incorporation of two distinct ncAAs, both accessible for click chemistry, by dual suppression in stable cell lines. Clonal selection allows for isolation of cells with high dual suppression efficiency and dual site-specific fluorescent labeling of a cell surface receptor using bioorthogonal click chemistries on live mammalian cells. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.26.534268v1?rss=1 Authors: Li, S., Wang, Y., Stoel, M. v. d., Zhou, X., Madhusudan, S., Kanerva, K., Nguyen, V. D., Eskici, N., Olkkonen, V. M., Zhou, Y., Raivio, T., Ikonen, E. Abstract: Auxin-inducible degron (AID) technology is powerful for chemogenetic control of proteolysis. However, generation of human cell lines to deplete endogenous proteins with AID remains challenging. Typically, homozygous degron-tagging efficiency is low and overexpression of an auxin receptor requires additional engineering steps. Here, we establish a one-step genome editing procedure with high-efficiency homozygous tagging and auxin receptor expression. We demonstrate its application in 5 human cell lines, including embryonic stem (ES) cells. The method allowed isolation of AID single-cell clones in 10 days for 11 target proteins with greater than 80% average homozygous degron-tagging efficiency in A431 cells, and greater than 50% efficiency for 5 targets in H9 ES cells. The tagged endogenous proteins were inducibly degraded in all cell lines, including ES cells and ES-cell derived neurons, with robust expected functional readouts. This method facilitates the application of AID for studying endogenous protein functions in human cells, especially in stem cells. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.27.534444v1?rss=1 Authors: Chou, C.-C., Vest, R., Prado, M. A., Wilson-Grady, J., Paulo, J. A., Shibuya, Y., Moran-Losada, P., Lee, T.-T., Luo, J., Gygi, S. P., Kelly, J. W., Finley, D., Wernig, M., Wyss-Coray, T., Frydman, J. Abstract: The role of proteostasis and organelle homeostasis dysfunction in human aging and Alzheimers disease (AD) remains unclear. Analyzing proteome-wide changes in human donor fibroblasts and their corresponding transdifferentiated neurons (tNeurons), we find aging and AD synergistically impair multiple proteostasis pathways, most notably lysosomal quality control (LQC). In particular, we show that ESCRT-mediated lysosomal repair defects are associated with both sporadic and PSEN1 familial AD. Aging- and AD-linked defects are detected in fibroblasts but highly exacerbated in tNeurons, leading to enhanced neuronal vulnerability, unrepaired lysosomal damage, inflammatory factor secretion and cytotoxicity. Surprisingly, tNeurons from aged and AD donors spontaneously develop amyloid-{beta} inclusions co-localizing with LQC markers, LAMP1/2-positive lysosomes and proteostasis factors; we observe similar inclusions in brain tissue from AD patients and APP-transgenic mice. Importantly, compounds enhancing lysosomal function broadly ameliorate these AD-associated pathologies. Our findings establish cell-autonomous LQC dysfunction in neurons as a central vulnerability in aging and AD pathogenesis. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.27.534337v1?rss=1 Authors: Anjum, S., Srivastava, S., Panigrahi, L., Ansari, U. A., Trivedi, A. K., Ahmed, S. Abstract: The mTOR complexes play a fundamental role in mitochondrial biogenesis and cellular homeostasis. Wat1, an ortholog of mammalian Lst8 is an important component of TOR complex and is essential for the regulation of downstream signaling. Earlier we reported the role of Wat1 in oxidative stress response. Here, we show that the inactivation of wat1 leads to respiratory defects and mitochondrial depolarization leading to decrease in ATP production. The confocal and electron microscopy in wat1{Delta} cells revealed the fragmented mitochondrial morphology implying its role in mitochondrial fission. Furthermore, we also showed its role in autophagy and the maintenance of calcium ion homeostasis. Additionally, tor2-287 mutant cells also exhibit defects in mitochondrial integrity indicating the TORC1-dependent involvement of Wat1 in the maintenance of mitochondrial homeostasis. The interaction studies of Wat1 and Tor2 with Por1 and Mmm1 proteins revealed a cross-talk between mitochondria and endoplasmic reticulum through the Mitochondria-associated membranes (MAM) and endoplasmic reticulum-mitochondria encounter structure (ERMES) complex, involving TORC1. Taken together, this study demonstrates involvement of Wat1/mLst8 in harmonizing various mitochondrial functions, redox status, and Ca2+ homeostasis. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.27.534325v1?rss=1 Authors: Nagano, M., Aoshima, K., Shimamura, H., Siekhaus, D. E., Toshima, J. Y., Toshima, J. Abstract: Clathrin-mediated vesicle trafficking plays central roles in the post-Golgi transport pathways from the trans-Golgi network (TGN) to endosomes. In yeast, two clathrin adaptors - AP-1 complex and GGA proteins (GGAs) - are predicted to generate distinct transport vesicles at the TGN, and epsin-related Ent3p/Ent5p act as accessories for these adaptors. Recently, we showed that vesicle transport from the TGN, rather than from the plasma membrane, is crucial for Rab5-mediated endosome formation, and that Ent3p/5p are crucial for this process, whereas AP-1 and GGAs are dispensable. However, these observations were incompatible with previous studies showing that these adaptors are required for Ent3p/5p recruitment to the TGN, and thus the overall mechanism responsible for regulation of Rab5 activity remains ambiguous. Here we investigated the functional relationships between clathrin adaptors in post-Golgi-mediated Rab5 activation. We were able to show that AP-1 disruption in ENT3/ENT5-deleted mutant impairs Rab5-GEF Vps9p transport to the Rab5 compartment, and severely reduces Rab5 activity. Additionally, GGAs, Golgi-resident PI4 kinase Pik1p and Rab11 GTPases Ypt31p/32p were found to have partially overlapping functions for recruitment of AP-1 and Ent3p/5p to the TGN. These findings suggest a distinct role of clathrin adaptors for Rab5 activation in the TGN-endosome trafficking pathway. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.26.534252v1?rss=1 Authors: Diaz-Barreiro, A., Cereghetti, G., Tonacini, J., Talabot-Ayer, D., Kieffer-Jaquinod, S., Huard, A., Swale, C., Coute, Y., Peter, M., Frances-Monerris, A., Palmer, G. Abstract: Cytokines of the interleukin (IL)-1 family are widely expressed in epithelial surfaces, including the epidermis, where they play a key role in the maintenance of barrier integrity and host defense. A recent report associated the IL-1 family member IL-33 with stress granules (SGs) in epithelial cells. Formation of SGs is promoted by the aggregation of proteins harboring low complexity regions (LCRs). In this study, using computational analyses, we predicted the presence of LCRs in six of the eleven IL-1 family members. Among these, IL-38 contained a long LCR and localized to Ras GTPase-activating protein binding protein 1 (G3BP1) positive SGs, as well as to G3BP1 negative intracellular protein condensates in keratinocytes exposed to oxidative stress (OS). In addition, we identified two highly aggregation-prone amyloid core (AC) motifs in the IL-38 LCR and detected the formation of amyloid IL-38 aggregates in response to OS in cells and in vitro. Disulfide bond mapping, in silico modelling and the analysis of specific cysteine mutants supported a model in which specific oxidation-sensitive cysteines act as redox switches to modify the conformation of IL-38 and thus the surface exposure of its ACs, shuttling it from a soluble state into biomolecular condensates. Finally, the presence of IL-38 granules in human epidermal layers highly exposed to environmental OS suggests that oxidation-induced formation of amyloid aggregates, as a previously unrecognized intrinsic biological property of IL-38, may be physiologically relevant at this epithelial barrier. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.26.534249v1?rss=1 Authors: Sugiyama, H., Goto, Y., Kondo, Y., Coudreuse, D., Aoki, K. Abstract: Cyclin-dependent kinase (CDK) plays an essential role in determining the temporal ordering of the cell cycle phases. However, despite significant progress in studying regulators of CDK, it remains elusive how they coordinately affect CDK activity at the single-cell level and how CDK controls the temporal order of cell cycle events. This could be due to the lack of tools to monitor CDK activity in living cells. Here, we elucidate the dynamics of CDK activity in fission yeast and mammalian cells by using a newly developed CDK activity biosensor, Eevee-spCDK, based on Forster Resonance Energy Transfer (FRET). Taking advantage of this system, we unravel the profile of CDK activity in vegetatively growing S. pombe cells. Thus, we detect a transient increase in S phase followed by a gradual increment during G2 phase. CDK activity then reaches its maximum in early M phase and rapidly decreases at mitotic exit. During G2 phase, CDK activity exhibits a biphasic pattern, i.e., an early slow increase and a late fast rise prior to the G2/M phase transition, as predicted from mathematical studies. Remarkably, although CDK activity does not necessarily correlate with cyclin levels, we find that it converges to the same level around mitotic onset in several mutant backgrounds, including pom1{Delta} cells and wee1 or cdc25 overexpressing cells. These data provide the first direct evidence that cells enter M phase when CDK activity reaches a high threshold, consistent with the quantitative model of cell cycle progression in fission yeast. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.25.534233v1?rss=1 Authors: Virga, D. M., Hamilton, S., Osei, B., Morgan, A., Zamponi, E., Park, N. J., Hewitt, V. L., Zhang, D., Gonzalez, K. C., Bloss, E., Polleux, F., Lewis, T. L. Abstract: Neuronal mitochondria play important roles beyond ATP generation, including Ca2+ uptake, and therefore have instructive roles in synaptic function and neuronal response properties. Mitochondrial morphology differs significantly in the axon and dendrites of a given neuronal subtype, but in CA1 pyramidal neurons (PNs) of the hippocampus, mitochondria within the dendritic arbor also display a remarkable degree of subcellular, layer-specific compartmentalization. In the dendrites of these neurons, mitochondria morphology ranges from highly fused and elongated in the apical tuft, to more fragmented in the apical oblique and basal dendritic compartments, and thus occupy a smaller fraction of dendritic volume than in the apical tuft. However, the molecular mechanisms underlying this striking degree of subcellular compartmentalization of mitochondria morphology are unknown, precluding the assessment of its impact on neuronal function. Here, we demonstrate that this compartment-specific morphology of dendritic mitochondria requires activity-dependent, Camkk2-dependent activation of AMPK and its ability to phosphorylate two direct effectors: the pro-fission Drp1 receptor Mff and the recently identified anti-fusion, Opa1-inhibiting protein, Mtfr1l. Our study uncovers a new activity-dependent molecular mechanism underlying the extreme subcellular compartmentalization of mitochondrial morphology in dendrites of neurons in vivo through spatially precise regulation of mitochondria fission/fusion balance. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.24.534106v1?rss=1 Authors: Esser, L., Springer, R., Dreissen, G., Lövenich, L., Konrad, J., Hampe, N., Merkel, R., Hoffmann, B., Noetzel, E. Abstract: Cell contractility regulates epithelial tissue geometry development and homeostasis. The underlying mechanobiological regulation circuits are poorly understood and experimentally challenging. We developed an elastomeric pillar cage (EPC) array to quantify cell contractility as a mechanoresponse of epithelial microtissues to substrate stiffness and topography. The spatially confined EPC geometry consisted of 24 circularly arranged slender pillars (1.2 MPa, height: 50 {micro} m; diameter: 10 {micro} m; distance: distance: 5 {micro}m). These high-aspect-ratio pillars were confined at both ends by planar substrates with different stiffness (0.15-1.2 MPa). Analytical modeling and finite elements simulation retrieved cell forces from pillar displacements. For evaluation, highly contractile myofibroblasts and cardiomyocytes were assessed to demonstrate that the EPC device can resolve static and dynamic cellular force modes. Human breast (MCF10A) and skin (HaCaT) cells grew as adherence junction-stabilized 3D microtissues within the EPC geometry. Planar substrate areas triggered the spread of monolayered clusters with substrate stiffness-dependent actin stress fiber (SF)-formation and substantial single-cell actomyosin contractility (150-200 nN). Within same continuous microtissues, the pillar-ring topography induced bilayered cell tube growth. Here, low effective pillar stiffness overwrote local substrate stiffness sensing and induced SF-lacking roundish cell shapes with extremely low cortical actin tension (11-15 nN). This work introduced a versatile biophysical tool to explore mechanobiological regulation circuits driving low- and high-tensional states in developing and homeostatic microtissues. EPC arrays facilitate simultaneously analyzing the impact of planar substrate stiffness and topography on microtissue contractility hence microtissue geometry and function. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.24.534147v1?rss=1 Authors: Boutry, M., DiGiovanni, L. F., Demers, N., Fountain, A., Mamand, S., Botelho, R., Kim, P. K. Abstract: Formation and fission of tubules from lysosomal organelles, such as autolysosomes, endolysosomes or phagolysosomes, are required for lysosome reformation. However, the mechanisms governing these processes in the different forms of lysosomal organelles are poorly understood. For instance, the role of phosphatidylinositol-4-phosphate (PI(4)P) is unclear as it was shown to promote the formation of tubules from phagolysosomes but was proposed to inhibit tubule formation on autolysosomes because the loss of PI4KIII{beta} causes extensive lysosomal tubulations. Using super-resolution live-cell imaging, we show that Arf1-PI4KIII{beta} positive vesicles are recruited to tubule fission sites from autolysosomes, endolysosomes and phagolysosomes. Moreover, we show that PI(4)P is required to form autolysosomal tubules and that increased lysosomal tubulation caused by loss of PI4KIII{beta} represents impaired tubule fission. At the site of fission, we propose that Arf1-PI4KIII{beta} positive vesicles mediate a PI(3)P signal on lysosomes in a process requiring the lipid transfer protein SEC14L2. Our findings indicate that Arf1-PI4KIII{beta} positive vesicles and their regulation of PI(3)P are critical components of the lysosomal tubule fission machinery. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.24.534108v1?rss=1 Authors: Hoffstadt, J. G., Wotring, J. W., Porter, S., Halligan, B. S., O'Meara, M. J., Tai, A. W., Sexton, J. Z. Abstract: Dengue Virus (DENV) causes dengue fever, a pandemic-potential disease with currently no FDA-approved antivirals. Additionally, the available vaccine for DENV can increase the risk of severe dengue fever for those who have never had a DENV infection due to antibody-dependent enhancements. Thus, there is an urgent need to identify dengue virus antivirals. Antivirals that target NS4B, the replication compartment forming protein of DENV and the flavivirus family, are a promising new drug class that minimize cytotoxic effects to host cells. Drug-repurposing and high-content screening were leveraged to efficiently identify antivirals likely to inhibit NS4B. Using high-content screening, we quantified the morphological patterns of NS4B and envelope (E) protein expression versus time and developed a viral pseudotime model that was able to predict the infection progression to enable drug screening. We then developed a single-cell infection classifier for antiviral efficacy and performed high-throughput drug screening of 960 compounds. We identified four concentration-dependent inhibitors of DENV with nanomolar potencies including: Nexium, Pralatrexate, GW4064, and LY411575. LY411575, a gamma secretase inhibitor, exhibited an IC50 of 72nM and reduced percent infection to levels indistinguishable from the mock infection control. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.25.532215v1?rss=1 Authors: Zamalloa, L. G., Pruitt, M. M., Hermance, N. M., Gali, H., Flynn, R. L., Manning, A. L. Abstract: The retinoblastoma tumor suppressor protein (RB) interacts physically and functionally with a number of epigenetic modifying enzymes to control transcriptional regulation, respond to replication stress, promote DNA damage response and repair pathways, and regulate genome stability. To better understand how disruption of RB function impacts epigenetic regulation of genome stability and determine whether such changes may represent exploitable weaknesses of RB-deficient cancer cells, we performed an imaging-based screen to identify epigenetic inhibitors that promote DNA damage and compromise viability of RB-deficient cells. We found that loss of RB alone leads to high levels of replication-dependent poly-ADP ribosylation (PARylation) and that preventing PARylation through inhibition of PARP enzymes enables RB-deficient cells to progress to mitosis with unresolved replication stress and under-replicated DNA. These defects contribute to high levels of DNA damage, decreased proliferation, and compromised cell viability. We demonstrate this sensitivity is conserved across a panel of inhibitors that target both PARP1 and PARP2 and can be suppressed by re-expression of the RB protein. Together, these data indicate that inhibitors of PARP1 and PARP2 may be clinically relevant for RB-deficient cancers. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.25.534211v1?rss=1 Authors: Mariano, N. C., Rusin, S. F., Nasa, I., Kettenbach, A. N. Abstract: Protein phosphorylation is an essential regulatory mechanism that controls most cellular processes, including cell cycle progression, cell division, and response to extracellular stimuli, among many others, and is deregulated in many diseases. Protein phosphorylation is coordinated by the opposing activities of protein kinases and protein phosphatases. In eukaryotic cells, most serine/threonine phosphorylation sites are dephosphorylated by members of the Phosphoprotein Phosphatase (PPP) family. However, we only know for a few phosphorylation sites which specific PPP dephosphorylates them. Although natural compounds such as calyculin A and okadaic acid inhibit PPPs at low nanomolar concentrations, no selective chemical PPP inhibitors exist. Here, we demonstrate the utility of endogenous tagging of genomic loci with an auxin-inducible degron (AID) as a strategy to investigate specific PPP signaling. Using Protein Phosphatase 6 (PP6) as an example, we demonstrate how rapidly inducible protein degradation can be employed to identify dephosphorylation SITES and elucidate PP6 biology. Using genome editing, we introduce AID-tags into each allele of the PP6 catalytic subunit (PP6c) in DLD-1 cells expressing the auxin receptor Tir1. Upon rapid auxin-induced degradation of PP6c, we perform quantitative mass spectrometry-based proteomics and phosphoproteomics to identify PP6 substrates in mitosis. PP6 is an essential enzyme with conserved roles in mitosis and growth signaling. Consistently, we identify candidate PP6c-dependent phosphorylation sites on proteins implicated in coordinating the mitotic cell cycle, cytoskeleton, gene expression, and mitogen-activated protein kinase (MAPK) and Hippo signaling. Finally, we demonstrate that PP6c opposes the activation of large tumor suppressor 1 (LATS1) by dephosphorylating Threonine 35 (T35) on Mps One Binder (MOB1), thereby blocking the interaction of MOB1 and LATS1. Our analyses highlight the utility of combining genome engineering, inducible degradation, and multiplexed phosphoproteomics to investigate signaling by individual PPPs on a global level, which is currently limited by the lack of tools for specific interrogation. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.24.534081v1?rss=1 Authors: Roeck, B. F., Vorndran, M. R. H., Garcia-Saez, A. J. Abstract: Ferroptosis is an iron-dependent form of regulated cell death characterized by accumulation of peroxidized lipids and plasma membrane disruption, whose molecular mechanism of execution remains poorly understood. Here, we developed a new optogenetic system, Opto-GPX4Deg, for light-induced degradation of the lipid reducing protein GPX4, which allows controlled ferroptosis induction with high precision in time and space. By using Opto-GPX4Deg to study cell death dynamics within the cellular population, we found that lipid peroxidation, followed by ferroptotic death, spread to neighboring cells in a distance-dependent manner. Remarkably, ferroptosis propagation showed a strong dependency on cell confluence and preferentially affected adjacent cells. Our findings establish cell death propagation as a feature of ferroptosis and provide new understanding of the mechanism involved. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.22.533773v1?rss=1 Authors: Liffner, B., Cepeda Diaz, A. K., Blauwkamp, J., Anaguano, D., Frolich, S., Muralidharan, V., Wilson, D. W., Dvorin, J., Absalon, S. Abstract: Apicomplexan parasites exhibit tremendous diversity in much of their fundamental cell biology, but study of these organisms using light microscopy is often hindered by their small size. Ultrastructural expansion microscopy (U-ExM) is a microscopy preparation method that physically expands the sample ~4.5x. Here, we apply U-ExM to the human malaria parasite Plasmodium falciparum during the asexual blood stage of its lifecycle to understand how this parasite is organized in three-dimensions. Using a combination of dye-conjugated reagents and immunostaining, we have catalogued 13 different P. falciparum structures or organelles across the intraerythrocytic development of this parasite and made multiple observations about fundamental parasite cell biology. We describe that the microtubule organizing center (MTOC) and its associated proteins anchor the nucleus to the parasite plasma membrane during mitosis. Furthermore, the rhoptries, Golgi, basal complex, and inner membrane complex, which form around this anchoring site while nuclei are still dividing, are concurrently segregated and maintain an association to the MTOC until the start of segmentation. We also show that the mitochondrion and apicoplast undergo sequential fission events while maintaining an MTOC association during cytokinesis. Collectively, this study represents the most detailed ultrastructural analysis of P. falciparum during its intraerythrocytic development to date, and sheds light on multiple poorly understood aspects of its organelle biogenesis and fundamental cell biology. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.23.533672v1?rss=1 Authors: Buckley, N., Craxton, A., Sun, X.-M., Panatta, E., Pinon, L., Llodra, J., Morone, N., Amelio, I., Melino, G., Martins, L. M., MacFarlane, M. Abstract: Dysregulated mitochondrial fusion and fission has been implicated in the pathogenesis of numerous diseases. We have identified a novel function of the p53 family protein TAp73 in regulating mitochondrial dynamics. TAp73 regulates the expression of Optic atrophy 1, a protein responsible for controlling mitochondrial fusion, cristae biogenesis and electron transport chain function. Disruption of this axis results in a fragmented mitochondrial network and an impaired capacity for energy production via oxidative phosphorylation. Owing to the role of OPA1 in modulating cytochrome c release, TAp73-/- cells also display an increased sensitivity to apoptotic cell death, e.g., via BH3-mimetics. We also show that the TAp73/OPA1 axis has functional relevance in the upper airway, where TAp73 expression is essential for multiciliated cell differentiation and function. Consistently, ciliated epithelial cells of Trp73-/- (global p73 KO) mice display decreased expression of OPA1 and perturbations of the mitochondrial network, which may drive multiciliated cell loss. In support of this, Trp73 and OPA1 expression is decreased in COPD patients, a disease characterised by alterations in mitochondrial dynamics. We therefore highlight a potential mechanism involving the loss of p73 in COPD pathogenesis. This work also adds to the growing body of evidence for growth-promoting roles of TAp73 isoforms. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.23.533959v1?rss=1 Authors: King, S. D., Cai, D., Fraunfelder, M. M., Chen, S.-Y. Abstract: BACKGROUND: Atherosclerosis is a progressive inflammatory disease where macrophage foam cells play a central role in the pathogenesis. Surfactant protein A (SPA) is a lipid-associating protein involved with regulating macrophage function in various inflammatory diseases. However, the role of SPA in atherosclerosis and macrophage foam cell formation has not been investigated. METHODS: Primary resident peritoneal macrophages were extracted from wild-type (WT) and SPA deficient (SPA-/-) mice to determine the functional effects of SPA in macrophage foam cell formation. SPA expression was assessed in healthy vessels and atherosclerotic aortic tissue from the human coronary artery and WT or apolipoprotein e-deficient (ApoE-/-) mice brachiocephalic arteries fed high fat diets (HFD) for 4 weeks. Hypercholesteremic WT and SPA-/- mice fed a HFD for 6 weeks were investigated for atherosclerotic lesions in vivo. RESULTS: In vitro experiments revealed that global SPA deficiency reduced intracellular cholesterol accumulation and macrophage foam cell formation. Mechanistically, SPA-/- dramatically decreased CD36 cellular and mRNA expression. SPA expression was increased in atherosclerotic lesions in humans and ApoE-/- mice. In vivo SPA deficiency attenuated atherosclerosis and reduced the number of lesion-associated macrophage foam cells. CONCLUSIONS: Our results elucidate that SPA is a novel factor for atherosclerosis development. SPA enhances macrophage foam cell formation and atherosclerosis through increasing scavenger receptor cluster of differentiation antigen 36 (CD36) expression. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.23.533840v1?rss=1 Authors: Perocheau, D. P., Baruteau, J., Gurung, S., Touramanidou, L., Duff, C., Sharma, G., Sebire, N., Finn, P., Cavedon, A., Siddiqui, S., Rice, L., Martini, P., Frassetto, A. Abstract: In academic research and the pharmaceutical industry, in vitro single cell line cultures and in vivo animal models are considered as gold standards in modelling diseases and assessing therapeutic efficacy. However, both models have limitations, with incomplete reproduction of pathophysiological characteristics and absence of 3-dimensional architecture with cell lines or the use of live animals brings ethical considerations, limiting the experimental scale and design. The use of precision-cut tissue slices can bridge the gap between these mainstream models as this technique combines the advantages of studying all cell sub-types whilst preserving the tissue-matrix architecture, thereby closely mimicking a mini-organ. Here, we describe an optimised and easy-to-implement protocol for the culture of sections from mouse livers. We show that precision-cut liver sections can be a reliable model for recapitulating the biological phenotype of inherited metabolic diseases, exemplified by common urea cycle defects citrullinemia type 1 and argininosuccinic aciduria, caused by argininosuccinic synthase (ASS1) and argininosuccinic lyase (ASL) deficiencies respectively. Therapeutic response to gene therapy such as messenger RNA replacement delivered via lipid nanoparticles can be monitored, demonstrating that precision-cut liver sections can be used as a preclinical screening tool to assess therapeutic response and toxicity in monogenic liver diseases. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.22.533832v1?rss=1 Authors: Nixon-Abell, J., Ruggeri, F. S., Qamar, S., Herling, T. W., Czekalska, M. A., Shen, Y., Wang, G., King, C., Fernandopulle, M. S., Sneideris, T., Watson, J. L., Pillai, V. V., Meadows, W., Henderson, J., Chambers, J. E., Wagstaff, J. L., Williams, S. H., Coyle, H., Lu, Y., Zhang, S., Marciniak, S. J., Freund, S. M., Derivery, E., Ward, M. E., Vendruscolo, M., Knowles, T. P., St George Hyslop, P. Abstract: Phase transitions of cellular proteins and lipids play a key role in governing the organisation and coordination of intracellular biology. The frequent juxtaposition of proteinaceous biomolecular condensates to cellular membranes raises the intriguing prospect that phase transitions in proteins and lipids could be co-regulated. Here we investigate this possibility in the ribonucleoprotein (RNP) granule-ANXA11-lysosome ensemble, where ANXA11 tethers RNP granule condensates to lysosomal membranes to enable their co-trafficking. We show that changes to the protein phase state within this system, driven by the low complexity ANXA11 N-terminus, induce a coupled phase state change in the lipids of the underlying membrane. We identify the ANXA11 interacting proteins ALG2 and CALC as potent regulators of ANXA11-based phase coupling and demonstrate their influence on the nanomechanical properties of the ANXA11-lysosome ensemble and its capacity to engage RNP granules. The phenomenon of protein-lipid phase coupling we observe within this system offers an important template to understand the numerous other examples across the cell whereby biomolecular condensates closely juxtapose cell membranes. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.23.533914v1?rss=1 Authors: Fedry, J., Silva, J., Vanevic, M., Fronik, S., Mechulam, Y., Schmitt, E., des Georges, A., Faller, W., Förster, F. Abstract: Aberrantly slow mRNA translation leads to ribosome stalling and subsequent collision with the trailing neighbor. Ribosome collisions have recently been shown to act as stress sensors in the cell, with the ability to trigger stress responses balancing survival and apoptotic cell-fate decisions depending on the stress level. However, we lack a molecular understanding of the reorganization of translation processes over time in mammalian cells exposed to an unresolved collision stress. Here we visualize the effect of a persistent collision stress on translation using in situ cryo electron tomography. We observe that low dose anisomycin collision stress leads to the stabilization of Z-site bound tRNA on elongating 80S ribosomes, as well as to the accumulation of an off-pathway 80S complex possibly resulting from collision splitting events. We visualize collided disomes in situ, occurring on compressed polysomes and revealing a stabilized geometry involving the Z-tRNA and L1 stalk on the stalled ribosome, and eEF2 bound to its collided rotated-2 neighbor. In addition, non-functional post-splitting 60S complexes accumulate in the stressed cells, indicating a limiting Ribosome associated Quality Control clearing rate. Finally, we observe the apparition of tRNA-bound aberrant 40S complexes shifting with the stress timepoint, suggesting a succession of different initiation inhibition mechanisms over time. Altogether, our work visualizes the changes of translation complexes under persistent collision stress in mammalian cells, indicating how perturbations in initiation, elongation and quality control processes contribute to an overall reduced protein synthesis. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.24.534085v1?rss=1 Authors: Fuelle, J. B., de Almeida, R. A., Lawless, C., Yanes, B., Lane, E. B., Garrod, D. R., Ballestrem, C. Abstract: Desmosomes are multiprotein adhesion complexes that link intermediate filaments to the plasma membrane, ensuring the mechanical integrity of cells across tissues, but how they participate in the wider signalling network to exert their full function is unclear. To investigate this we carried out multiplexed protein proximity mapping using biotinylation (BioID). The combined interactomes of the essential desmosomal proteins desmocollin 2a, plakoglobin and plakophilin 2a (Pkp2a) in Madin-Darby canine kidney epithelial cells were mapped and their differences and commonalities characterised as desmosome matured from Ca2+-dependence to the mature, Ca2+-independent, hyper-adhesive state, which predominates in tissues. Results suggest that individual desmosomal proteins have distinct roles in connecting to cellular signalling pathways and that these roles alter substantially when cells change their adhesion state. The data provide further support for a dualistic concept of desmosomes in which the properties of Pkp2a differ from those of the other, more stable proteins. This body of data provides an invaluable resource for analysis of desmosome function. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.23.534015v1?rss=1 Authors: Prevo, B., Cheerambathur, D., Earnshaw, W. C., Desai, A. Abstract: Multiple microtubule-directed activities concentrate on chromosomes during mitosis to ensure their accurate distribution to daughter cells. These activities include couplers and dynamics regulators localized at the kinetochore, the specialized microtubule interface built on centromeric chromatin, as well as motor proteins recruited to kinetochores and to mitotic chromatin. Here, we describe an in vivo reconstruction approach in which the effect of removing the major microtubule-directed activities on mitotic chromosomes is compared to the selective presence of individual activities. This approach revealed that the kinetochore dynein module, comprised of the minus end-directed motor cytoplasmic dynein and its kinetochore-specific adapters, is sufficient to biorient chromosomes and to remodel outer kinetochore composition following microtubule attachment; by contrast, the kinetochore dynein module is unable to support chromosome congression. The chromosome-autonomous action of kinetochore dynein, in the absence of the other major microtubule-directed factors on chromosomes, rotates and orients a substantial proportion of chromosomes such that their sister chromatids attach to opposite spindle poles. In tight coupling with orientation, the kinetochore dynein module drives removal of outermost kinetochore components, including the dynein motor itself and spindle checkpoint activators. The removal is independent of the other major microtubule-directed activities and kinetochore-localized protein phosphatase 1, suggesting that it is intrinsic to the kinetochore dynein module. These observations indicate that the kinetochore dynein module has the ability coordinate chromosome biorientation with attachment state-sensitive remodeling of the outer kinetochore that facilitates cell cycle progression. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC