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Identification of omega-3 oxylipins in human milk-derived extracellular vesicles with pro-resolutive actions in gastrointestinal inflammation.

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
8/5/20230
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Super-resolution optical imaging reveals accumulation of small mitochondria in the nucleoli of mouse embryos and stem cells

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
8/5/20230
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A fibroblast-derived TGFβ/sFRP2 noncanonical Wnt signaling axis underlies epithelial metaplasia in idiopathic pulmonary fibrosis

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
8/5/20230
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Cell growth and nutrient availability control the mitotic exit signaling network in budding yeast

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
8/4/20230
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Tight junction membrane proteins regulate the mechanical resistance of the apical junctional complex

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
8/4/20230
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The mechanics of bronchoconstriction cause airway epithelial damage and inflammation by excess crowding-induced extrusion

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
8/4/20230
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RBL2 represses the transcriptional activity of Multicilin to inhibit multiciliogenesis

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
8/4/20230
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Optogenetic control of lysosome function

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
8/4/20230
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Multi-color live-cell optical nanoscopy using phasor analysis

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
8/4/20230
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Positive feedback regulation of Pparγ1sv and Pparγ2 during adipogenesis in 3T3-L1 cells

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
8/4/20230
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A force-sensitive mutation reveals a spindle assembly checkpoint-independent role for dynein in anaphase progression

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
8/4/20230
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FAM210A mediates an inter-organelle crosstalk essential for protein synthesis and muscle growth in mouse

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
8/4/20230
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Aggregation of Disrupted in Schizophrenia 1 arises from a central region of the protein

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
8/4/20230
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Recovery of proteasome activity in cells pulse-treated with proteasome inhibitors is independent of DDI2

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
8/4/20230
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A Bright, Photostable Dye that Enables Multicolor, Time Lapse, and Super-Resolution Imaging of Acidic Organelles

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
8/4/20230
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Oligomerization and feedback on membrane recruitment stabilize PAR-3 asymmetries in C. elegans zygotes.

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
8/4/20230
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Determining the structure of protein-bound ceramides, essential lipids for skin barrier function

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
8/4/20230
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Correlative single-cell X-ray tomography and X-ray fluorescence imaging

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
8/4/20230
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Discovery of a multipotent cell type from the term human placenta

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
8/4/20230
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Parallel proteomics and phosphoproteomics defines starvation signal specific processes in cell quiescence

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
8/4/20230
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Diverse mechanisms control amino acid-dependent environmental alkalization by Candida albicans

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
8/4/20230
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Optogenetic strategies for optimizing the performance of biosensors of membrane phospholipids in live cells

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
8/4/20230
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Sulfolobus acidocaldarius adhesion pili power twitching motility in the absence of a dedicated retraction ATPase

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
8/4/20230
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Lactate activates the mitochondrial electron transport chain independent of its metabolism

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
8/4/20230
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Comprehensive genome editing confers "off-the-shelf" CAR-T cells superior efficacy against solid tumors

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
8/4/20230
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hGRAD - a versatile 'one-fits-all' system for the acute depletion of RNA binding proteins in nuclear condensates

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
8/4/20230
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Presence of the ER-Golgi intermediate compartment that matures into the Golgi in yeast

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
8/3/20230
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Inhibition of Topoisomerase 2 catalytic activity impacts the integrity of heterochromatin and repetitive DNA and leads to interlinks between clustered repeats

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
8/3/20230
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Lysosomal release of amino acids at ER three-way junctions regulates transmembrane and secretory protein mRNA translation

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
8/3/20230
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Immunoregulatory macrophages modify local pulmonary immunity and ameliorate hypoxic-pulmonary hypertension

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
8/3/20230
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Chaperone Complexes From The Endoplasmic Reticulum (ER) And The Cytosol Inhibit wt-p53 By Activation The ER To Cytosol Signaling.

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
8/3/20230
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Discretised flux balance analysis for reaction-diffusion simulation of single-cell metabolism

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
8/3/20230
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IST1 regulates select endosomal recycling pathways

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
8/2/20230
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The ciliary MBO2 complex targets assembly of inner arm dynein b and reveals additional doublet microtubule asymmetries

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
8/2/20230
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The Crk4-Cyc4 complex regulates G2 phase of apicomplexan endodyogeny

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
8/2/20230
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Myogenetic oligodeoxynucleotide induces myocardial differentiation of murine pluripotent stem cells

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
8/2/20230
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Disordered C-terminal domain drives spatiotemporal confinement of RNAPII to enhance search for chromatin targets

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
8/2/20230
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Dynamic proximity interaction profiling suggests that YPEL2 is involved in cellular stress surveillance

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
8/2/20230
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Induced pluripotent stem cell-derived human macrophages as an infection model for Leishmania donovani

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
8/2/20230
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Mitochondrial Ca2+ uniporter (MCU) variants form plasma-membrane channels

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
8/2/20230
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Multivalent GU-rich oligonucleotides sequester TDP-43 in the nucleus by inducing high molecular weight RNP complexes

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
8/2/20230
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Growth-dependent concentration gradient of the oscillating Min system in Escherichia coli

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
8/2/20230
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Single-cell and spatial transcriptomics reveals the human liver immunological landscape and myeloid dysfunction in PSC.

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
8/1/20230
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Therapeutic effect of dietary interventions on senescence in animals and humans: A systematic review

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
8/1/20230
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Nanoscale analysis of human G1 and metaphase chromatin in situ

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
7/31/20230
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Long-chain fatty acyl-coenzyme A activates the mitochondrial fission factors MiD49 and MiD51 by inducing their oligomerization

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
7/31/20230
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Single tyrosine mutation in VE-cadherin modulates gene lung expressions: evidence for FOXF1 mediated S1PR1 upregulation to stabilize vessels in mice

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
7/31/20230
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Actin-Driven Nanotopography Enhances Integrin Molecular Clutch in Developing Tissue

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
7/30/20230
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DCLRE1B/hSNM1B (Apollo) is not acutely required for human pluripotent stem cell survival

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
7/30/20230
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Mutual dependence of Osbp and PI4KII in the maturation of regulated secretory granules

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
7/30/20230
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A mechanical modelling framework to study endothelial permeability

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
7/30/20230
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Evaluating cytotoxicity and genotoxicity of oil extracted from visceral fat of Caiman yacare (Daudin, 1802) in chinese hamster lung fibroblast in vitro.

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
7/30/20230
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Targeted Photoconvertible BODIPYs Based on Directed Photooxidation Induced Conversion for Applications in Photoconversion and Live Super Resolution Imaging

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
7/30/20230
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Conserved long noncoding RNA TILAM promotes liver fibrosis through interaction with PML in hepatic stellate cells

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
7/30/20230
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Distinct human stem cell subpopulations drive adipogenesis and fibrosis in musculoskeletal injury

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
7/29/20230
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Epigenetic modifications driving ground state pluripotency exit require an NF-κB-independent chromatin IκBα function

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
7/29/20230
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Centrosome amplification primes for apoptosis and favors the response to chemotherapy in ovarian cancer beyond multipolar divisions

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
7/29/20230
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Unbiased method for spectral analysis of cells with great diversity of autofluorescence spectra

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
7/29/20230
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Neuroprotective role of lactate in a human in vitro model of the ischemic penumbra

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
7/29/20230
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Rho-associated kinase regulates Langerhans cell morphology and responsiveness to tissue damage

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
7/29/20230
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Enhancing gene transfer to renal tubules and podocytes by context-dependent selection of AAV capsids

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
7/29/20230
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An adaptive biomolecular condensation response is conserved across environmentally divergent species

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
7/29/20230
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Microtubule association of TRIM3 revealed by differential extraction proteomics

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
7/28/20230
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A new mechanism of posttranslational polyglutamylation regulates phase separation and signaling of the Wnt pathway protein Dishevelled.

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
7/28/20230
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Universal length fluctuations of actin structures found in cells

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
7/28/20230
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Human receptive endometrial organoid for deciphering the implantation window

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
7/28/20230
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Ribosomal Protein SA (RPSA) is required for localized translation and sarcomere maintenance

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
7/28/20230
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The nuclei of human adult stem cells can move within the cell and generate cellular protrusions to contact other cells

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
7/27/20230
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The membrane-actin linkers ezrin, radixin, and moesin are dispensable for macrophage migration and cortex mechanics.

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
7/27/20230
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Disease-associated gut microbiome and metabolome changes in chronic low back pain patients with bone marrow lesions

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
7/27/20230
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Functional analysis of Cdc20 reveals a critical role of CRY box in mitotic checkpoint signaling

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
7/27/20230
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RAD54L regulates replication fork progression and nascent strand degradation in BRCA1/2-deficient cells

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
7/27/20230
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Megakaryocyte maturation involves activation of the IRE1α-dependent adaptive unfolded protein response

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
7/27/20230
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Heparan sulfate promotes TRAIL-induced tumor cell apoptosis

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
7/27/20230
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Downregulation of lysosomal trafficking in ARPE19 cells leads to decreased transfection efficiency at high passage

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
7/27/20230
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Apical annuli are specialised sites of post-invasion secretion of dense granules in Toxoplasma

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
7/27/20230
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Beyond 40 fluorescent probes for deep phenotyping of blood mononuclear cells, using spectral technology.

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
7/27/20230
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IF1 controls UCP1-dependent mitochondrial bioenergetics in brown adipocytes

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
7/27/20230
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Leishmania major-induced alteration of host cellular and systemic copper homeostasis drives the fate of infection

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
7/26/20230
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Primary human lung fibroblasts exhibit trigger- but not disease-specific cellular senescence and impair alveolar epithelial cell progenitor function

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
7/26/20230
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The maintenance of centriole appendages and motile cilia basal body anchoring relies on TBCCD1

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
7/26/20230
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The use of imaging flow cytometry for rapid, high-throughput and automated analysis of the Leishmania mexicana promastigote cell cycle provides new insights into cell cycle events of short duration

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
7/25/20230
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Novel centriolar defects underlie a primary ciliary dyskinesia phenotype in adenylate kinase 7 deficient ciliated epithelium.

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
7/25/20230
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FAM104 proteins promote the nuclear localization of p97/VCP

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
7/25/20230
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The Hippo kinase cascade regulates a contractile cell behavior and cell density in a close unicellular relative of animals

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
7/25/20230
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RAB3 phosphorylation by pathogenic LRRK2 impairs trafficking of synaptic vesicle precursors

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
7/25/20230
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TTLL12 is required for primary ciliary axoneme formation in polarized epithelial cells

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
7/25/20230
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pHusion: A robust and versatile toolset for automated detection and analysis of exocytosis

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
7/25/20230
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Mechano-dependent sorbitol accumulation supports biomolecular condensate

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
7/25/20230
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Collective dynamics of formin and microtubule and its crosstalk mediated by FHDC1

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
7/25/20230
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The Arf-GAP Age2 localizes to the late-Golgi via a conserved amphipathic helix

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
7/24/20230
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Evolution of cullin E3 ubiquitin ligases and function in trypanosomes

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
7/24/20230
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Downregulation of a cell polarity protein potentiates Chikungunya Virus infection in host cells

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
7/24/20230
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Characterization of Pik1 function in fission yeast reveals its conserved role in lipid synthesis and not cytokinesis

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
7/24/20230
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Coronin-1 controls directional cell rearrangement in Drosophila wing epithelium

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
7/24/20230
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Mecp2 Fine-tunes Quiescence Exit by Targeting Nuclear Receptors

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
7/24/20230
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SCN9A gene therapy: Novel mechanism to inhibit cellular senescence in astrocytes in vitro

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
7/24/20230
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A genome-wide single-cell 3D genome atlas of lung cancer progression

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
7/24/20230
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MiniBAR/KIAA0355 is a dual Rac and Rab effector required for ciliogenesis

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
7/24/20230
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Metabolomic rearrangement controls the intrinsic microbial response to temperature changes

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
7/24/20230
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Endocytic vesicles act as vehicles for glucose uptake in response to growth factor stimulation

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
7/24/20230
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Essential role of the CCL2-CCR2 axis in Mayaro virus-induced disease

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
7/24/20230
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Regulation of mitochondrial calcium uniporter expression and calcium signalling by lncRNA Tug1 in cardiomyocytes.

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
7/24/20230
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Dynamic regulatory phosphorylation of mouse CDK2 occurs during meiotic prophase I

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
7/24/20230
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Helical motors and formins synergize to compact chiral filopodial bundles: a theoretical perspective.

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
7/24/20230
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Resolution enhancement with deblurring by pixel reassignment (DPR)

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
7/24/20230
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The Role of the Tyrosine-Based Sorting Signals of the ORF3a Protein of SARS-CoV-2 on Intracellular Trafficking, Autophagy, and Apoptosis

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
7/24/20230
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An mTOR/RNA pol I axis shapes chromatin architecture in response to fasting

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
7/22/20230
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Extracting herbicide and antibiotic natural products from a plant-microbe interaction system

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
7/22/20230
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ISGylation-independent protection of cell growth by USP18 following interferon stimulation

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
7/21/20230
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Generation of an induced pluripotent stem cell line from a healthy adult indigenous Nigerian participant

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
7/21/20230
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The interactome of the UapA transporter reveals putative new players in anterograde membrane cargo trafficking

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
7/21/20230
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Identification of Periostin as a critical niche for myofibroblast dynamics and fibrosis during tendon healing

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
7/21/20230
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Cdc42 mobility and membrane flows regulate fission yeast cell shape and survival

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
7/21/20230
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Dual regulation of Misshapen by Tao and Rap2l promotes collective cell migration

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
7/21/20230
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Enucleation of the C. elegans embryo revealed the mechanism of dynein-dependent spacing between microtubule asters

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
7/21/20230
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Identification and Characterization of Calcium Binding Protein, Spermatid Associated 1 (CABS1) in Selected Human Tissues and Fluids

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
7/21/20230
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Transcription factor-mediated direct cellular reprogramming yields cell-type specific DNA methylation signature

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
7/21/20230
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Validation of GCN5L1/BLOC1S1/BLOS1 Antibodies Using Knockout Cells and Tissue

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
7/21/20230
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Autophagy maintains the homeostatic environment in the male reproductive accessory organs playing a key role in fertility

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
7/21/20230
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Spatiotemporal precise optical manipulation of intracellular molecular activities

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
7/20/20230
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A molecular toolbox to study progesterone receptor signaling

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
7/20/20230
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AMPK targets PDZD8 to trigger carbon source shift to glutamine

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
7/20/20230
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Quantitative Measurement of Secretory Protein Mistargeting by Proximity Labeling and Parallel Reaction Monitoring

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
7/20/20230
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Post-translational regulation of the Numb/Notch pathway in neurogenesis and cancer by Dlk2

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
7/20/20230
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Continuous endosomes form functional subdomains and orchestrate rapid membrane trafficking in trypanosomes

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
7/20/20230
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Pancreatic islet cell stress induced by insulin-degrading enzyme deficiency promotes islet regeneration and protection from autoimmune diabetes

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
7/20/20230
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Bioactive effects of natural and novel unnatural tropolone sesquiterpenoids in a murine cell model of renal interstitial fibroblasts

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
7/19/20230
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Dynamics of pax7 expression during development, muscle regeneration, and in vitro differentiation of satellite cells in the trout.

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
7/19/20230
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High resolution spatial investigation of intracellular oxygen in muscle cells.

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
7/19/20230
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Effects of Alternative Splicing-Specific Knockdown of Tjp1 α+ by Rbm47 on Tight Junctions Assembly during Blastocyst Development

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
7/19/20230
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Analysis of small EV proteomes reveals unique functional protein networks regulated by VAP-A

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
7/19/20230
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The cellular landscape of the endochondral bone highlights its multipotent and immunomodulatory features during the transition to extrauterine life

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
7/19/20230
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Single cell RNA analysis uncovers the cell differentiation trajectories and functionalization for air breathing of frog lung

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
7/19/20230
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Cold temperature induces a TRPM8-independent calcium release from the endoplasmic reticulum in human platelets

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
7/19/20230
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Trabid patient mutations impede the axonal trafficking of adenomatous polyposis coli to disrupt neurite growth

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
7/19/20230
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A Plant Flavonol Rescues a Pathogenic Mutation Associated with Kinesin in Neurons

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
7/19/20230
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Cardiomyocyte mechanical memory is regulated through the talin interactome and DLC1 dependent regulation of RhoA

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
7/19/20230
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Perturbations of glutathione and sphingosine metabolites in Port Wine Birthmark patient-derived induced pluripotent stem cells

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
7/19/20230
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Analysis of Drosophila and mouse mutants reveals that Peroxidasin is required for tissue mechanics and full viability

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
7/19/20230
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A signalling rheostat controls chromosome segregation fidelity during early lineage specification and neurogenesis by modulating DNA replication stress

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
7/18/20230
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S100A11 promotes focal adhesion disassembly via myosin II-driven contractility and Piezo1-mediated Ca2+ entry

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
7/18/20230
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ICAM-1 nanoclusters regulate hepatic epithelial cell polarity by leukocyte adhesion-independent control of apical actomyosin

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
7/18/20230
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Non-cell autonomous cardiomyocyte regulation complicates gene supplementation therapy for LMNA cardiomyopathy

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
7/18/20230
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Cell networks in the mouse liver during partial hepatectomy

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
7/18/20230
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Monitoring hPSC genomic stability in the chromosome 20q region by ddPCR

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
7/18/20230
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Tepsin binds LC3B to promote ATG9A export and delivery at the cell periphery

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
7/18/20230
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Three-step docking by WIPI2, ATG16L1 and ATG3 delivers LC3 to the phagophore

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
7/17/20230
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Multi-monoubiquitination controls VASP-mediated actin dynamics

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
7/17/20230
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Increased beta2-adrenergic signaling is a targetable stimulus essential for bone healing by promoting callus neovascularization

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
7/16/20230
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Loss of Progranulin Results in Increased Pan-Cathepsin Activity and Reduced LAMP1 Lysosomal Protein

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
7/16/20230
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From Pixels to Phenotypes: Integrating Image-Based Profiling with Cell Health Data Improves Interpretability

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
7/16/20230
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Ubiquitination-mediated Golgi-to-endosome sorting determines the poison-antidote duality of wtf meiotic drivers

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
7/16/20230
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Refrigerated storage and cryopreservation of hormone induced sperm in a threatened frog

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
7/16/20230
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Disrupting actin filaments enhances glucose-stimulated insulin secretion independent of the cortical actin cytoskeleton

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
7/16/20230
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Cryo-electron tomography of eel sperm flagella reveals a molecular "minimum system" for motile cilia

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
7/16/20230
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Cryo-electron tomography of eel sperm flagella reveals a molecular "minimum system" for motile cilia

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
7/16/20230
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Cryo-electron tomography of eel sperm flagella reveals a molecular "minimum system" for motile cilia

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
7/16/20230
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Cardiomyocyte-specific adenylyl cyclase type-8 overexpression induces cell-autonomous activation of RelA and non-cell-autonomous myocardial and systemic inflammation

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
7/16/20230
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Role of trafficking protein particle complex 2 in medaka development

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
7/14/20230
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Pooled endogenous protein tagging and recruitment for scalable discovery of effectors for induced proximity therapeutics

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
7/14/20230
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Changes in cell morphology and function induced by NRAS Q61R mutation in lymphatic endothelial cells

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
7/14/20230
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Polyubiquitinated PCNA promotes alternative lengthening of telomeres by inducing break-induced replication

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
7/14/20230
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DNA damage and nuclear morphological changes in cardiac hypertrophy are mediated by SNRK through actin depolymerization

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
7/14/20230
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Pooled tagging and hydrophobic targeting of endogenous proteins for unbiased mapping of unfolded protein responses

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
7/14/20230
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Trafficking Machinery is Rapidly Primed to Facilitate Polarised IL-6 Secretion in Dendritic Cells

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
7/13/20230
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A Size Filter Regulates Apical Protein Sorting

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
7/13/20230
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Treatment of advanced atherosclerotic mice with the senolytic agent ABT-263 is associated with reduced indices of plaque stability and increased mortality

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
7/13/20230
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Unlocking the Role of Endothelial MPL Receptor and JAK2V617F Mutation: Insights into Cardiovascular Dysfunction in MPNs and CHIP

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
7/13/20230
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Multiple genetic variants at the SLC30A8 locus affect a local super-enhancer cluster to influence pancreatic β-cell survival and function

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
7/13/20230
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Eye lens organoids going simple: characterization of a new 3-dimensional organoid model for lens development and pathology

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
7/12/20230
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The Septin Cytoskeleton is Required for Plasma Membrane Repair

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
7/12/20230
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Polarized localization of kinesin-1 and RIC-7 drives axonal mitochondria anterograde transport

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
7/12/20230
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BNIP3-mediated mitophagy boosts the competitive dominant growth of lenvatinib resistant cells via reprogramming energy metabolism in HCC

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
7/12/20230
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EMDiffuse: a diffusion-based deep learning method augmenting ultrastructural imaging and volume electron microscopy

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
7/12/20230
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Oncostatin M is a Master Regulator of an Inflammatory Network in Dnmt3a-Mutant Hematopoietic Stem Cells

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
7/12/20230
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Natural transformation specific DprA coordinate DNA double strand break repair pathways in heavily irradiated D. radiodurans

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
7/12/20230
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Multi-tissue proteomics identifies a link between satellite DNA organization and transgenerational transposon repression in Drosophila

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
7/12/20230
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Endocytosis Inhibitors Block SARS-CoV-2 Pseudoparticle Infection of Mink Lung Epithelium

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
7/12/20230
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OrgaMapper: A robust and easy-to-use workflow for analyzing organelle positioning

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
7/11/20230
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Cofilin-Driven Nuclear Deformation Drives Dendritic Cell Migration through the Extracellular Matrix

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
7/11/20230
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Pathogenic aggregates alter actin organization and cellular viscosity resulting in stalled clathrin mediated endocytosis

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
7/11/20230
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Mitochondrial proteostasis mediated by CRL5Ozz and Alix maintains skeletal muscle function

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
7/11/20230
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Exportin-mediated nucleocytoplasmic transport maintains Pch2 homeostasis during meiosis

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
7/10/20230
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ZIF-1-mediated degradation of endogenous and heterologous zinc finger proteins in the C. elegans germ line

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
7/10/20230
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Remodeling of the secretory pathway is coordinated with de novo membrane formation in budding yeast gametogenesis

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
7/10/20230
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The Drosophila Tumour Suppressor Lgl and Vap33 activate the Hippo pathway by a dual mechanism, involving RtGEF/Git/Arf79F and inhibition of the V-ATPase.

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
7/10/20230
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miR-324 mediates bone homeostasis through the regulation of osteoblast and osteoclast differentiation and activity

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
7/10/20230
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An amino acid-resolution interactome for motile cilia illuminates the structure and function of ciliopathy protein complexes

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
7/10/20230
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Label-free ghost cytometry for manufacturing of cell therapy products

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
7/10/20230
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Spatial organization and function of RNA molecules within phase-separatedcondensates are controlled by Dnd1

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
7/9/20230
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ScaleFExSM: a lightweight and scalable method to extract fixed features from single cells in high-content imaging screens

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
7/9/20230
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Peripheral positioning of lysosomes supports melanoma aggressiveness

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
7/9/20230
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Mitochondrial-Derived Compartments Remove Surplus Proteins from the Outer Mitochondrial Membrane

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
7/8/20230
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Affinity hierarchies underlie the co-assembly of nucleolar and heterochromatin condensates in Drosophila embryos

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
7/8/20230
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Neuromorphic Cytometry: Implementation on cell counting and size estimation

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
7/7/20230
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Near infrared-light treatment alters mitochondrial homeostasis to induce senescence in breast cancer cells

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
7/7/20230
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Filter-aided extracellular vesicle enrichment (FAEVEr)

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
7/7/20230
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Activation of goblet cell stress sensor IRE1β is controlled by the mucin chaperone AGR2

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
7/7/20230
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A role for Vps13-mediated lipid transfer at the ER-endosome contact site in ESCRT-mediated sorting

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
7/7/20230
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Mon1a and FCHO2 are required for maintenance of Golgi architecture

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
7/7/20230
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Complex Sphingolipid Profiling and Identification of an Inositol Phosphorylceramide Synthase in Dictyostelium discoideum

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
7/7/20230
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Mitochondrial-Derived Compartments are Multilamellar Domains that Encase Membrane Cargo and Cytosol

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
7/7/20230
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Distinct structural motifs are necessary for targeting and import of Tim17 in Trypanosoma brucei mitochondrion

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
7/7/20230
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Nuclear envelope assembly relies on CHMP-7 in the absence of BAF-LEM-mediated hole closure

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
7/7/20230
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Aβ-aggregation-generated blue autofluorescence illuminates senile plaques, complex blood and vascular pathologies in the Alzheimer's disease

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
7/7/20230
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Aβ-aggregation-generated blue autofluorescence illuminates senile plaques, complex blood and vascular pathologies in the Alzheimer's disease

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
7/7/20230
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Aβ-aggregation-generated blue autofluorescence illuminates senile plaques, complex blood and vascular pathologies in the Alzheimer's disease

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
7/7/20230
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Aβ-aggregation-generated blue autofluorescence illuminates senile plaques, complex blood and vascular pathologies in the Alzheimer's disease

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
7/7/20230
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Augmented Super-Resolution Radial Fluctuations (aSRRF) Pushing the Limits of Structured Illumination Microscopy

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
7/6/20230
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Systemic Lupus Erythematosus Serum Stimulation of Human Intestinal Organoids Induces Changes in Goblet Cell Differentiation and Mitochondrial Fitness

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
7/5/20230
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Phosphorylation-dependent interactions of VAPB and ELYS contribute to the temporal progression of mitosis

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
7/5/20230
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Cell contacts and pericellular matrix in the Xenopus gastrula chordamesoderm

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
7/5/20230
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Control of C. elegans growth arrest by stochastic, yet synchronized DAF-16/FOXO nuclear translocation pulses

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
7/5/20230
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Metabolism regulates muscle stem cell self-renewal by connecting the microenvironment and histone acetylation

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
7/5/20230
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A matrisome atlas of germ cell development

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
7/4/20230
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The longer the better: longer acrosomes contain more proteins involved in sperm-egg interactions

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
7/4/20230
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A scalable, data analytics workflow for image-based morphological profiles

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
7/4/20230
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CaaX-motif adjacent residues control G protein prenylation under suboptimal conditions

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
7/4/20230
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Dysregulation of N-terminal acetylation causes cardiac arrhythmia and cardiomyopathy

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
7/3/20230
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Effect of Aging on the Human Myometrium at Single-Cell Resolution

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
7/3/20230
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Establishing RNAge to score cellular aging and rejuvenation paradigms and identify novel age-modulating compounds

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
7/3/20230
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Dynein and dynactin move long-range but are delivered separately to the axon tip

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
7/3/20230
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Direct recruitment of Mis18 to interphase spindle poles promotes CENP-A chromatin assembly

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
7/3/20230
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Sub-lethal exposure to chlorfenapyr reduces the probability of developing Plasmodium falciparum parasites in surviving Anopheles mosquitoes

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
7/3/20230
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Novel phosphatidylinositol flippases contribute to phosphoinositide homeostasis in the plasma membrane

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
7/3/20230
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Diseased human pancreas and liver microphysiological system for preclinical diabetes research

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
7/3/20230
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Differential expression of tension-sensitive HOX genes in fibroblasts is associated with different scar types

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
7/3/20230
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Extracellular vesicles promote autophagy in human microglia through lipid raft-dependent mechanisms

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
7/3/20230
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ROM1 is redundant to PRPH2 as a molecular building block of photoreceptor disc rims

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
7/2/20230
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Hippo and Wnt pathways are impaired in Port Wine Birthmark-derived induced pluripotent stem cells and endothelial cells

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
7/2/20230
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BRD4 directs mitotic cell division by inhibiting DNA damage

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
7/2/20230
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Raman micro-spectroscopy reveals the spatial distribution of fumarate in cells and tissues.

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
7/2/20230
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Fusion of histone variants to Cas9 enhances homology-directed repair

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
7/2/20230
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A Serum- and Feeder-Free System to Generate CD4 and Regulatory T Cells from Human iPSCs

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
7/2/20230
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Mitigation of the effect of high light on the photosynthetic apparatus of Rhodobacter alkalitolerans when grown in an alkaline environment

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
7/1/20230
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The efficient induction of human retinal ganglion-like cells provides a platform for studying optic neuropathies

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
7/1/20230
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Preferential transport of synaptic vesicles across neuronal branches is regulated by the levels of the anterograde motor UNC-104/KIF1A

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
7/1/20230
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Interaction between PSD 95 and TRPV4 through PDZ domain controls TRPV4's localization and activity.

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
7/1/20230
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Orchestration of differential mesodermal fate choice from ESCs by Wnt-USP3 link and H2A/H2B contextual deubiquitination.

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
7/1/20230
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Structural insights into dual-antagonize mechanism of AB928 on adenosine A2 receptors

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
7/1/20230
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Interaction between PSD 95 and TRPV4 through PDZ domain controls TRPV4's localization and activity.

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
7/1/20230
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Interaction between PSD 95 and TRPV4 through PDZ domain controls TRPV4's localization and activity.

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
7/1/20230
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The super-healing MRL strain promotes muscle growth in muscular dystrophy through a regenerative extracellular matrix

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
6/30/20230
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Single cell-resolution in situ sequencing elucidates spatial dynamics of multiple sclerosis lesion and disease evolution

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
6/30/20230
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Transcription factor Nrf1 regulates proteotoxic stress-induced autophagy

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
6/30/20230
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Mechanical coupling coordinates microtubule growth

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
6/30/20230
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Mechanistic constraints on the trade-off between photosynthesis and respiration in response to warming

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
6/30/20230
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Matrix stiffening promotes perinuclear clustering of mitochondria

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
6/30/20230
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Post-Transplant Administration of G-CSF Impedes Engraftment of Gene Edited Human Hematopoietic Stem Cells by Exacerbating the p53-Mediated DNA Damage Response

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
6/30/20230
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A partial Drp1 knockout improves autophagy flux independent of mitochondrial function

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
6/30/20230
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C-terminal amides mark proteins for degradation via SCF/FBXO31

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
6/30/20230
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TREX reveals proteins that bind to specific RNA regions in living cells

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
6/30/20230
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Interaction between host G3BP and viral nucleocapsid protein regulates SARS-CoV-2 replication

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
6/30/20230
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Sse1, Hsp110 chaperone of yeast, controls the cellular fate during Endoplasmic Reticulum-stress

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
6/29/20230
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Proteins differentially expressed between pathogenic and non-pathogenic Entamoeba histolytica clones influence pathogenicity by different mechanisms

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
6/29/20230
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A rapid, inexpensive, non-lethal method for detecting disseminated neoplasia in a bivalve

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
6/29/20230
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Liver-specification of human iPSC-derived endothelial cells transplanted into mouse liver

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
6/29/20230
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Nuclear quality control of non-imported secretory proteins attenuates proteostasis decline in the cytosol

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
6/28/20230
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A novel role for the peptidyl-prolyl cis-trans isomerase Cyclophilin A in DNA-repair following replication fork stalling via the MRE11-RAD50-NBS1 complex.

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
6/28/20230
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p53 controls the nuclear entry and epigenetic modification of H3.1 by downregulating nuclear phosphatidic acid

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
6/28/20230
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The Tubulin Nano-Code: a protofilament-specific pattern of tubulin post-translational modifications regulates ciliary beating mechanics

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
6/28/20230
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Decline of DNA damage response along with myogenic differentiation

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
6/28/20230
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Identification of small molecule inhibitors of G3BP-driven stress granule formation

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