PaperPlayer biorxiv cell biology: Recent Episodes
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Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.28.514275v1?rss=1
Authors: Plazen, L., Khadra, A.
Abstract: Mesenchymal cell motility is mainly regulated by two members of the Rho-family of GTPases, called Rac and Rho. The mutual inhibition exerted by these two proteins on each other's activation and the promotion of Rac activation by an adaptor protein called paxillin have been implicated in driving cellular polarization comprised of front (high active Rac) and back (high active Rho) during cell migration. Mathematical modeling of this regulatory network has previously shown that bistability is responsible for generating a spatiotemporal pattern underscoring cellular polarity called wave-pinning when diffusion is included. We previously developed a 6D reaction-diffusion model of this network to decipher the role of Rac, Rho and paxillin (along with other auxiliary proteins) in generating wave-pinning. In this study, we simplify this model through a series of steps into an excitable 3D ODE model comprised of one fast variable (the scaled concentration of active Rac), one slow variable (the maximum paxillin phosphorylation rate - turned into a variable) and a very slow variable (a recovery rate - also turned into a variable). We then explore, through slow-fast analysis, how excitability is manifested by showing that the model can exhibit relaxation oscillations (ROs) as well as mixed-mode oscillations (MMOs) whose underlying dynamics are consistent with a delayed Hopf bifurcation. By reintroducing diffusion and the scaled concentration of inactive Rac into the model, we obtain a 4D PDE model that generates several unique spatiotemporal patterns that are relevant to cell motility. These patterns are then characterized and their impact on cell motility are explored by employing the cellular potts model (CPM). Our results reveal that wave pinning produces purely very directed motion in CPM, while MMOs allow for meandering and non-motile behaviours to occur. This highlights the role of MMOs as a potential mechanism for mesenchymal cell motility.
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Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.28.513298v1?rss=1
Authors: Nguyen Ngoc, K. V., Sai, S., Jun, Y., Bender, R. H. F., Kravets, V., Zhu, H., Hatch, C. J., Schlichting, M., Gaetani, R., Mallick, M., Hachey, S. J., Christman, K., George, S. C., Hughes, C. C., Sander, M.
Abstract: Blood vessels play a critical role in pancreatic islet health and function, yet current culture methods to generate islet organoids from human pluripotent stem cells (SC-islets) lack a vascular component. Here, we engineered 3D vascularized SC-islet organoids by assembling SC-islet cells, human primary endothelial cells (ECs) and fibroblasts both in a non-perfused model and a microfluidic device with perfused vessels. Vasculature improved stimulus-dependent Ca2+ influx into SC-{beta}-cells, a hallmark of {beta}-cell function that is blunted in non-vascularized SC-islets. We show that an islet-like basement membrane is formed by vasculature and contributes to the functional improvement of SC-{beta}-cells. Furthermore, cell-cell communication networks based on scRNA-seq data predicted BMP2/4-BMPR2 signaling from ECs to SC-{beta}-cells. Correspondingly, BMP4 augmented the SC-{beta}-cell Ca2+ response and insulin secretion. These vascularized SC-islet models will enable further studies of crosstalk between {beta}-cells and ECs and can serve as in vivo-mimicking platforms for disease modeling and therapeutic testing.
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Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.28.514255v1?rss=1
Authors: Chioccioli, M., Magruder, S., McDonough, J. E., Nouws, J., Gonzalez, D., Borriello, L., Traub, B., Ye, X., Hendry, C. E., Entenberg, D., Kaminski, N., Krishnaswamy, S., Sauler, M.
Abstract: Tissue repair requires a highly coordinated cellular response to ensure the correct balance of replacement cells to lost cells. In the lung, alveolar type 2 (AT2) cells act as stem cells and can replace both themselves and alveolar type 1 cells (AT1); however, the complex orchestration of AT2 stem cell activity following lung injury is poorly understood owing to the inability to track individual stem cells and their dynamic behavior over time. Here, we apply live time lapse imaging to ex vivo mouse precision cut lung slice (PCLS) culture and in vivo mouse lung to track individual GFP-labeled AT2 cells for 72h following intra-tracheal administration of bleomycin. We observe highly dynamic movement of AT2 cells, including migration within and between alveoli, as well as the emergence of at least three distinct morphokinetic AT2 cell states. Small molecule-based inhibition of Rho-associated protein kinase (ROCK) pathway significantly reduced motility of AT2 stem cells following injury and reduced expression of Krt8, a known marker of intermediate progenitor cells. Together, our results uncover motility of alveolar stem cells as a new injury response mechanism in the lung and uncover properties of stem cell motility at high cellular resolution.
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Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.28.514302v1?rss=1
Authors: Radousky, Y. A., Hague, M. T. J., Fowler, S., Paneru, E., Codina, A., Rugamas, C., Hartzog, G., Cooper, B. S., Sullivan, W.
Abstract: A broad array of endosymbionts radiate through host populations via vertical transmission, yet much remains unknown concerning the cellular basis, diversity and routes underlying this transmission strategy. Here we address these issues, by examining the cellular distributions of Wolbachia strains that diverged up to 50 million years ago in the oocytes of 18 divergent Drosophila species. This analysis revealed three Wolbachia distribution patterns: 1) a tight clustering at the posterior pole plasm (the site of germline formation); 2) a concentration at the posterior pole plasm, but with a significant bacteria population distributed throughout the oocyte; 3) and a distribution throughout the oocyte, with none or very few located at the posterior pole plasm. Examination of this latter class reveals Wolbachia accesses the posterior pole plasm during the interval between late oogenesis and the blastoderm formation. We also find that one Wolbachia strain in this class concentrates in the posterior somatic follicle cells that encompass the pole plasm of the developing oocyte, suggesting these are the source of Wolbachia that ultimately occupy the germline. In contrast, strains in which Wolbachia concentrate at the posterior pole plasm generally exhibit no or few Wolbachia in the follicle cells associated with the pole plasm. Phylogenomic analysis indicates that closely related Wolbachia strains tend to exhibit similar patterns of posterior localization, suggesting that specific localization strategies are a function of Wolbachia-associated factors. Previous studies revealed that endosymbionts rely on one of two distinct routes of vertical transmission: continuous maintenance in the germline (germline-to-germline) or a more circuitous route via the soma (germline-to-soma-to-germline). Here we demonstrate that Wolbachia strains infecting Drosophila species maintain the diverse arrays of cellular mechanisms necessary for both of these distinct transmission routes. This characteristic may account for its ability to infect and spread globally through a vast range of host insect species.
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Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.28.514178v1?rss=1
Authors: Jiravejchakul, N., Abe, G. L., Loza, M., Park, S., Matangkasombut, P., Sasaki, J.-I., Imazato, S., Diez, D., Standley, D. M.
Abstract: In-depth knowledge of the cellular and molecular composition of dental pulp (DP) and the crosstalk between DP cells that drive tissue homeostasis or regeneration are not well understood. To address these questions, we performed data analysis of publicly available single-cell transcriptomes of DP. This analysis revealed that DP resident fibroblasts have a unique gene expression profile when compared with fibroblasts from 5 other reference tissues: blood, bone marrow, adipose tissue, lung, and skin. Genes coded for heparin-binding growth-factors, pleiotrophin (PTN) and midkine (MDK), possessed the highest differential expression levels in DP fibroblasts. In addition, we identified extensive crosstalk between DP fibroblasts and several other DP cells, including Schwann cells, MSCs and odontoblasts. These findings suggest that fibroblast-derived growth factors regulate DP niches, and thus have a potential role as dental therapeutic targets.
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Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.27.514151v1?rss=1
Authors: Botman, D., Kanagasabapathi, S., Teusink, B.
Abstract: Budding yeast uses the well-conserved TORC1-Sch9 and cAMP-PKA signalling pathways to regulate adaptations to changing nutrient environments. Dynamic and single-cell measurements of the activity of these two cascades will improve our understanding of cellular adaptation of yeast. Here, we employed the AKAR3-EV biosensor developed for mammalian cells to measure the cellular phosphorylation status determined by Sch9 and PKA activity in budding yeast. Using various mutant strains and inhibitors, we show that AKAR3-EV robustly measures the Sch9- and PKA-dependent phosphorylation status in intact yeast cells. At the single-cell level, we found that the phosphorylation responses are homogenous for glucose, sucrose and fructose, but highly heterogeneous for mannose. The Sch9 and PKA pathways have a relatively high affinity for glucose (K0.5 of 0.24 mM) under glucose derepressed conditions. Lastly, steady-state FRET levels of AKAR3-EV seem to be independent of growth rates, suggesting that the Sch9- and PKA-dependent phosphorylation activity are transient responses to nutrient transitions. We believe that the AKAR3-EV sensor is an excellent addition to the biosensor arsenal for illuminating cellular adaptation in single yeast cells.
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Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.27.514052v1?rss=1
Authors: Whitlock, J. M., de Castro, L. F., Collins, M. T., Chernomordik, L. V., Boyce, A. M.
Abstract: Metabolic bone diseases are a collection of disorders resulting in diminished skeletal integrity and changes in bone mass due to perturbations in the life-long process of bone remodeling. Perturbations in the number, size and nuclear multiplicity of osteoclasts underpin the development of diverse metabolic bone diseases that impact greater than 13% of adults over age 50 world-wide. Each metabolic bone disease (e.g., osteoporosis, Pagets disease, fibrous dysplasia (FD), osteopetrosis) presents with unique phenotypes, rises from distinct etiologies and progresses with disparate severities, but all are underpinned by a breakdown in osteoclast formation/function. These perturbations of osteoclast formation/function either stem from or cause dysfunctional osteoclast-osteoblast coordination. Unfortunately, a mechanistic understanding of osteoclast-osteoblast coordination and communication is lacking and represents a major barrier to understanding the biology underpinning bone remodeling and the development of effective treatments targeting this process. Here we have developed an inducible ex vivo culture model that models osteoclast-osteoblast coordination in the bone remodeling compartment. Doxycycline addition to cultures activates GsR201C expression and RANKL release from osteoprogenitors, which elicits the differentiation and fusion of neighboring preosteoclasts. In turn, multinucleated osteoclast formation promotes the proliferation of osteoprogenitors, accompanied by the robust release of RANK+ extracellular vesicles, all within ~4 days. This system recapitulates many aspects of the complex osteoclast-osteoblast coordination required for the function of the bone remodeling compartment in both health and diseases underpinned by excessive osteoclast formation. Moreover, based on the ease of isolation, culture, reproducibility and the general adaptability of these cultures to a variety of assays, we expect that this new model will expedite the investigation of osteoclast-osteoblast coordination and osteoclast fusion in bone remodeling and offer a powerful tool for evaluating signaling cascades and novel therapeutic interventions in osteoclast-linked skeletal disease.
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Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.27.514111v1?rss=1
Authors: Farmer, T., Han, K.-J., Vaeth, K., Taliaferro, M., Prekeris, R.
Abstract: Midbodies (MBs) have been shown to function during telophase as a recruiting hub, especially for ESCRT-III complex subunits, including CHMP4B, to regulate the abscission step of cytokinesis. However, the molecular machinery governing specific protein targeting and activation at the MB remains poorly understood. Until recently, it was thought that abscission regulating proteins, such as ESCRT-III complex subunits, accumulate at the MB by directly or indirectly binding to the MB resident protein, CEP55. However, recent studies have shown that depletion of CEP55 does not fully block ESCRT-III targeting to the MB, and cells in CEP55 knock-out mice divide normally. Additionally, since MBs are microtubule-rich, proteinaceous structures, it is conceptually hard to imagine how large protein complexes, such as the ESCRT-III complex, can successfully diffuse into the MB from the cytosol in a rapid and highly regulated manner. Here, we show that MBs contain mRNAs and that these MB-associated mRNAs can be locally translated, thus, resulting in the accumulation of abscission-regulating proteins. We also demonstrate that localized MB-associated translation of CHMP4B is required for its targeting to the abscission site. Finally, we demonstrate that 3-UTR-dependent CHMP4B mRNA targeting to the MB is required for successful completion of cytokinesis. Based on all this data, we propose a novel method of regulating cytokinesis and abscission by MB-associated targeting and localized translation of selective mRNAs.
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Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.27.513965v1?rss=1
Authors: Liu, X., Xu, L., Song, Y., Li, X., Wong, C.-Y., Rong, C., Feng, J., Chow, H.-M., Yao, S., Gao, S., Liu, X., Duan, L.
Abstract: Mitochondria are membrane-bound organelles that perform diverse critical biological functions. They undergo constant fission and fusion, which are important for mitochondrial inheritance, functions, and quality control. While tremendous efforts have identified many factors governing mitochondria dynamics, emerging evidence indicates the involvement of various intracellular or extracellular mechanical cues. However, how mechanical stress directly modulates mitochondrial dynamics remains largely unknown. Here utilizing an optogenetic mitochondria-specific mechanostimulator to apply pulling forces to intracellular mitochondria, we find that mechanostimulation can promote mitochondrial fission, with sustained mechanostimulation triggering fission more effectively than transient one. Asymmetrical fission can occur at different sub-mitochondrial sites after force-induced mitochondrial elongation. Such force-induced fission is dependent on DRP1 and involves the wrapping of ER tubules. Moreover, mechanical force generates mitochondrial fragments without mtDNA which recruit Parkin proteins. Our results prove the mechanosensitivity and mechanoresponsiveness of mitochondria and reveal the role of mechanical cues in directly regulating mitochondrial dynamics.
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Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.25.513802v1?rss=1
Authors: Ouyang, Y., Cunningham, C. N., Berg, J. A., Toshniwal, A. G., Hughes, C. E., Van Vranken, J. G., Jeong, M.-Y., Cluntun, A. A., Lam, G., Winter, J. M., Akdogan, E., Dove, K. K., Gygi, S. P., Dunn, C. D., Winge, D. R., Rutter, J.
Abstract: Mitochondrial membrane potential directly powers many critical functions of mitochondria, including ATP production, mitochondrial protein import, and metabolite transport. Its loss is a cardinal feature of aging and mitochondrial diseases, and cells closely monitor membrane potential as an indicator of mitochondrial health. Given its central importance, it is logical that cells would modulate mitochondrial membrane potential in response to demand and environmental cues, but there has been little exploration of this question. We report that loss of the Sit4 protein phosphatase in yeast increases mitochondrial membrane potential, both through inducing the electron transport chain and the phosphate starvation response. Indeed, a similarly elevated mitochondrial membrane potential is also elicited simply by phosphate starvation or by abrogation of the Pho85-dependent phosphate sensing pathway. This enhanced membrane potential is primarily driven by an unexpected activity of the ADP/ATP carrier. We also demonstrate that this connection between phosphate limitation and enhancement of the mitochondrial membrane potential is also observed in primary and immortalized mammalian cells as well as in Drosophila. These data suggest that mitochondrial membrane potential is subject to environmental stimuli and intracellular signaling regulation and raise the possibility for therapeutic enhancement of mitochondrial functions even with defective mitochondria.
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Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.27.513735v1?rss=1
Authors: Petersen, J. D., Mekhedov, E., Kaur, S., Roberts, D. D., Zimmerberg, J.
Abstract: Extracellular vesicles (EVs) released by resting endothelial cells support vascular homeostasis. To better understand endothelial cell EV biogenesis, we examined cultured human umbilical vein endothelial cells (HUVECs) prepared by rapid freezing, freeze-substitution, and serial thin section electron microscopy. Thin sections of HUVECs revealed clusters of membrane protrusions on the otherwise smooth cell surface. The protrusions contained membrane-bound organelles, including multivesicular bodies (MVBs), and appeared to be on the verge of pinching off to form microvesicles. Beyond cell peripheries, membrane-bound vesicles with internal MVBs were observed, and serial sections confirmed that they were not connected to cells. These observations are consistent with the notion that these multi-compartmented microvesicles (MCMVs) pinch-off from protrusions. Remarkably, omega figures formed by fusion of MVBs with the MCMV limiting membrane were directly observed, apparently caught in the act of releasing exosomes from the MCMV. In summary, MCMVs are a novel form of EV that bud from membrane protrusions on the HUVEC surface, contain MVBs and release exosomes. These observations suggest that exosomes can be harbored within and released from transiting microvesicles after departure from the parent cell, constituting a new site of exosome biogenesis occurring from endothelial and potentially additional cell types.
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Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.25.513729v1?rss=1
Authors: Del Toro, K., Sayaman, R. W., Thi, K., Licon-Munoz, Y., Hines, W. C.
Abstract: A fundamental question in biology, central to our understanding of cancer and other pathologies, is determining how different cell types coordinate to form and maintain tissues. Recognizing the distinct features and capabilities of the cells that compose these tissues is critical. Unfortunately, the complexity of tissues often hinders our ability to distinguish between neighboring cell types and, in turn, scrutinize their transcriptomes and generate reliable and tractable cell models for studying their inherently different biologies. In a companion article, we introduced a novel method that permits the identification and purification of the twelve cell types that compose the human breast-nearly all of which could be reliably propagated in the laboratory. Here, we explore the nature of these cell types. We sequence mRNAs from each purified population and investigate transcriptional patterns that reveal their distinguishing features. We describe the differentially expressed genes and enriched biological pathways that capture the essence of each cell type, and we highlight transcripts that display intriguing expression patterns. These data, analytic tools, and transcriptional analyses form a rich resource whose exploration provides remarkable insights into the inner workings of the cell types composing the breast, thus furthering our understanding of the rules governing normal cell and tissue function.
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Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.25.513730v1?rss=1
Authors: Rossi, M., Anerillas, C., Idda, M. L., Munk, R., Shin, C. H., Donega, S., Tsitsipatis, D., Herman, A. B., Martindale, J. L., Yang, X., Piao, Y., Mazan-Mamczarz, K., Fan, J., Ferrucci, L., De, S., Abdelmohsen, K., Gorospe, M.
Abstract: Senescent cells release a variety of cytokines, proteases, and growth factors collectively known as the senescence-associated secretory phenotype (SASP). Sustained SASP contributes to a pattern of chronic inflammation associated with aging and implicated in many age-related diseases. Here, we investigated the expression and function of the immunomodulatory cytokine BAFF (B-cell activating factor), a SASP protein, in multiple senescence models. We first characterized BAFF production across different senescence models, including senescent human diploid fibroblasts (WI-38, IMR-90) and monocytic leukemia cells (THP-1), and tissues of mice induced to undergo senescence. We then identified IRF1 (interferon response factor 1) as a transcription factor required for promoting BAFF mRNA transcription in senescence. We discovered that suppressing BAFF production decreased the senescent phenotype of both fibroblasts and monocyte-derived THP-1 cells, overall reducing IL6 secretion, SA-{beta}-Gal staining, and {gamma}-H2AX accumulation. Importantly, however, the influence of BAFF on the senescence program was cell type-specific: in monocytes, BAFF promoted the early activation of NF-{kappa}B and general SASP secretion, while in fibroblasts, BAFF contributed to the production and function of TP53 (p53). We propose that BAFF is elevated across senescence models and is a potential target for senotherapy.
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Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.27.513991v1?rss=1
Authors: Garcia, G., Zhang, H., Moreno, S., Tsui, C. K., Webster, B. M., Higuchi-Sanabria, R., Dillin, A.
Abstract: Changes in lipid metabolism are associated with aging and age-related diseases, including proteopathies. The endoplasmic reticulum (ER) is uniquely a major hub for protein and lipid synthesis, making its function essential for both protein and lipid homeostasis. However, it is less clear how lipid metabolism and protein quality may impact each other. Here, we identity let-767, a putative hydroxysteroid dehydrogenase, as an essential gene for both lipid and ER protein homeostasis. Knockdown of let-767 reduces lipid stores, alters ER morphology in a lipid-dependent manner, and also blocks induction of the Unfolded Protein Response of the ER (UPRER). Interestingly, a global reduction in lipogenic pathways restores UPRER induction in animals with reduced let-767. Specifically, we find that supplementation of 3-oxoacyl, the predicted metabolite directly upstream of let-767, is sufficient to block induction of the UPRER. This study highlights a novel interaction through which changes in lipid metabolism can alter a cell's response to protein-induced stress.
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Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.27.513982v1?rss=1
Authors: Segui-Perez, C., Stapels, D., Ma, Z., Su, J., Passchier, E., Westendorp, B., Wu, W., van Putten, J. P., Strijbis, K.
Abstract: Regulation and adaptation of intestinal epithelial barrier function is essential for human health. The transmembrane mucin MUC13 is an abundant intestinal glycoprotein with important functions for mucosal maintenance that are not yet completely understood. We demonstrate that in intestinal epithelial monolayers MUC13 localized to both the apical surface and the tight junction (TJ) region on the lateral membrane. MUC13 deletion resulted in increased transepithelial resistance (TEER) and reduced translocation of small solutes. TJ proteins including claudins and occludin were highly increased in membrane fractions of MUC13 knockout cells. Removal of the MUC13 cytoplasmic tail (CT) also altered TJ composition but did not result in increased TEER. The increased buildup of TJ complexes in {Delta}MUC13 and MUC13-{Delta}CT cells was dependent on PKC, which is in line with a predicted PKC motif in the MUC13 cytoplasmic tail. The responsible PKC member might be PKC{delta} based on elevated protein levels in the absence of MUC13. Our results identify MUC13 as a central player in TJ complex stability and intestinal barrier permeability.
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Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.27.514035v1?rss=1
Authors: Wang, N., Shibata, Y., Paulo, J. A., Gygi, S., Rapoport, T. A.
Abstract: Autophagy is essential for cellular homeostasis and begins with the formation of a phagophore, a cup-like membrane sheet consisting of two closely apposed lipid bilayers connected by a highly curved rim. How the membrane sheet forms, bends, and eventually generates an autophagosome that enwraps cargo remains enigmatic. Specifically, it is unclear how the high membrane curvature of the phagophore rim, an energetically unfavorable state, is stabilized. Here, we demonstrate that phagophore formation requires the conserved, membrane curvaturegenerating REEP1 proteins. The REEP1 family proteins (REEP1-4 in vertebrates) differ from the related endoplasmic reticulum-shaping REEPs in abundance and membrane topology. In fission yeast, the single REEP1 ortholog is involved in both bulk and selective autophagy. It is recruited at early stages of phagophore formation and required for their maturation into autophagosomes. The function of REEP1 relies on its ability to generate high membrane curvature and its localization to the phagophore rim. Mammalian REEP1 proteins also associate with phagophores upon induction of autophagy and colocalize with early autophagic markers. We propose that REEP1 proteins stabilize the phagophore's highly curved rim so that the two membrane sheets are kept in close proximity to form the autophagosome. Defective autophagy may underlie the effect of curvature-compromising mutations in human REEP1 proteins linked to neurological disease.
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Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.26.513928v1?rss=1
Authors: chattopadhyay, c., Bhattacharya, R., Roszik, J., Khan, F., Wells, G. A., Villanueva, H., Qin, Y., Bhattacharya, R., Patel, S., Grimm, E. A.
Abstract: Uveal melanoma (UM) originating in the eye and metastasizing to the liver is associated with poor prognosis. Here, we investigated whether the IGF-1/IGF-1R signaling axis is involved in UM growth and metastasis. TCGA dataset analysis reveals that UM has high IRS-1 expression, which is the first substrate of IGF-1R. Furthermore, IRS-1 is over-expressed in all UM cell lines tested (relative to non -cancer/normal cells) and in matched eye and liver UM tumors. Therefore, we targeted IRS-1/2 in UM cells as well as UM tumors developed on a chicken egg chorioallantoic membrane (CAM) model, and subcutaneous (subQ) UM tumors grown in mice using NT157, a small molecule inhibitor of IRS-1/2. NT157 treatment in UM cells resulted in reduced cell survival and cell migration, and increased apoptosis. NT157 treatment also significantly inhibited UM tumor growth in the in vivo chicken egg CAM and subQ mouse models, validating the in vitro effect. Moreover, NT157 appears more effective than a monoclonal antibody-based approach to block IGF-1R signaling. Mechanistically, through reverse phase protein array (RPPA) analysis, we identified significant proteomic changes in the PI3K/AKT pathway with NT157 treatment. Together, these results suggest that NT157 inhibits cell survival, migration in vitro and tumor growth in vivo via inhibiting IGF-1 signaling in UM cells.
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Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.25.513669v1?rss=1
Authors: Swinkels, M., Hordijk, S., Bürgisser, P. E., Slotman, J. A., Carter, T., Leebeek, F. W. G., Jansen, A. J. G., Voorberg, J., Bierings, R.
Abstract: Background: Platelet alpha-granules contain Von Willebrand factor (VWF), which is stored in eccentric alpha-granule nanodomains, and VWF propeptide (VWFpp). Differential release of VWF and VWFpp has been reported from endothelial cells. It is unclear if this also occurs during platelet alpha-granule exocytosis. We have recently developed a 3D super-resolution imaging workflow for quantification of platelet alpha-granule content based on Structured Illumination Microscopy (SIM). With this we can study alpha-granule cargo release following platelet activation in hundreds of platelets simultaneously. Aims: To study release of VWF and VWFpp from alpha-granules using quantitative super-resolution microscopy. Methods: Platelets were activated with PAR-1 activating peptide (PAR-1 ap) or collagen-related peptide (CRP-XL). Alpha-tubulin, VWF, VWFpp, SPARC and fibrinogen were imaged using 3D-SIM, followed by semi-automated analysis in FIJI. Uptake of anti-VWF nanobody during degranulation was used to identify alpha-granules that partially released content. Results: VWF+ and VWFpp+ structures overlapped nearly completely (~90%) in resting platelets, implying they are stored in similar eccentric alpha-granule nanodomains. A subset of VWF+/VWFpp+-structures was released completely at 0.6 M PAR-1-ap, but at higher concentration (20 M) significantly more VWFpp (85.3{+/-}1.6%) was released than VWF (37.6{+/-}1.4%). Release of other cargo was intermediate at 20 M (SPARC: 62.2{+/-}1.4% ; fibrinogen: 51.9{+/-}2.9%), providing further evidence for differential cargo release. Similar results were obtained using CRP-XL. Anti-VWF nanobody was taken up by VWF+/VWFpp- structures and increased with stimulus strength, demonstrating these were post-exocytotic structures. Conclusions: VWF and VWFpp are differentially released from alpha-granules. This may affect how platelet-derived VWF and VWFpp contribute to formation and stabilization of hemostatic clots.
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Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.24.513614v1?rss=1
Authors: Lewis, B. M., Cho, C. Y., Her, H.-L., Hunter, T., Yeo, G. W.
Abstract: Mitochondrial associated RNA-binding proteins (RBPs) have emerged as key contributors to mitochondrial biogenesis and homeostasis. With few examples described, we set out to identify RBPs that regulate nuclear-encoded mitochondrial mRNAs (NEMmRNAs). Our systematic analysis of RNA-targets of 150 RBPs identified RBPs with a preference for binding NEMmRNAs, including LARP4, a La RBP family member. We show that LARP4s targets are particularly enriched in mRNAs that encode respiratory chain complex proteins (RCCPs) and mitochondrial ribosome proteins (MRPs) across multiple human cell lines. Quantitative proteomics of cells lacking LARP4 show that protein levels of RCCPs and MRPs are significantly reduced. Furthermore, we show that LARP4 depletion reduces mitochondrial function, and that this phenotype is rescued by LARP4 re-expression. Our findings shed light onto a novel function for LARP4 as an RBP that binds to NEMmRNAs to promote mitochondrial respiratory function.
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Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.24.513629v1?rss=1
Authors: Wang, Y.-J., Di, X.-J., Han, D.-Y., Nashmi, R., Henderson, B. J., Moss, F. J., Mu, T.-W.
Abstract: Protein homeostasis (proteostasis) deficiency is recognized as a contributing factor to many neurodegenerative, neurological, and metabolic diseases. However, how the proteostasis network orchestrates the folding and assembly of multi-subunit membrane proteins is not completely understood. In this investigation, we focus on characterizing the biogenesis pathway of a multi-subunit neuroreceptor, the gamma-aminobutyric acid type A (GABAA) receptor. Previous proteomics studies identified Hsp47 (Gene: SERPINH1), a heat shock protein in the endoplasmic reticulum lumen, as the most enriched GABAA receptor-interacting chaperone. Here, we show that Hsp47 enhances neuronal GABAA receptor functional surface expression, acting after Binding immunoglobulin Protein (BiP), to preferentially bind the folded conformation of GABAA receptors. Therefore, Hsp47 promotes the subunit-subunit interaction, the receptor assembly process, and the anterograde trafficking of GABAA receptors. These Hsp47 properties are also extended to other Cys-loop receptors, including nicotinic acetylcholine receptors. Therefore, in addition to its known function as a collagen chaperone, this work establishes that Hsp47 also plays a critical and general role in the maturation of multi-subunit neuroreceptors.
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Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.24.513621v1?rss=1
Authors: Hao, S., Fuehrer, H., Flores, E., Demmerle, J., Lippincott-Schwartz, J., Liu, Z., Sukenik, S., Cai, D.
Abstract: YAP/TEAD signaling is essential for organismal development, cell proliferation, and cancer progression. As a transcriptional coactivator, how YAP activates its downstream target genes is incompletely understood. YAP forms biomolecular condensates in response to hyperosmotic stress, concentrating transcription-related factors to activate downstream target genes. However, whether YAP forms condensates under other signals, how YAP condensates organize and function, and how YAP condensates activate transcription in general are unknown. Here, we report that endogenous YAP forms sub-micron scale condensates in response to Hippo pathway regulation and actin cytoskeletal tension. The transcription factor TEAD1 actively stabilizes YAP condensates, which also recruit BRD4, a coactivator that is enriched at active enhancers. Using single molecule tracking, we found that YAP condensates slowed YAP diffusion within condensate boundaries, a possible mechanism for promoting YAP target search. These results reveal that YAP condensate formation is a highly regulated process that is critical for YAP/TEAD target gene expression.
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Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.24.513472v1?rss=1
Authors: Giovanazzi, A., van Herwijnen, M. J. C., van der Meulen, G. N., Wauben, M. H. M.
Abstract: The promise of extracellular vesicles (EVs)-based liquid biopsy resides in the identification of specific signatures of EVs of interest. Knowing the EV profile of a body fluid can facilitate the identification of EV-based biomarkers of diseases. To this end, we characterised purified EVs from paired human milk and serum by surface protein profiling of cellular markers in association with gold standard EV markers (tetraspanins CD9, CD63 and CD81). By using the MACSPlex bead-based flow-cytometry assay with pan-tetraspanin detection (i.e. simultaneous CD9, CD63 and CD81 detection), besides specific breast epithelial cell signatures in milk EVs and platelet signatures in serum EVs, we also identified body fluid-specific markers of immune cells and stem cells. Interestingly, comparison of pan-tetraspanin and single tetraspanin detection unveiled both body fluid-specific tetraspanin distributions and specific tetraspanin distributions associated with certain cellular markers, which were used to model the potential biogenesis route of different EV subsets and their cellular origin.
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Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.24.513534v1?rss=1
Authors: Prusen Mota, I., Galova, M., Schleiffer, A., Nguyentan, T.-t., Kovacikova, I., Nishiyama, T., Gregan, J., Peters, J.-M., Schlogelhofer, P.
Abstract: Cohesin mediates sister chromatid cohesion to enable chromosome segregation and DNA damage repair. To perform these functions, cohesin needs to be protected from WAPL, which otherwise releases cohesin from DNA. It has been proposed that cohesin is protected from WAPL by SORORIN. However, in vivo evidence for this antagonism is missing and SORORIN is only known to exist in vertebrates and insects. It is therefore unknown how important and widespread SORORINs functions are. Here we report the identification of SORORIN orthologs in Schizosaccharomyces pombe (Sor1) and Arabidopsis thaliana (AtSORORIN). sor1 delta mutants display cohesion defects, which are partially alleviated by wpl1 delta. Atsororin mutant plants display dwarfism, tissue specific cohesion defects and chromosome mis-segregation. Furthermore, Atsororin mutant plants are sterile and separate sister chromatids prematurely at anaphase I. The somatic, but not the meiotic deficiencies can be alleviated by loss of WAPL. These results provide in vivo evidence for SORORIN antagonizing WAPL, reveal that SORORIN is present in organisms beyond the animal kingdom and indicate that it has acquired tissue specific functions in plants.
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Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.24.513548v1?rss=1
Authors: Nguyen, T. T., Voeltz, G. K.
Abstract: Mitochondria are dynamic organelles that undergo cycles of fission and fusion at a unified platform defined by endoplasmic reticulum (ER)-mitochondria membrane contact sites (MCSs). These MCSs or nodes co-localize fission and fusion machinery. We set out to identify how ER-associated mitochondrial nodes can regulate both fission and fusion machinery assembly. We have used a promiscuous biotin ligase linked to the fusion machinery, Mfn1, and proteomics to identify an ER membrane protein, Aphyd, as a major regulator of node formation. In the absence of Aphyd, fission and fusion machineries fail to recruit to ER-associated mitochondrial nodes and fission and fusion rates are significantly reduced. Aphyd contains an acyltransferase motif and an /{beta} hydrolase domain and point mutations in critical residues of these regions fail to rescue the formation of ER-associated mitochondrial hot spots. These data suggest a mechanism whereby Aphyd functions by altering phospholipid composition at ER mitochondria MCSs. Our data present the first example of an ER membrane protein that regulates the recruitment of both fission and fusion machineries to mitochondria.
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Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.24.513582v1?rss=1
Authors: Krajnik, A., Nimmer, E., Sullivan, A., Joseph, B. A., Heo, Y., Krug, A., Kolega, J., Heo, S.-J., Lee, K., Weil, B. R., Kim, D.-H., Bae, Y.
Abstract: Vascular dysfunction is a common cause of cardiovascular diseases characterized by the narrowing and stiffening of arteries, such as atherosclerosis, restenosis, and hypertension. Arterial narrowing results from the aberrant proliferation of vascular smooth muscle cells (VSMCs) and their increased synthesis and deposition of extracellular matrix (ECM) proteins. These, in turn, are modulated by arterial stiffness, but the mechanism for this is not fully understood. We found that survivin (an inhibitor of apoptosis) is an important regulator of stiffness-mediated ECM synthesis and intracellular stiffness in VSMCs. Whole-transcriptome analysis and cell culture experiments showed that survivin expression is upregulated in injured femoral arteries in mice and in human VSMCs cultured on stiff fibronectin-coated hydrogels. Suppressed expression of survivin in human VSMCs and mouse embryonic fibroblasts decreased the stiffness-mediated expression of ECM components implicated in arterial stiffness, namely, collagen-I, fibronectin, and lysyl oxidase. By contrast, expression of these proteins was upregulated by the overexpression of survivin in human VSMCs cultured on soft hydrogels. Atomic force microscopy analysis showed that suppressed or enhanced expression of survivin decreases or increases intracellular stiffness, respectively. These findings suggest a novel mechanism by which survivin modulates arterial stiffness.
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Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.24.513562v1?rss=1
Authors: Guerber, L., Pangou, E., Vuidel, A., Liao, Y., Kleiss, C., Grandgirard, E., Sumara, I.
Abstract: PLK1 is a key regulator of mitosis whose protein levels and activity fluctuate during cell cycle. PLK1 dynamically localizes to distinct mitotic structures to regulate proper chromosome segregation. However, the molecular mechanisms linking localized PLK1 activity to its protein stability remain elusive. Here, we identify the Ubiquitin-Binding Protein 2-Like (UBAP2L) protein that regulates both dynamic removal of PLK1 from kinetochores and PLK1 protein stability during mitosis. We demonstrate that UBAP2L localizes to kinetochores in a PLK1-dependent manner and that UBAP2L depletion leads to the abnormal retention of PLK1 at kinetochores and segregation defects. We show that C-terminal domain of UBAP2L mediates its function on PLK1 and that UBAP2L specifically regulates PLK1 and no other mitotic factors. We demonstrate that inhibited kinetochore removal of PLK1 in UBAP2L-depleted cells, increases PLK1 stability after mitosis completion and results in aberrant PLK1 kinase activity in interphase and cellular death. Overall, our data suggest that UBAP2L is required to fine-tune PLK1 signaling in human cells.
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Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.23.513385v1?rss=1
Authors: Hammad, A. S., Yu, F., Horgen, F. D., Machaca, K.
Abstract: Store-operated calcium entry (SOCE) contributes to several physiological and pathological conditions including transcription, secretion, immunodeficiencies, and cancer. SOCE has been shown to be important for breast cancer cell migration where knockdown of SOCE components (STIM1 or Orai1) decreases cancer metastasis. Here we show unexpectedly that STIM1 knockout (KO) metastatic MDA-MB-231 breast cancer cells migrate faster and have enhance invasion capacity compared to parental cells. In contrast, Orai1-KO cells, which have similar levels of SOCE inhibition as STIM1-KO, migrate slower than the parental cell line. This shows that the enhanced migration phenotype of STIM1-KO cells is not due to the loss of a Ca2+ entry through SOCE, rather it involves transcriptional remodeling. Interestingly, NFATC2 is significantly downregulated in STIM1-KO cells and overexpression of NFATC2 reversed the enhanced migration of STIM1-KO cells. This demonstrates that STIM1 modulates NFATC2 expression independently of its role in SOCE.
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Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.23.513322v1?rss=1
Authors: Chen, X., Rong, K., Han, W., Pang, Y., Chai, G.
Abstract: In previous research, miR-148a-3p deficiency was observed in bone malformation in hemifacial microsomia. Herein, in this article, we probed into the role of miR-148a-3p in bone physiology by utilizing miR-148a knock-out (KO) mice. Compared with wild-type (WT) or heterozygotic (HE) littermates, miR-148a knock-out mice manifested lower body weight, bone dysplasia with increased bone mass. Through in-vitro experiments, in terms of miR-148a-3p overexpression (miRNA mimic transfection) and knockout (primary cells from WT and KO littermates), we found that miR-148a-3p can suppress osteogenesis, either in the ALP activity or bone nodules formation. Afterward, by means of proteomics, combined with RNA-sequencing and prediction databases of microRNA targets (miRDB and TargetScan), nine candidate genes targeted by miR-148a-3p were identified. Among them, only Itga11 was regulated by mRNA degradation, while the others were modulated via post-transcriptional inhibition. Based on several online databases (GenePaint, BioGPS, STRING), Integrin Subunit Alpha 11 (Itga11) was suggested to play an essential role in osteogenesis and it was confirmed as one direct target of miR-148a-3p by dual-luciferase reporter assay. Meanwhile, gene set enrichment analysis (GSEA) indicated activation of PI3K-Akt signaling pathway and WNT signaling pathway in miR-148a KO mice. The thereafter western blot confirmed that PI3K/Akt/GSK3/{beta}-catenin signaling pathway was involved. Taken together, we demonstrated that miR-148a-3p can inhibit osteogenesis by targeting Itga11 via PI3K/Akt/GSK3/{beta}-catenin pathway.
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Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.20.513083v1?rss=1
Authors: El Yakoubi, W., Akera, T.
Abstract: Reproductive isolation occurs when the genomes of two populations accumulate genetic incompatibilities that prevent inter-breeding. Cell biological understanding of such hybrid incompatibility is limited, especially for hybrid female sterility. Here we find that species divergence in condensin regulation and centromere organization between two mouse species, Mus musculus and Mus spretus, drives chromosome de-condensation and mis-segregation in their F1 hybrid oocytes, reducing female fertility. The chromosome condensation defects in hybrid oocytes were especially prominent at Mus musculus centromeres due to their highly abundant major satellite DNA, leading to species-specific chromosome mis-segregation. This study provides the first mechanistic insights into hybrid incompatibility in female meiosis and demonstrates that condensin mis-regulation can be a reproductive isolating barrier in mammals.
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Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.20.512932v1?rss=1
Authors: Gomez, R., Page, J., Garcia-Gonzalo, F. R., Hidalgo, I., Perez-Martin, S., Lopez-Jimenez, P.
Abstract: Cilia are hair-like projections of the plasma membrane with an inner microtubule skeleton known as axoneme. Motile cilia and flagella beat to displace extracellular fluids, playing important roles in the airways and reproductive system, among others. Instead, primary cilia function as cell type-dependent sensory organelles, detecting chemical, mechanical or optical signals from the extracellular environment. Cilia dysfunction is associated with genetic diseases called ciliopathies, and with some types of cancer. Cilia have been recently identified in zebrafish gametogenesis as an important regulator of the bouquet conformation and recombination. However, there is very little information about the structure and functions of cilia in mammalian meiosis. Here we describe the presence of cilia in male mouse meiotic cells. These solitary cilia form transiently in 20% of zygotene spermatocytes and reach considerable lengths (up to 15 um). CEP164 and CETN3 localization studies indicate that these cilia emanate from the mother centriole, prior to centrosome duplication. In addition, the study of telomeric TFR2 suggests that these cilia are not directly related to the bouquet conformation during early male mouse meiosis. Instead, based on TEX14 labeling of intercellular bridges in spermatocyte cysts, we suggest that mouse meiotic cilia may have sensory roles affecting cyst function during prophase I.
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Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.21.513182v1?rss=1
Authors: Costa, D. F., Ricci, J. M. B., Rodrigues, M. S., Oliveira, M. A., Doretto, L., Nobrega, R. H.
Abstract: This study unravels the roles of TGF-{beta} (Transforming growth factor-{beta}) superfamily signaling pathway in the zebrafish spermatogonial activity (self-renewal vs. differentiation) by combining ex vivo and specific pathway inhibitors approaches. The TGF-{beta} superfamily signaling pathway is subdivided into TGF-{beta} and Bone morphogenetic proteins (BMP) subfamilies, and is ubiquitous among metazoans, regulating several biological processes, including spermatogenesis. In this study, we evaluated the function of the TGF-{beta} and BMP subfamily pathways in the zebrafish spermatogonial niche using A83-01 and DMH1 inhibitors, respectively. Our results showed that A83-01 potentiated the follicle-stimulating hormone (Fsh) effects on zebrafish spermatogenesis, reducing type A undifferentiated spermatogonia and increasing differentiated spermatogonia (type Adiff and type B spermatogonia) after 7 days of culture. In agreement with histomorphometrical data, the mRNA levels of dazl (marker of spermatogonial differentiation) and pro-differentiation growth factors, such as igf3 and insl3, were significantly augmented following A83-01. For the BMP signaling pathway, exposure to DMH1 inhibitor showed opposite effects as compared to TGF-{beta} superfamily signaling pathway inhibitor. Histomorphometrical analysis demonstrated an accumulation of type A undifferentiated spermatogonia, while the frequency of differentiated spermatogonia was significantly reduced following co-treatment of DMH1 with Fsh after 7 days of culture. To support this data, expression analysis revealed that BMP signaling pathway inhibitor also decreased the testicular mRNA levels of dazl, igf3 and insl3 when compared to control incubation (Fsh). In conclusion, our study demonstrated that TGF-{beta} and BMP subfamily pathways exert a role in zebrafish spermatogonial niche with antagonistic functions for the spermatogonia fate. The TGF-{beta} subfamily pathway is involved with spermatogonial self-renewal and inhibition of differentiation, whereas the BMP subfamily pathway promotes spermatogonial differentiation. These findings are not only relevant to understanding stem cell biology, but may also be useful in several in vitro assays, promoting control of self-renewal and differentiation by potentially directing these processes.
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Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.20.512880v1?rss=1
Authors: Cha, J., Tong, X., Walker, E. M., Dahan, T., Cochrane, V., Ashe, S., Russell, R., Osipovich, A. B., Mawla, A. M., Guo, M., Liu, J.-h., Huising, M. O., Magnuson, M. A., Hebrok, M., Dor, Y., Stein, R.
Abstract: Type 2 diabetes (T2D) is associated with compromised identity of insulin-producing pancreatic islet beta ({beta}) cells, characterized by inappropriate production of other islet cell-enriched hormones. Here we examined how hormone misexpression was influenced by the MAFA and MAFB transcription factors, closely related proteins that maintain islet cell function. Mice specifically lacking MafA in {beta} cells demonstrated broad, population-wide changes in hormone gene expression with an overall gene signature closely resembling islet gastrin (Gast)-positive cells generated under conditions of chronic hyperglycemia and obesity. A human {beta} cell line deficient in MAFB, but not one lacking MAFA, also produced a gastrin (GAST)-positive gene expression pattern. In addition, GAST was detected in human T2D {beta} cells with low levels of MAFB. Moreover, evidence is provided that human MAFB can directly repress GAST gene transcription. These results support a novel, species-specific role for MafA and MAFB in maintaining adult mouse and human {beta} cell identity, respectively, by repressing expression of Gast/GAST and other non-{beta} cell hormones.
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Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.20.513128v1?rss=1
Authors: Rahman, M. M., Pathak, A., Schueler, K. L., Alsharif, H. A., Michl, A. N., Alexander, J., Kim, J.-a., Chapman, E. R., Bhatnagar, S.
Abstract: Stimulus-coupled insulin secretion from beta-cells involves the fusion of insulin granules to the plasma membrane (PM) via SNARE complex formation--a cellular process key for maintaining whole-body glucose homeostasis. Optimal insulin secretion depends on how the clamping of SNAREs is released, rendering granules fusogenic. We show that an insulin granule protein synaptotagmin-9 (Syt9) deletion in lean mice increased glucose clearance, random-fed plasma insulin levels, and insulin secretion (in vivo and ex vivo islets) without affecting insulin sensitivity. These outcomes demonstrate that Syt9 has an inhibitory function in insulin secretion. Moreover, Syt9 interacts with PM-Stx1A and soluble Tomosyn-1 proteins to form non-fusogenic complexes between PM and insulin granules, preventing Stx1A-SNARE formation and insulin secretion. Furthermore, Syt9 inhibits SNARE-complex formation by posttranscriptional regulation of Tomosyn-1. We conclude that Syt9 and Tomosyn-1 are endogenous inhibitors that modulate Stx1A availability to determine beta-cell secretory capacity.
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Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.21.512903v1?rss=1
Authors: Masser, E. A., Noble, T. D., Siefert, J. C., Goins, D., Sansam, C. G., Sansam, C. L.
Abstract: Deregulated DNA replication causes human developmental disorders and cancer, but we know little about how DNA replication is coordinated with changes in transcription and chromatin structure. The initiation of replication forks follows a spatiotemporal pattern called the replication timing program. We have developed the zebrafish into a model system to study the mechanisms by which the replication timing program changes during the extensive changes in the cell cycle, transcription, chromatin organization, and nuclear structure that occur during development. Our previous studies identified changes in DNA replication timing patterns occurring from the onset of zygotic transcription through gastrulation in zebrafish embryos. Rif1 is required for DNA replication timing in a wide range of eukaryotes. The broader role of Rif1 in establishing the replication timing program and chromatin structure during early vertebrate development remains unknown. We have generated Rif1 mutant zebrafish and have performed RNA sequencing and whole-genome replication timing analyses on multiple developmental stages. Rif1 mutants were viable but had a defect in female sex determination. Surprisingly, Rif1 loss predominantly affected DNA replication timing after gastrulation, while its impacts on transcription were more substantial during zygotic genome activation. Our results indicate that Rif1 has distinct roles in DNA replication and transcription control that manifest at different stages of development.
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Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.20.513104v1?rss=1
Authors: Zhou, C. Y., Dekker, B., Liu, Z., Cabrera, H., Ryan, J., Dekker, J., Heald, R.
Abstract: During the rapid and reductive cleavage divisions of early embryogenesis, subcellular structures such as the nucleus and mitotic spindle scale to decreasing cell size. Mitotic chromosomes also decrease in size during development, presumably to coordinately scale with mitotic spindles, but underlying mechanisms are unclear. Here we combine in vivo and in vitro approaches using eggs and embryos from the frog Xenopus laevis to show that mitotic chromosome scaling is mechanistically distinct from other forms of subcellular scaling. We found that mitotic chromosomes scale continuously with cell, spindle and nuclear size in vivo. However, unlike for spindles and nuclei, mitotic chromosome size cannot be re-set by cytoplasmic factors from earlier developmental stages. In vitro, increasing nucleo-cytoplasmic (N/C) ratio is sufficient to recapitulate mitotic chromosome scaling, but not nuclear or spindle scaling, through differential loading of maternal factors during interphase. An additional pathway involving importin scales mitotic chromosomes to cell surface area/volume (SA/V) during metaphase. Finally, single-chromosome immunofluorescence and analysis of Hi-C data suggest that mitotic chromosomes scale through decreased recruitment of condensin I, resulting in major rearrangements of DNA loop architecture to accommodate the same amount of DNA on a shorter axis. Together, our findings demonstrate that mitotic chromosome size is set by spatially and temporally distinct developmental cues in the early embryo.
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Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.20.513094v1?rss=1
Authors: Taylor, M. F., Black, M. A., Hampton, M. B., Ledgerwood, E. C.
Abstract: Hydrogen peroxide (H2O2) is a ubiquitous oxidant produced in a regulated manner by various enzymes in mammalian cells. H2O2 reversibly oxidises thiol groups of cysteine residues to mediate intracellular signalling. Whilst examples of H2O2 dependent signalling have been reported, the exact molecular mechanism(s) of signalling and the pathways affected are not well understood. Here, the transcriptomic response of Jurkat T cells to H2O2 was investigated to determine global effects on gene expression. With a low H2O2 concentration (10 M) that did not induced an oxidative stress response or cell death, extensive changes in gene expression occurred after 4 hours (6803 differentially expressed genes). Of the genes with greater then 2-fold change in expression, 85% were upregulated suggesting that in a physiological setting H2O2 predominantly activates gene expression. Pathway analysis identified gene expression signatures associated with FOXO and NTRK signalling. These signatures were associated with an overlapping set of transcriptional regulators. Overall, our results provide a snapshot of gene expression changes in response to H2O2, which, along with further studies, will lead to new insights into the specific pathways that are activated in response to endogenous production of H2O2, and the molecular mechanisms of H2O2 signalling.
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Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.20.513059v1?rss=1
Authors: Myburgh, E., Geoghegan, V., Alves-Ferreira, E. V. C., Nievas, Y. R., Grewal, J. S., Brown, E., McLuskey, K., Mottram, J. C.
Abstract: Leishmania parasites undergo differentiation between various proliferating and non-dividing forms to adapt to changing host environments. The mechanisms that link environmental cues with the parasite's developmental changes remain elusive. Here, we report that Leishmania TORC1 is a key environmental sensor for parasite differentiation in the sand fly-stage promastigotes and for replication of mammalian-stage amastigotes. We show that Leishmania RPTOR1, interacts with TOR1 and LST8. We investigate TORC1 function by conditional deletion of RPTOR1, where under nutrient rich conditions RPTOR1 depletion results in decreased protein synthesis and growth, G1 cell cycle arrest and premature differentiation from proliferative promastigotes to non-dividing mammalian-infective metacyclic forms. These parasites cannot develop into proliferative amastigotes in the mammalian host, or respond to nutrients to differentiate to proliferative retroleptomonads, which are required for their blood-meal induced amplification in sand flies and enhanced mammalian infectivity. RPTOR1-dependent TORC1 functionality represents a critical mechanism for driving parasite growth and proliferation.
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Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.20.513071v1?rss=1
Authors: LeBlanc-Straceski, J., Williams, R., Ward, K., Bates, A., Duran, C., Anderson, L., Murray, M., PereiraBadji, J., Shoushani, C., Thibault, J.
Abstract: In a cellular model of Down Syndrome, hTERT immortalized RPE-1 (human retinal pigment epithelial-1) cells carrying an extra copy of chromosome 21 exhibit reduced fitness, in part, as an increase in doubling time (or a reduction in cell proliferation rate) in culture. ROCK2 (Rho associated coiled-coil containing kinase 2) was identified in a whole genome CRISPR knockout (KO) screen designed to identify genes and pathways that could be therapeutically targeted to improve cell proliferation (Replogle JM, et al. manuscript in preparation). ROCK2 KO cell lines of both RPE-1 euploid and trisomy 21 aneuploid cells were created using CRISPR. Trisomy 21 ROCK2 KO cell lines showed a modest increase in cell proliferation rate compared to the parental aneuploid cells, similar to the relative effect that ROCK2 knockout had in the whole genome CRISPR screen. Euploid ROCK2 KO cell lines showed no difference in growth rate vs their ROCK2 expressing counterparts. Changes in doubling time in response to two pharmaceutical ROCK inhibitors, Fasudil and Y27632, also showed the same modest increase in cell proliferation rate in the trisomy cells. The actin cytoskeleton, a target of ROCK2 regulation, exhibited long stress fibers that aligned across multiple contiguous cells in confluent trisomy 21 ROCK2 KO cells compared to the disorganized stress fibers of the parental trisomy 21 cells with normal ROCK2 expression.
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Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.19.512916v1?rss=1
Authors: Perl, A. L., Koetsier, J. L., Green, K. J.
Abstract: Critical for the maintenance of epidermal integrity and function are attachments between intermediate filaments (IF) and intercellular junctions called desmosomes. The desmosomal cytoplasmic plaque protein desmoplakin (DP) is essential for anchoring IF to the junction. DP-IF interactions are regulated by a phospho-regulatory motif within the DP C-terminus controlling keratinocyte intercellular adhesion. Here we identify the protein phosphatase 2A (PP2A)-B55 holoenzyme as the major serine/threonine phosphatase regulating DPs C-terminus and consequent intercellular adhesion. Using a combination of chemical and genetic approaches, we show that the PP2A-B55 holoenzyme interacts with DP at intercellular membranes in 2D- and 3D- epidermal models and human skin samples. Our experiments demonstrate that PP2A-B55 regulates the phosphorylation status of junctional DP and is required for maintaining strong desmosome mediated intercellular adhesion. These data identify PP2A-B55 as part of a regulatory module capable of tuning intercellular adhesion strength and a candidate disease target in desmosome related disorders of the skin and heart.
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Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.20.513009v1?rss=1
Authors: Zhao, Z., Hong, L., Huang, G., He, Y., Zuo, X., Han, W.
Abstract: Cells sense physical cues, such as changes in extracellular matrix (ECM) stiffness, and translate these stimuli into biochemical signals that control various aspects of cellular behavior, thereby facilitating physiological and pathological processes in various organs. Evidence from multiple studies suggests that the anterior vaginal wall stiffness is higher in POP patients than in non-POP patients. Our experiments found that the expression of -smooth muscle actin (-SMA) in the anterior vaginal wall of patients with POP was increased, and the expression of DNMT1 was decreased. We used polyacrylamide gel to simulate matrix stiffening in vitro, and substrate stiffening induced the high expression of myofibroblast markers -SMA and CTGF in L929 cells. Inhibition of DNMT1 promotes fibroblast differentiation into myofibroblasts in vitro. The results of bioinformatics analysis showed that the expression of DNMT1 was significantly correlated with microtubule polymerization-related proteins. The experiment showed that the microtubule polymerization inhibitor nocodazole could eliminate the decrease of DNMT1 expression in fibroblasts induced by high stiffness. We conclude that fibroblasts sense an increase in the stiffness of the surrounding matrix and regulate fibroblast differentiation by regulating the expression of DNA methyltransferase 1 (DNMT1) through the regulation of microtubule polymerization. This study may help to elucidate the complex crosstalk between vaginal fibroblasts and their surrounding matrix in both healthy and pathological conditions, and provide new insights into the implications of potentially targeted phenotypic regulation mechanisms in material-related therapeutic applications.
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Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.19.512511v1?rss=1
Authors: Stamateris, R. E., Sharma, R. B., Landa-Galvan, H. V., Darko, C., Redmond, D., Rane, S. G., Alonso, L. C.
Abstract: Expanding beta cell mass is a critical goal in the fight against diabetes. CDK4, an extensively characterized cell cycle activator, is required to establish and maintain beta cell number. Beta cell failure in the IRS2-deletion mouse type 2 diabetes model is in part due to loss of CDK4 regulator Cyclin D2. We set out to determine whether replacement of endogenous CDK4 with the inhibitor-resistant mutant CDK4-R24C rescued the loss of beta cell number in Irs2-deficient mice. Surprisingly, not only beta cell number but also beta cell dedifferentiation status was effectively rescued, despite no improvement in insulin sensitivity. Ex vivo studies in primary islet cells revealed a novel mechanism in which CDK4 intervened downstream in the insulin signaling pathway to prevent FOXO1-mediated transcriptional repression of critical beta cell transcription factor Pdx1. FOXO1 inhibition was not related to E2F1 activity, to FOXO1 phosphorylation, or even to FOXO1 subcellular localization, but rather was related to deacetylation of FOXO1 and reduced FOXO1 abundance. Taken together, these results demonstrate a novel differentiation-promoting activity of the classical cell cycle activator CDK4 and support the concept that beta cell mass can be expanded without compromising function.
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Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.19.512957v1?rss=1
Authors: Jiu, Y., Zhang, Y., Zhang, X., Li, Z., Yang, H., Tang, D., Zhao, S., Zhang, Q., Li, B., Lappalainen, P., Cui, Z., Liu, H., Li, H., Zhao, W.
Abstract: Emerging COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses a great threat to human health and economics. Although SARS-CoV-2 entry mechanism has been explored, little is known about how SARS-CoV-2 regulates the host cell remodeling to facilitate virus invasion process. Here we unveil that SARS-CoV-2 boosts and repurposes filopodia for entry to the target cells. Using SARS-CoV-2 virus-like particle (VLP), real-time live-cell imaging and simulation of active gel model, we reveal that VLP-induced Cdc42 activation leads to the formation of filopodia, which reinforce the viral entry to host cells. By single-particle tracking and sparse deconvolution algorithm, we uncover that VLP particles utilize filopodia to reach the entry site in two patterns, surfing and grabbing, which are more efficient and faster than entry via flat plasma membrane regions. Furthermore, the entry process via filopodia is dependent on the actin cytoskeleton and actin-associated proteins fascin, formin, and Arp2/3. Importantly, either inhibition the actin cross-linking protein fascin or the active level of Cdc42 could significantly hinders both the VLP and the authentic SARS-CoV-2 entry. Together, our results highlight that the spatial-temporal regulation of the actin cytoskeleton by SARS-CoV-2 infection makes filopodia as a highway for virus entry, which emerges as an antiviral target.
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Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.20.513002v1?rss=1
Authors: Oliveira Mendes, B., Alameh, M., Ollivier, B., Montnach, J., Bidere, N., Souaze, F., Escriou, N., Charpentier, F., Baro, I., De Waard, M., Loussouarn, G.
Abstract: Controversial reports have suggested that SARS-CoV E and 3a proteins may be viroporins that conduct currents through the plasma membrane of the infected cells. If true, these proteins would represent accessible targets for the development of new antiviral drugs by using high-throughput patch-clamp techniques. Here we aimed at better characterizing the cell responses induced by E or 3a protein with a particular focus on the ion conductances measured at the cell surface. First, we show that expression of SARS-CoV-2 E or 3a protein in CHO cells gives rise to cells with newly-acquired round shape, tending to detach from the Petri dish. This suggests that cell death is induced upon expression of E or 3a protein. We confirmed this hypothesis by using flow cytometry, in agreement with earlier reports on other cell types. In adhering cells expressing E or 3a protein, whole-cell currents were in fact not different from the control condition indicating that E and 3a proteins are not plasma membrane viroporins. In contrast, recording currents on detached cells uncovered outwardly-rectifying currents, much larger than those observed in control. The current characteristics are reminiscent of what was previously observed in cells expressing SARS-CoV-1 E or 3a proteins. Herein, we illustrate for the first time that carbenoxolone blocks these outward currents suggesting that they are conducted by pannexin channels, mostly likely activated by cell morphology change and/or cell death. Alongside we also demonstrate that truncation of the C-terminal PDZ binding motifs reduces the proportion of dying cells but does not prevent pannexin currents suggesting distinct pathways for cell death and pannexin currents induced by E and 3a proteins. We conclude that SARS-CoV-2 E and 3a proteins are not acting as viroporins expressed at the plasma membrane.
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Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.20.512863v1?rss=1
Authors: Westerich, K. J., Tarbashevich, K., Gupta, A., Zhu, M., Hull, K., Romo, D., Gross-Thebing, T., Raz, E.
Abstract: Germ granules, condensates of phase-separated RNA and protein, are essential for germline development, but how these molecules are organized within the granules and whether such an organization is relevant for germ cell fate is unclear. Combining three-dimensional in vivo structural and functional analyses, we study the dynamic spatial organization of molecules within zebrafish germ granules. We find that the vertebrate-specific Dead end protein is essential for positioning nanos3 RNA at the periphery of the condensates, where ribosomes are located. Without Dead end, or when translation is inhibited, nanos3 RNA translocates into granule interiors, far from the location of the ribosomes. These findings reveal the molecular mechanisms controlling the spatial organization of RNA within the phase-separated organelle and the importance of sub-granule RNA localization for preserving germ cell totipotency.
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Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.19.512819v1?rss=1
Authors: Rodriguez-Real, G., Prados-Carvajal, R., Bayona-Feliu, A., Balestra, F. R., Huertas, P.
Abstract: The centrosome is a cytoplasmic organelle with roles in microtubule organization which has also been proposed to act as a hub for cellular signaling. For example, it has been suggested that some centrosomal component are required for full activation of the DNA Damage Response, the cellular signaling network that is activated upon the appearance of chromosome breaks. However, if the centrosome and/or some of its components regulate specific DNA repair pathways is not known. Double strand breaks are mostly repaired by two alternative mechanisms, the homology-independent non-homologous end-joining and the homology-driven homologous recombination. Here, we show that centrosomes presence is required to fully activate recombination, specifically to completely license its initial step, the so-called DNA end resection. Additionally, loss of centrosome upregulates the non-homologous end-joining repair pathway. Furthermore, we identify a centriolar structure, the subdistal appendages, and a specific factor, CEP170, as the critical centrosomal component involved in the regulation of recombination and resection, albeit it does not control end-joining repair. Cells lacking centrosomes or depleted for CEP170 are, consequently, hyper-sensitive to DNA damaging agents. Moreover, low levels of CEP170 in multiple cancer types correlate with an increase of the mutation burden associated with specific mutational signatures and a better prognosis, suggesting this protein can act as a driver mutation but also could be targeted to improve current oncological treatments.
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Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.19.512755v1?rss=1
Authors: Sarkar, A., Khandelwal, S., Kim, H., Gruel, Y., Rollin, J., Wool, G. D., Arepally, G. M., Cines, D. B., Rauova, L., Poncz, M.
Abstract: Heparin-induced thrombocytopenia (HIT) is characterized by mild thrombocytopenia associated with a highly prothrombotic state due to the development of pathogenic antibodies that recognize human (h) platelet factor 4 (PF4) complexed with various polyanions. While non-heparin anticoagulants and intravenous immunoglobulin (IVIG) are the mainstay of care, bleeding may develop, and risk of new thromboembolic events remain. We had described a mouse IgG{kappa}2b antibody KKO that mimics the sentinel features of pathogenic HIT antibodies, including binding to the same neoepitope on hPF4:polyanion complexes. KKO, like HIT IgGs, activate platelets through Fc{gamma}RIIA and induces complement activation. We now asked whether Fc-modified KKO can be used as a novel therapeutic to prevent or treat HIT. Using the endoglycosidase EndoS, we created deglycosylated KKO (DGKKO). DGKKO bound to PF4-polyanion complexes, and blocked Fc{gamma}RIIA-dependent activation of PF4 treated platelets by KKO, 5B9 (another HIT-like monoclonal antibody), and isolated IgGs from HIT patients. DGKKO also decreased complement activation and deposition of C3c on platelets. Injection of DGKKO into ''HIT mice'' lacking mouse PF4, but transgenic for hPF4 and Fc{gamma}RIIA, prevented and reversed thrombocytopenia when injected before or after KKO, 5B9 or HIT IgG, respectively, in a microfluidic system. DGKKO reversed antibody-induced thrombus growth in HIT mice. In contrast, DGKKO was ineffective in preventing thrombosis by IgG from a patient with the HIT-related disorder, vaccine-induced immune thrombotic thrombocytopenia. Thus, DGKKO may represent a new class of therapeutics for targeted treatment of patients with HIT.
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Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.18.512792v1?rss=1
Authors: Sun, L., York, S. B., Pate, B., Zhang, Y., Meckes, D. G.
Abstract: Current extracellular vesicle (EV) isolation methods depend on large expensive equipment like ultracentrifuges and are laborious and time consuming. There is also currently no method available for high throughput isolation to meet clinical demands. Here, we present a method that combines our previous published ExtraPEG method and magnetic beads. Western blot and nanoparticle tracking analysis (NTA) of the purified EVs revealed higher or equivalent recovery and purity with this method compared to ExtraPEG or size exclusion chromatography (SEC) methods. With this newly developed workflow and automated liquid handling instrument, we have successfully isolated up to 96 EV samples from 5 L pre-cleared serum in 45 minutes. Moreover, DNA / small RNA / protein purification and profiling steps could be seamlessly integrated into the isolation workflow. To profile EV protein markers, EVs were lysed from the binding step and covalently bound to the surface of the beads. TotalSeq or ELISA antibody can be applied with under a standard protocol. With this extended protocol, researchers can easily complete EV isolation and protein profiling experiment within 8 hours. Taken together, we provide a high throughput method for EV isolation and molecular analyses that may be used for sensitive biomarker detection from biological fluids.
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Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.18.512791v1?rss=1
Authors: Truong, T., Johnston, S. M., Webber, K., Boekweg, H., Lundgren, C., Liang, Y., Nydegger, A., Xie, X., Payne, S. H., Kelly, R. T.
Abstract: The sensitivity of single-cell proteomics (SCP) has increased dramatically in recent years due to advances in experimental design, sample preparation, separations and mass spectrometry instrumentation. Further increasing the sensitivity of SCP methods and instrumentation will enable the study of proteins within single cells that are expressed at copy numbers too small to be measured by current methods. Here we combine efficient nanoPOTS sample preparation and ultra-low-flow liquid chromatography with a newly developed data acquisition and analysis scheme termed wide window acquisition (WWA) to quantify greater than 3,000 proteins from single cells in fast label-free analyses. WWA is based on data-dependent acquisition (DDA) but employs larger precursor isolation windows to intentionally co-isolate and co-fragment additional precursors along with the selected precursor. The resulting chimeric MS2 spectra are then resolved using the CHIMERYS search engine within Proteome Discoverer 3.0. Compared to standard DDA workflows, WWA employing isolation windows of 8-12 Th increases peptide and proteome coverage by ~28% and ~39%, respectively. For a 40-min LC gradient operated at ~15 nL/min, we identified an average of 2,150 proteins per single-cell-sized aliquots of protein digest directly from MS2 spectra, which increased to an average of 3,524 proteins including proteins identified with MS1-level feature matching. Reducing the active gradient to 20 min resulted in a modest 10% decrease in proteome coverage. We also compared the performance of WWA with DIA. DIA underperformed WWA in terms of proteome coverage, especially with faster separations. Average proteome coverage for single HeLa and K562 cells was respectively 1,758 and 1,642 based on MS2 identifications with 1% false discovery rate and 3042 and 2891 with MS1 feature matching. As such, WWA combined with efficient sample preparation and rapid separations extends the depths of the proteome that can be studied at the single-cell level.
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Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.17.512558v1?rss=1
Authors: Hirschhäuser, A., Molitor, D., Salinas, G., Grosshans, J., Rust, K., Bogdan, S.
Abstract: Drosophila blood cells called hemocytes form an efficient barrier against infections and tissue damage. During metamorphosis, hemocytes undergo tremendous changes in their shape and behavior preparing them for tissue clearance. Yet, the diversity and functional plasticity of pupal blood cells have not been explored. Here, we combine single-cell transcriptomics and high-resolution microscopy to dissect the heterogeneity and plasticity of pupal hemocytes. We identified precursor and effector hemocytes with distinct molecular signatures and cellular functions clearly distinct from other stages of hematopoiesis. Strikingly, we identified that PSC cells, which function as lymph gland niche, are highly migratory and immune responsive cells in the pupa. PSC cells can transdifferentiate to lamellocytes triggered by wasp infection. Altogether, our data highlight a remarkable cell heterogeneity, and identifies a cell population that acts not only as a stem cell niche in larval hematopoiesis, but functions as cell reservoir to pupal and adult blood cells.
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Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.18.512697v1?rss=1
Authors: Leclech, C., Krishnamurthy, A., Muller, L., Barakat, A. I.
Abstract: In many tissues, cell shape and orientation are controlled by a combination of internal and external biophysical cues. Anisotropic substrate topography is a ubiquitous cue that leads to cellular elongation and alignment, a process termed contact guidance, whose underlying mechanisms remain incompletely understood. Additionally, whether contact guidance responses are similar in single cells and in cellular monolayers is unknown. Here, we address these questions in vascular endothelial cells (ECs) that in vivo form a monolayer that lines blood vessels. Culturing single ECs on microgrooved substrates that constitute an idealized mimic of anisotropic basement membrane topography elicits a strong, groove depth-dependent contact guidance response. Interestingly, this response is greatly attenuated in confluent monolayers. While contact guidance in single cells is principally driven by persistence bias of cell protrusions in the direction of the grooves and is surprisingly insensitive to actin stress fiber disruption, cell shape and alignment in dense EC monolayers are driven by the organization of the basement membrane secreted by the cells, which leads to a loss of interaction with the microgrooves. The findings of distinct contact guidance mechanisms in single ECs and in EC monolayers promise to inform strategies aimed at designing topographically patterned endovascular devices.
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Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.19.512717v1?rss=1
Authors: Harvey, K. E., Tang, S., LaVigne, E. K., Pratt, E. P. S., Hockerman, G. H.
Abstract: The ER Ca2+ channel ryanodine receptor 2 (RyR2) is required for maintenance of insulin content and glucose-stimulated insulin secretion, in part, via regulation of the protein IRBIT in the insulinoma cell line INS-1. Here, we examined store-operated and depolarization-dependent Ca2+entry using INS-1 cells in which either RyR2 or IRBIT were deleted. Store-operated Ca2+ entry (SOCE) stimulated with thapsigargin was reduced in RyR2KO cells compared to controls, but was unchanged in IRBITKO cells. STIM1 protein levels were not different between the three cell lines. Basal and stimulated (500 M carbachol) phospholipase C (PLC) activity was also reduced specifically in RyR2KO cells. Insulin secretion stimulated by tolbutamide was reduced in RyR2KO and IRBITKO cells compared to controls, but was potentiated by an EPAC-selective cAMP analog in all three cell lines. Cellular PIP2 levels were increased and cortical f-actin levels were reduced in RyR2KO cells compared to controls. Whole-cell Cav channel current density was increased by 65% in RyR2KO cells compared to controls, and barium current was reduced by acute activation of the lipid phosphatase pseudojanin preferentially in RyR2KO cells over control INS-1 cells. Action potentials stimulated by 18 mM glucose were more frequent in RyR2KO cells compared to controls, and insensitive to the SK channel inhibitor apamin. Taken together, these results suggest that RyR2 plays a critical role in regulating PLC activity and PIP2 levels via regulation of SOCE. RyR2 also regulates {beta}-cell electrical activity by controlling Cav current density, via regulation of PIP2 levels, and SK channel activation.
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Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.19.512855v1?rss=1
Authors: Duan, J., Liu, H., Ji, Y., Yuan, Q., Li, X., Wu, K., Gao, T., Zhu, S., Jiang, Y., Yin, W., Xu, H. E.
Abstract: Phosphorylation of G protein-coupled receptors (GPCR) by GPCR kinases (GRKs) desensitizes G protein signaling and promotes arrestin signaling, which is also modulated by biased ligands. Molecular assembly of GRKs to GPCRs and the basis of GRK-mediated biased signaling remain largely unknown due to the weak GPCR-GRK interactions. Here we report the complex structure of neurotensin receptor 1 (NTSR1) bound to GRK2, Gaq, and an arrestin-biased ligand, SBI-553, at a resolution of 2.92 Angstrom. The high-quality density map reveals the clear arrangement of the intact GRK2 with the receptor, with the N-terminal helix of GRK2 docking into the open cytoplasmic pocket formed by the outward movement of the receptor TM6, analogous of the binding of G protein to the receptor. Strikingly, the arrestin-biased ligand is found at the interface between GRK2 and NTSR1 to enhance GRK2 binding. The binding mode of the biased ligand is compatible with arrestin binding but is clashed with the binding of a G protein, thus provide an unambiguous mechanism for its arrestin-biased signaling capability. Together, our structure provides a solid model for understanding the details of GPCR-GRK interactions and biased signaling.
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Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.19.512772v1?rss=1
Authors: Zheng, S., Dadina, N., Mozumdar, D., Lesiak, L., Martinez, K., Miller, E., Schepartz, A.
Abstract: The densely packed inner mitochondrial membrane (IMM) is vital for bioenergy generation and its dynamics control mitochondrial health and cellular homeostasis. IMM structure is complex, however, and imaging its dynamics with high temporal and spatial resolution is complicated by the photosensitivity of IMM-resident enzymes. Here we describe the cell-permeant, lipid-like acridine orange derivative MAO-N3 and use it to assemble high-density, environmentally sensitive (HIDE) probes that selectively label and image the IMM in live cells. MAO-N3 pairs with multiple SPAAC-reactive fluorophores to support HIDE imaging via confocal, Structured Illumination, Single Molecule Localization, and Stimulated Emission Depletion microscopy, all with significantly improved resistance against photobleaching. The HIDE probes generated using MAO-N3 require no genetic manipulations, are non-toxic in model cell lines and primary cardiomyocytes, even under conditions that amplify the effects of mitochondrial toxins, and visualize the IMM for up to 12.5 hours with unprecedented spatial and temporal resolution.
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Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.17.512459v1?rss=1
Authors: Shi, B.
Abstract: Epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) positively affect the initial control of non-small cell lung cancer (NSCLC). The rapidly acquired TKIs resistance accounts for a major hurdle in successful treatment. However, the mechanisms controlling EGFR-TKIs resistance remain largely unknown. RNA structures have widespread and crucial roles in various biological processes; but, their role in regulating cancer drug resistance remains unclear. Here, the PARIS method is used to establish the higher-order RNA structure maps of EGFR-TKI resistant- and sensitive-cells of NSCLC. According to our results, RNA structural regions are enriched in UTRs and correlate with translation efficiency. Moreover, YRDC facilitates resistance to EGFR-TKIs in NSCLC cells, and RNA structure formation in YRDC 3'UTR suppress ELAVL1 binding leading to EGFR-TKIs sensitivity by impairing YRDC translation. A potential cancer therapy strategy is provided by using antisense oligonucleotide (ASO) to perturb the interaction between RNA and protein. Our study reveals an unprecedented mechanism in which the RNA structure switch modulates EGFR-TKIs resistance by controlling YRDC mRNA translation in an ELAVL1-dependent manner.
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Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.16.512413v1?rss=1
Authors: Telles, B. d. S., Rebelato, H. J., Esquisatto, M. A., Catisti, R.
Abstract: Poor nutrition during pregnancy causes permanent metabolic and/or structural adaptation in offspring. The adrenal gland produces various steroid hormones during pregnancy. Thus, this study aimed to evaluate the influence of diet during pregnancy on the adrenal glands of Wistar rats. For this, 10-week-old pregnant Wistar rats (p, n=15) and non-pregnant rats (np, n=15) were divided into three groups and received a normoproteic control diet (C, 17% casein, n=5), isocaloric low-protein diet (PR, 6% casein, n=5), or 50% calorie restriction (CR, 50% of the diet consumed by group C), over a period of 21 days. On the 21st day of gestation (21dG, p groups) or on the 21st day of diet (np groups), after anesthetic deepening, the right adrenal gland was collected, weighed (total mass), and prepared for inclusion in Paraplast for histomorphometric and immunohistochemical analysis (Ki-67, glucocorticoid receptors (GR), and mineralocorticoid receptor (MR)) in the different areas of the gland. Data, expressed as the mean and SD, were evaluated by one-way analysis of variance with Tukey's post-test (p less than 0.05). CR in pregnancy increased the amount of GR, MR, and Ki-67 receptors in the adrenal gland. The npRC group showed highest GR staining compared to the animals that received a normal diet. Protein restriction in pregnancy decreases adrenal MR. The results allowed us to conclude that even without altering the weight of the adrenal glands, the pRC group suffered the most from stress during the study, suggesting that CR associated with pregnancy can cause morphofunctional changes in the adrenal glands.
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Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.17.512504v1?rss=1
Authors: Meyer, K., Lammers, N. C., Bugaj, L. J., Garcia, H. H., Weiner, O. D.
Abstract: YAP is a transcriptional regulator that controls pluripotency, germ layer specification, and proliferation. Different subsets of YAP target genes are engaged in each physiological setting, but how YAP selectively regulates different effectors in different contexts is not known. Here we use optogenetics to investigate how the levels and dynamics of YAP activation control its pluripotency effectors Oct4 and Nanog. We observe different thresholds for repression of Oct4 and Nanog, enabling differential control of both genes through YAP levels. Pluripotency factors also decode YAP dynamics. Oct4 preferentially responds to oscillatory YAP inputs that mimic endogenous pulsatile YAP dynamics. Using single-cell live imaging of Oct4 transcription and computational-theoretical analysis of transcriptional regulation, we demonstrate that YAP dynamics are decoded by an adaptive change sensor that modulates Oct4 transcription burst frequency. Our results reveal how the levels and timing of YAP activation enable multiplexing of information transmission for key regulators of cellular differentiation and pluripotency.
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Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.17.512486v1?rss=1
Authors: Basier, C., Nurse, P.
Abstract: Proliferating eukaryotic cells grow and undergo cycles of cell division. Growth is continuous whilst the cell cycle consists of discrete events. How the production of biomass is controlled as cells increase in size and proceed through the cell cycle is important for understanding the regulation of global cellular growth. This has been studied for decades but has not yielded consistent results. Previous studies investigating how cell size, the amount of DNA, and cell cycle events affect the global cellular production of proteins and RNA molecules have led to highly conflicting results, probably due to perturbations induced by the synchronisation methods used. To avoid these perturbations, we have developed a system to assay unperturbed exponentially growing populations of fission yeast cells. We generated thousands of single-cell measurements of cell size, of cell cycle stage, and of the levels of global cellular translation and transcription. This has allowed us to determine how cellular changes arising from progression through the cell cycle and cells growing in size affect global cellular translation and transcription. We show that translation scales with size, and additionally increases at late S-phase/early G2, then increases early in mitosis and decreases later in mitosis, suggesting that cell cycle controls are operative over global cellular translation. Transcription increases with both size and the amount of DNA, suggesting that the level of transcription of a cell may be the result of a dynamic equilibrium between the number of RNA polymerases associating and disassociating from DNA.
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Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.17.512471v1?rss=1
Authors: Godinho, S. A., Monteiro, P., Yeon, B., Wallis, S. S.
Abstract: Intracellular organelle organisation is conserved in eukaryotic cells and is primarily achieved through active transport by motor proteins along the microtubule cytoskeleton. Microtubule posttranslational modifications (PTMs) contribute to microtubule diversity and differentially regulate motor-mediated transport. Here we show that centrosome amplification induces a global change in organelle positioning towards the cell periphery and facilitates nuclear migration through confined spaces. This reorganisation requires kinesin-1 and is analogous to loss of dynein. Cells with amplified centrosomes display increased levels of acetylated tubulin, a PTM known to enhance kinesin-1 mediated transport. Depletion of -tubulin acetyltransferase 1 (TAT1) to block tubulin acetylation, which has no impact on control cells, rescues the displacement of centrosomes, mitochondria and vimentin, but not Golgi or endosomes. Analyses of the distribution of acetylated microtubules indicates that the polarisation of modified microtubules, rather than levels alone, plays an important role in organelle positioning. We propose that tubulin acetylation differentially impacts kinesin-1-mediated organelle displacement, suggesting that each organelle must have its own sensing and response mechanisms to ensure fine-tuning of its distribution in cells.
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Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.17.512434v1?rss=1
Authors: Siegfried, H., Le Borgne, R., Durieu, C., De Azevedo Laplace, T., Verraes, A., Daunas, L., Verbavatz, J.-M., Heuze, M. L.
Abstract: Cell motility processes highly depend on the membrane distribution of Phosphoinositides, giving rise to cytoskeleton reshaping and membrane trafficking events. Membrane contact sites serve as platforms for lipid exchange and calcium fluxes between two organelles. Here, we show that VAPA, an ER-resident contact site tether, plays a crucial role during cell motility. CaCo2 adenocarcinoma epithelial cells depleted for VAPA exhibit several collective and individual motility defects, disorganized actin cytoskeleton and altered protrusive activity. During migration, VAPA is required for maintaining high levels of PI(4,5)P2 at the plasma membrane (PM) but not in other compartments. In addition, VAPA plays a local function at focal adhesions (FA) where it anchors and stabilizes ER-PM contact sites, thus mediating microtubule-dependent FA disassembly. Our study reveals unprecedented functions for VAPA in cell motility processes through the regulation of Phosphoinositides homeostasis and local anchoring of ER-PM contact sites to FA.
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Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.17.512513v1?rss=1
Authors: Jiang, Y., He, Y., Liu, S., Li, G., Chen, D., Deng, W., Li, P., Zhang, Y., Wu, J., Li, J., Wang, L., Lin, J., Wang, H., Kong, S., Shi, G.
Abstract: Decidualization, denoting the transformation of endometrial stromal cells into specialized decidual cells, is a prerequisite for normal embryo implantation and a successful pregnancy in human. Here we demonstrated that knockout of Gaq lead to an aberrantly enhanced inflammatory state during decidualization. Furthermore, we showed that deficiency of Gaq resulted in over-activation of nuclear factor (NF)-{kappa}B signaling, due to the decreased expression of NF{kappa}BIA, which encode the I{kappa}B protein and is the negative regulator for NF{kappa}B. Mechanistically, Gaq deficiency decreased the PKD/PKC phosphorylation levels, so leading to attenuated HDAC5 phosphorylation and thus its nuclear export. Aberrantly high level of nuclear HADC5 retarded histone acetylation to inhibit NF{kappa}BIA transcription during decidualization. Consistently, pharmacological activation of the PKD/PKC or inhibition of the HDAC5 signaling restored the inflammatory state and proper decidual response. Finally, we disclosed that over-active inflammatory state in Gaq deficient decidua deferred the blastocyst hatching and adhesion in vitro, and the decidual expression of Gq was significantly lower in women with recurrent pregnancy loss compared with normal pregnancy. In brief, we showed here that Gq as a key regulator of the inflammatory cytokine's expression and decidual homeostasis in response to differentiation cues, which is required for successful implantation and early pregnancy.
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Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.16.512400v1?rss=1
Authors: Yu, Y., Gao, S. M., Guan, Y., Hu, P., Zhang, Q., Liu, J., Jing, B., Zhao, Q., Sabatini, D. M., Abu-Remaileh, M., Jung, S. Y., Wang, M. C.
Abstract: Lysosomes are active sites to integrate cellular metabolism and signal transduction. A collection of proteins enriched at lysosomes mediate these metabolic and signaling functions. Both lysosomal metabolism and lysosomal signaling have been linked with longevity regulation; however, how lysosomes adjust their protein composition to accommodate this regulation remains unclear. Using large-scale proteomic profiling, we systemically profiled lysosome-enriched proteomes in association with different longevity mechanisms. We further discovered the lysosomal recruitment of AMPK and nucleoporin proteins and their requirements for longevity in response to increased lysosomal lipolysis. Through comparative proteomic analyses of lysosomes from different tissues and labeled with different markers, we discovered lysosomal heterogeneity across tissues as well as the specific enrichment of the Ragulator complex on Cystinonsin positive lysosomes. Together, this work uncovers lysosomal proteome heterogeneity at different levels and provides resources for understanding the contribution of lysosomal proteome dynamics in modulating signal transduction, organelle crosstalk and organism longevity.
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Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.17.512236v1?rss=1
Authors: Galea, G., Kuodyte, K., Khan, M. M., Thul, P. J., Neumann, B., Lundberg, E., Pepperkok, R.
Abstract: Cells are constantly exposed to a multitude of DNA-damaging agents that can lead to mutation, dysregulation, and possibly cell death. To ensure genomic integrity, DNA Damage Response (DDR) mechanisms are set in motion to repair and mitigate any damage to the DNA structure. Although these pathways are well-studied in the context of nuclear function, relatively little is known of the regulatory function of cytoplasmic organelles. Here we show the first example of DDR regulation at the Golgi complex, coordinating Homologous Recombination (HR)-mediated DNA repair. We found that RAD51C, a regulatory HR protein, localises to the Golgi and nuclear compartments and in response to double-strand DNA breaks, the Golgi protein population of RAD51C redistributes to form nuclear foci. Furthermore, we found that the Golgi localisation of RAD51C is dependent on the Golgin Giantin. Depletion of Giantin induces the redistribution of the RAD51C Golgi pool to form nuclear foci, independent of DNA damage induction, and concurrent with a significant increase in genomic instability and inhibition of HR signalling regulators. This study presents evidence for a novel pathway where the Golgi is a central regulatory hub for HR DDR and potentially other repair pathways, a finding with important therapeutic implications.
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Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.17.512492v1?rss=1
Authors: Martin, S. D., Connor, T., Sanigorski, A., McEwen, K. A., Henstridge, D. C., Nijagal, B., De Souza, D. P., Tull, D., Meikle, P., Kowalski, G. M., Bruce, C., Gregorevic, P., Febbraio, M., Collier, F., Walder, K. R., McGee, S.
Abstract: Lipotoxicity, the accumulation of lipids in non-adipose tissues, alters the metabolic transcriptome and mitochondrial metabolism in skeletal muscle. The mechanisms involved remain poorly understood. Here we show that lipotoxicity increased histone deacetylase 4 (HDAC4) and histone deacetylase 5 (HDAC5), which reduced the expression of metabolic genes and oxidative metabolism in skeletal muscle. This metabolic reprogramming was linked with reduced expression of p53-dependent genes that mediate apoptosis and ferroptosis, which preserved cell viability in response to lipotoxicity. Mechanistically, impaired mitochondrial metabolism reduced acetylation of p53 at K120, a modification required for transcriptional activation of apoptosis, while redox drivers of ferroptosis were also reduced. Overexpression of loss-of-function HDAC4 and HDAC5 mutants in skeletal muscle of obese db/db mice enhanced oxidative capacity, increased apoptosis and ferroptosis and reduced muscle mass. This study identifies HDAC4 and HDAC5 as repressors of the oxidative state of skeletal muscle, and that this metabolic reprogramming, considered deleterious for normal metabolism, is critical to preserve muscle integrity in response to lipotoxicity.
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Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.17.512253v1?rss=1
Authors: Mahlandt, E. K., Palacios Martinez, S., Arts, J. J. G., Tol, S., van Buul, J. D., Goedhart, J.
Abstract: The inner layer of blood vessels consists of endothelial cells, which form the physical barrier between blood and tissue. This vascular barrier is tightly regulated to allow the passage of essential molecules like oxygen, carbon-dioxide, water, ions, and nutrients. The vascular endothelial barrier is defined by cell-cell contacts through adherens and tight junctions. To further investigate the signaling in the endothelium that regulates vascular barrier strength, we focused on Rho GTPases, regulators of the actin cytoskeleton and known to control junction integrity. Rho GTPase signaling is confined in space and time. To manipulate the signaling in a temporal and spatial manner we applied optogenetics. Guanine exchange factor (GEF) domains from ITSN1, TIAM1 and p63RhoGEF, activating Cdc42, Rac and Rho respectively, were integrated into the optogenetic recruitment tool iLID. This tool allows for activation at the subcellular level in a reversible and non-invasive manner and thereby to recruit a GEF to local areas at the plasma membrane, enabling the local activation of specific Rho GTPases. The membrane tag of iLID was optimized and a HaloTag was applied to gain more flexibility for multiplex imaging. The resulting Opto-RhoGEFs were tested in an endothelial cell monolayer and demonstrated precise temporal control of vascular barrier strength by a cell-cell overlap-dependent, VE-cadherin-independent, mechanism. Furthermore, Opto-RhoGEFs enabled precise optogenetic control in endothelial cells over morphological features such as cell-size, -roundness, local extension, and cell contraction. In conclusion, we have optimized and applied the optogenetic iLID GEF recruitment tool i.e. Opto-RhoGEFs, to study the role of Rho GTPases in the vascular barrier of the endothelium and found that membrane protrusions at the junction region can rapidly increase barrier integrity independent of VE-cadherin.
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Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.17.512616v1?rss=1
Authors: Cadart, C., Bartz, J., Oaks, G., Liu, M., Heald, R.
Abstract: A positive correlation between genome size and cell size is well documented, but impacts on animal physiology are poorly understood. In Xenopus frogs, the number of genome copies (ploidy) varies across species and can be manipulated within a species. Here we show that triploid tadpoles contain fewer, larger cells than diploids and consume oxygen at a lower rate. Treatments that altered cell membrane stability or electrical potential abolished this difference, suggesting that a decrease in total cell surface area reduces basal energy consumption in triploids. Comparison of Xenopus species that evolved through polyploidization revealed that metabolic differences emerged during development when cell size scaled with genome size. Thus, ploidy affects metabolism by altering the cell surface area to volume ratio in a multicellular organism.
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Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.17.512562v1?rss=1
Authors: Vishwanatha, A., Princova, J., Hohos, P., Zach, R., Prevorovsky, M.
Abstract: Mitotic fidelity is crucial for the faithful distribution of genetic information into the daughter cells. Many fungal species, including the fission yeast Schizosaccharomyces pombe, undergo a closed form of mitosis, during which the nuclear envelope does not break down. In S. pombe numerous processes have been identified that contribute to successful completion of mitosis. Notably, perturbations of lipid metabolism can lead to catastrophic mitosis and the "cut" phenotype. It was suggested that these mitotic defects are caused by insufficient membrane phospholipid supply during the anaphase nuclear expansion. However, it is not clear whether additional factors are involved. In this study we characterized in detail the mitosis in an S. pombe mutant lacking the Cbf11 transcription factor, which regulates lipid metabolism genes. We show that in cbf11{Delta} cells mitotic defects appear already prior to anaphase, before the nuclear expansion begins. Moreover, we identify altered cohesin dynamics and centromeric chromatin structure as additional factors affecting mitotic fidelity in cells with disrupted lipid homeostasis, providing new insights into this fundamental biological process.
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Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.17.512548v1?rss=1
Authors: Lanz, M. C., Elias, J. E., Skotheim, J. M.
Abstract: Accurate measurements of the molecular composition of single cells will be key to elucidating the relationship between gene expression and function in diverse cell types. One of the most important phenotypes that differs between cells is their size, which was recently shown to be an important determinant of proteome composition in populations of similarly sized cells. We therefore sought to test if the effects of cell size on protein concentrations were also evident in single cell proteomics data. Using the relative concentrations of histone proteins to estimate a cell's DNA-to-cell volume ratio, we found that cell size correlated with the cell-to-cell variance in two single cell proteome datasets, each acquired using different preparation and measurement platforms. Moreover, the proteome differences between small and large single cells significantly correlated with how cell size affects the proteomes of cultured cells measured in bulk. We therefore conclude that cell size accounts for a substantial amount of proteome heterogeneity in single cells and should be considered particularly when comparing cells of a similar type.
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Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.14.512337v1?rss=1
Authors: Wu, F., Cheng, Y., Zhou, J., Ye, P., Liu, X., Zhang, L., Lin, R., Xiang, S., Liu, Z., Wang, C.
Abstract: A high concentration of oxalate is associated with an increased risk of kidney calcium oxalate (CaOx) stones, and the degradation of exogenous oxalate mainly depends on oxalate-degrading enzymes from the intestinal microbiome. We found that Zinc Gluconate supplement to patients with CaOx kidney stones could significantly improve the abundance of oxalate metabolizing bacteria in human body through clinical experiments on the premise of simultaneous antibiotic treatment and the imbalance of Lactobacillus and OxDC was involved in CaOx kidney stones through clinical sample analysis. Then, we identified that Zn2+ could be used as an external factor to improve the activity of OxDC and protect Lactobacillus, achieved the preventive effect on rats with stones aggravated by antibiotics. Finally, by analyzing the three-dimensional structure of OxDC and some in vitro experiments, we propose a hypothesis Zn2+ increases the metabolism of oxalate in humans through its positive effects on Lactobacillus and OxDC to reduce CaOx kidney stone symptoms in rats.
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Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.15.512353v1?rss=1
Authors: Lind, S., Wu, Y., Sundqvist, M., Forsman, H., Dahlgren, C.
Abstract: Allosterically modulated free fatty acid receptor 2 (FFA2R/GPR43) can be activated without the involvement of any orthosteric FFA2R agonist, by signals generated for example by P2Y2R, the G protein coupled receptor for ATP. An FFA2R specific positive allosteric modulator (PAM; Cmp58) was used to disclose the molecular mechanism by which signals generated by ATP/P2Y2R transactivates FFA2R. The P2Y2R induced signal that transactivates the allosterically modulated FFA2R was generated downstream of the Gq containing G protein that couple to P2Y2R. A receptor induced rise in the cytosolic concentration of ionized calcium ([Ca2+]i) was hypothesized to be the receptor transactivation signal. The Gq dependent transient rise in [Ca2+]i induced by the ATP activated P2Y2Rs was not affected by Cmp58. The hypothesis gained, however, support from the finding that the modulator transferred FFA2R to a Ca2+sensitive state. The rise in [Ca2+]i induced by the Ca2+ specific ionophore ionomycin, activated the allosterically modulated FFA2R. The response induced by ionomycin was rapidly terminated and the FFA2Rs could then no longer be activated by the orthosteric FFA2R agonist propionate or be transactivated by the signal generated by the activated ATP receptor. The desensitized/non-responding state of FFA2R was, however, revoked by an earlier described cross-sensitizing/activating allosteric FFA2R modulator. The receptor transactivation of the allosterically modulated FFA2Rs, represent a unique regulatory receptor cross-talk mechanism by which the activity of a G protein coupled receptor is controlled by a signaling system operating from the cytosolic side of the plasma membrane.
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Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.13.512159v1?rss=1
Authors: Chanez-Paredes, S. D., Abtahi, S., Zha, J., Zuo, L., He, W., Turner, J. R.
Abstract: Intestinal epithelia express two long MLCK splice variants, MLCK1 and MLCK2. We have previously shown that disruption of inflammation-induced MLCK1 recruitment to the perijunctional actomyosin ring prevents barrier loss and attenuates disease progression. Here we sought to define the domains responsible for distinct MLCK1 and MLCK2 behaviors. Quantitative analysis of human biopsies demonstrated specific increases in MLCK1 expression and perijunctional localization in Crohns disease. When expressed in cultured intestinal epithelial cells, we found, as expected, that MLCK1 is most concentrated at the perijunctional actomyosin ring. In contrast, MLCK2 is predominantly associated with basal F-actin stress fibers. Immunoglobulin-cell adhesion molecule domain 3 (IgCAM3) must be critical for MLCK1 recruitment, as that domain is incomplete in MLCK2. Consistent with this, truncation mutants consisting of N-terminal IgCAM domains 1-4, without C-terminal catalytic domains, localized similarly to full-length MLCK1 and MLCK2, respectively. Further mutagenesis allowed identification of IgCAM2 and IgCAM3 domains as the minimal region required for MLCK1 recruitment. Although IgCAM3 does not concentrate perijunctionally, it can act as a dominant negative effector that limits steady-state and TNF-induced MLCK1 recruitment and barrier loss. Together, the demonstration of selective MLCK1 upregulation and perijunctional recruitment in Crohns disease and identification of domains required for perijunctional MLCK1 recruitment provide a conceptual understanding and structural data needed for development of therapeutic means of blocking MLCK1-mediated barrier loss without the toxicity of enzymatic MLCK inhibition.
SIGNIFICANCE STATEMENTRecent work has demonstrated that long myosin light chain kinase isoform 1 (MLCK1) recruitment to the perijunctional actomyosin ring is a critical component of inflammation-induced intestinal barrier loss. Chanez-Paredes et al. show that this occurs in Crohns disease and define the essential structural elements that direct MLCK1 recruitment, thereby creating a foundation for therapeutic interruption of MLCK1 recruitment in disease.
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Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.13.512083v1?rss=1
Authors: Lanteri, M. L., Silveyra, M. X., Moran, M. M., Boutet, S., Solis-Gozar, D.-D., Perreau, F., Andreu, A. B.
Abstract: Andean potatoes (Solanum tuberosum L. ssp. andigena) are a good source of dietary antioxidant polyphenols. We have previously demonstrated that polyphenol extracts from Andean potato tubers exerted a dose-dependent cytotoxic effect in human neuroblastoma SH-SY5Y cells, being skin extracts more potent than flesh ones. In order to gain insight into the bioactivities of potato phenolics, we investigated the composition and the in vitro cytotoxic activity of total extracts and fractions of skin and flesh tubers of three Andean potato cultivars (Santa Maria, Waicha, and Moradita). Potato total extracts were subjected to liquid-liquid fractionation using ethyl acetate solvent in organic and aqueous fractions. We analyzed both fractions by HPLC-DAD, HPLC-ESI-MS/MS, and HPLC-HRMS. Results corroborated the expected composition of each fraction. Organic fractions were rich in hydroxycinnamic acids (principally chlorogenic acid isomers), whereas aqueous fractions contained mainly polyamines conjugated with phenolic acids, glycoalkaloids, and flavonoids. Organic fractions were not cytotoxic against SH-SY5Y cells, and indeed, some increased cellular metabolism compared to controls. Aqueous fractions were cytotoxic and even more potent than their respective total extracts. Treatment with a combination of both fractions showed a similar cytotoxic response to the corresponding extract. According to correlation studies, it is tempting to speculate that polyamines and glycoalkaloids are crucial in inducing cell death. Our findings indicate that the activity of Andean potato extracts is a combination of various compounds and contribute to the revalorization of potato as a functional food.
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Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.14.512213v1?rss=1
Authors: Khan, M. N., Diaz-Hernandez, M. E., Chihab, S., Priyadarshani, P., Bhattaram, P., Mortensen, L. J., Guzzo, R. M., Drissi, H.
Abstract: Induced pluripotent stem cells (iPSCs) are potential cell sources for regenerative medicine. The iPSCs exhibit a preference for lineage differentiation to the donor cell type indicating the existence of memory of origin. Although the intrinsic effect of the donor cell type on differentiation of iPSCs is well recognized, whether disease-specific factors of donor cells influence the differentiation capacity of iPSC remains unknown. Using viral based reprogramming, we demonstrated the generation of iPSCs from chondrocytes isolated from healthy (AC-iPSCs) and osteoarthritis cartilage (OA-iPSCs). These reprogrammed cells acquired markers of pluripotency and differentiated into uncommitted-mesenchymal progenitors. Interestingly, AC-iPSCs exhibited enhanced chondrogenic potential as compared OA-iPSCs and showed increased expression of chondrogenic genes. Pan-transcriptome analysis showed that chondrocytes derived from AC-iPSCs were enriched in molecular pathways related to energy metabolism and epigenetic regulation, together with distinct expression signature that distinguishes them from OA-iPSCs. The molecular tracing data demonstrated that epigenetic and metabolic marks were imprint of original cell sources from healthy and OA-chondrocytes. Our results suggest that the epigenetic and metabolic memory of disease may predispose OA-iPSCs for their reduced chondrogenic differentiation and thus regulation at epigenetic and metabolic level may be an effective strategy for controlling the chondrogenic potential of iPSCs.
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Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.13.512146v1?rss=1
Authors: Liu, Y., Trnka, M. J., He, L., Burlingame, A., Correia, M. A.
Abstract: We have previously documented that in liver cells, the multifunctional protein scaffold p62/SQSTM1 is closely associated with I{kappa}B, an inhibitor of the transcriptional activator NF-{kappa}B. Such an intimate p62-I{kappa}Bassociation we now document leads to a marked 18-fold proteolytic I{kappa}B-stabilization, enabling its nuclear entry and termination of the NF-{kappa}B-activation cycle. In p62-/--cells, such termination is abrogated resulting in the nuclear persistence and prolonged activation of NF-{kappa}B following inflammatory stimuli. Utilizing various approaches both classic (structural deletion, site-directed mutagenesis) as well as novel (in cell chemical crosslinking), coupled with proteomic analyses, we have defined the precise structural hotspots of p62-I{kappa}B association. Accordingly, we have identified such I{kappa}B hotspots to reside around N-terminal (K38, K47 and K67) and C-terminal (K238/C239) residues in its 5th ankyrin repeat domain. These sites interact with two hotspots in p62: One in its PB-1 subdomain around K13, and the other comprised of a positively charged patch (R183/R186/K187/K189) in the intervening region between its ZZ- and TB-subdomains. APEX proximity analyses upon I{kappa}B co-transfection of cells with and without p62 have enabled the characterization of the p62 influence on I{kappa}B-protein-protein interactions. Interestingly, consistent with p62 capacity to proteolytically stabilize I{kappa}B, its presence greatly impaired I{kappa}B interactions with various 20S/26S proteasomal subunits. Furthermore, consistent with p62-interaction with I{kappa}B on an interface opposite to that of its NF-{kappa}B-interacting interface, p62 failed to significantly affect I{kappa}B-NF-{kappa}B interactions. These collective findings together with the known dynamic p62 nucleocytoplasmic shuttling, leads us to speculate that it may be involved in piggy-back nuclear transport of I{kappa}B following its NF-{kappa}B-elicited transcriptional activation and de novo synthesis, required for the termination of the NF-{kappa}B-activation cycle. Consequently, mice carrying a liver specific deletion of p62-residues 68-252 harboring its positively charged patch, reveal age-dependent enhanced liver inflammation. Our findings reveal yet another mode of p62-mediated pathophysiologically relevant regulation of NF-{kappa}B.
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Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.14.512222v1?rss=1
Authors: Wong, W. K., Joglekar, M. V., Cheng, F., Jiang, G., Sorensen, A. E., Chew, Y. V., Loudovaris, T., Thomas, H. E., Ma, R. C., Hawthorne, W. J., Dalgaard, L. T., Hardikar, A. A.
Abstract: Human islets are widely used in research for understanding pathophysiological mechanisms leading to diabetes. Sex, age, and body mass index (BMI) are key donor traits influencing insulin secretion. Islet function is also regulated by an intricate network of microRNAs. Here, we profiled 754 microRNAs and 58,190 transcripts in up to 131 different human islet donor preparations (without diabetes) and assessed their association with donor traits. MicroRNA analyses identified miR-199a-5p and miR-214-3p associated with sex, age and BMI; miR-147b with sex and age; miR-378a-5p with sex and BMI; miR-542-3p, miR-34a-3p, miR-34a-5p, miR-497-5p and miR-99a-5p with age and BMI. There were 959 mRNA transcripts associated with sex (excluding those from sex-chromosomes), 940 with age and 418 with BMI. MicroRNA-199a-5p and miR-214-3p levels inversely associate with transcripts critical in islet function, metabolic regulation, and senescence. Our analyses identify human islet cell microRNAs influenced by donor traits.
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Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.14.512258v1?rss=1
Authors: MARR, N., ZAMBOULIS, D. E., WERLING, D., FELDER, A. A., DUDHIA, J., PITSILLIDES, A. A., THORPE, C. T.
Abstract: The interfascicular matrix (IFM) is critical to the mechanical adaptations and response to load in energy-storing tendons, such as the human Achilles and equine superficial digital flexor tendon (SDFT). We hypothesized that the IFM is a tendon progenitor cell niche housing an exclusive cell subpopulation. Immunolabelling of equine SDFT was used to identify the IFM niche, localising expression patterns of CD31 (endothelial cells), CD146 (IFM cells) and LAMA4 (IFM basement membrane marker). Magnetic-activated cell sorting was employed to isolate and compare in vitro properties of CD146+ and CD146- subpopulations. CD146 demarcated an exclusive interfascicular cell subpopulation that resides in proximity to a basal lamina which forms interconnected vascular networks. Isolated CD146+ cells exhibited limited mineralization (osteogenesis) and lipid pro-duction (adipogenesis). This study demonstrates that the IFM is a unique tendon cell niche, con-taining a vascular-rich network of basement membrane, CD31+ endothelial cells and CD146+ cell populations that are likely essential to tendon structure- and/or function. Interfascicular CD146+ subpopulations did not exhibit stem cell-like phenotypes and are more likely to represent a per-icyte lineage. Previous work has shown that tendon CD146 cells migrate to sites of injury, therefore mobilisation of endogenous tendon IFM cell populations may promote intrinsic repair.
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Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.14.512212v1?rss=1
Authors: Niethamer, T. K., Levin, L. I., Morley, M. P., Babu, A., Zhou, S., Morrisey, E. E.
Abstract: Following acute injury, the capillary vascular bed in the lung must be repaired to reestablish gas exchange with the external environment. Little is known about the transcriptional and signaling factors that drive pulmonary endothelial cell (EC) proliferation and subsequent regeneration of pulmonary capillaries, as well as their response to stress. Here, we show that the transcription factor Atf3 is essential for the regenerative response of the mouse pulmonary endothelium after influenza infection. Atf3 expression defines a subpopulation of capillary ECs enriched in genes involved in endothelial development, differentiation, and migration. During lung alveolar regeneration, this EC population expands and increases expression of genes involved in angiogenesis, blood vessel development, and cellular response to stress. Importantly, endothelial cell-specific loss of Atf3 results in defective alveolar regeneration, in part through increased apoptosis and decreased proliferation in the endothelium. This leads to the general loss of alveolar endothelium and persistent morphological changes to the alveolar niche, including an emphysema-like phenotype with enlarged alveolar airspaces lined with regions that lack vascular investment. Taken together, these data implicate Atf3 as an essential component of the vascular response to acute lung injury that is required for successful lung alveolar regeneration.
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Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.13.512167v1?rss=1
Authors: Shao, B., Panchenko, M.
Abstract: Members of the conserved subfamily, JADE1S and JADE1L isoforms, are expressed in epithelial cells, fibroblasts, and epithelial cell lining in vivo. JADE1 proteins interact with histone acetyl transferase HBO1 complex. The two consecutive PHD zinc fingers of JADE1 bind chromatin. We recently reported novel effects of JADE1S on cytokinesis progression. JADE1S depletion facilitated G2/M-to-G1 transition and increased polyploidy and aneuploidy. JADE1S over-expression arrested cells in late cytokinesis, an effect reversed by AURKB inhibitor. In late cytokinesis cells JADE1S protein localized to the midbody. Results suggested a JADE1S role in final abscission delay. Here we investigated the expression of JADE1 in the central spindle, interactions with HBO1, and the role of PHD fingers in late cytokinesis arrest. The midzone begins to assemble in anaphase and forms into a midbody in cytokinesis. The midbody structure connects two daughter cells and is thought to bear factors controlling the final abscission. We questioned whether, similar to established factors, JADE1S is targeted to the central spindle structures in anaphase. Indeed, in cells transitioning from mitosis to cytokinesis, JADE1S was sequentially targeted to early midzone, midbody flanking zone, and midbody. The step-wise increase of JADE1S expression in midzone and midbody of synchronously dividing cells suggested protein recruitment. The increase of late cytokinesis arrest caused by recombinant JADE1S correlated with increased expression in midbody. Spatial analysis of the members of the chromatin passenger complex, microtubule associated proteins, and centralspindlin, revealed transient co-localization with JADE1S and mapped JADE1S within the cytokinesis bridge. Deletion of the two PHD zinc fingers inactivated JADE1S ability to arrest cells in late cytokinesis but did not affect its midbody localization. Thus, PHD zinc fingers are required for JADE1S cytokinesis delay but not for midbody targeting. Recombinant HBO1 protein decreased the proportion of late cytokinesis cells, prevented late cytokinesis arrest by JADE1S as well as its midbody localization. Enzyme inactive HBO1 mutant recapitulated the wild type phenotype. The results demonstrate antagonistic relationship and suggest HBO1-mediated midbody dislocation of JADE1S. Our study supports the role of JADE1S in cytokinesis delay and implicates protein partners.
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Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.14.512247v1?rss=1
Authors: Francis, M., Sheshadri, P., Prasanna, J., Kumar, A.
Abstract: Diabetes is a metabolic disease caused majorly due to loss of insulin secreting {beta}-cells. Along with apoptosis, recent reports revealed dedifferentiation to be the added reason for the reduced {beta}-cell mass. The Ubiquitin Proteasome system comprising of E3 ligase and deubiquitinases (DUBs) control several key aspects of pancreatic {beta}-cell functions. The role of deubiquitinases in orchestrating the dedifferentiation process in several cancers have been well deciphered, but its role in dedifferentiation of pancreatic {beta}-cells remains elusive. In this study, screening for key DUBs that regulate dedifferentiation, identified USP1 to be specifically involved in the process. Inhibition of USP1 either by genetic intervention or small molecule inhibitor ML323 restored epithelial phenotype of {beta}-cells, but not with inhibition of other DUBs. Conversely overexpression of USP1 was sufficient to dedifferentiate {beta}-cells, even in absence of dedifferentiation inducing cues. Mechanistic insight showed USP1 to probably mediate its effect via modulating the expression of Inhibitor of Differentiation (ID) 2. Further, in an in vivo streptozotocin (STZ) induced dedifferentiation mouse model system, treatment with ML323 rescued the hyperglycaemic state. Overall, this study assigns a novel role to USP1 in dedifferentiation of {beta}-cells and its inhibition may have a therapeutic application of reducing the {beta}-cell loss during diabetes.
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Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.14.512240v1?rss=1
Authors: Lopez, L. A., Marra, M. F., Ibanez, J., Millan, M. E., Freites, C. L., Fernandez, D.
Abstract: The cells involved in spermatogenesis are germ-cells, called spermatogonia, classified as: type A-undifferentiated, type A-intermediate and type B. During the spermatogenesis, more than 75% of the germ-cells undergo apoptosis and most of them are phagocyted by Sertoli cells. Peritubular macrophages in adult mouse testis are macrophages that both stimulate the proliferation and differentiation of undifferentiated spermatogonia in the wall of the seminiferous tubule. They have long processes and ramified appearance that squished between the lateral sides of neighbor myoid cells. We show, that a population of peritubular macrophages, grouped in pairs and activated, phagocyted undifferentiated spermatogonia in apoptosis. In adult mouse testis, 3.3x 10E5 undifferentiated spermatogonia are in the germinal epithelium and 8,250 of them are in apoptosis. We counted in the testis 2,634 peritubular macrophages with phagocytic activity. If each one phagocyted one undifferentiated spermatogonia in apoptosis, it may indicated that peritubular macrophages phagocyted 31.9% of the total undifferentiated spermatogonia in apoptosis. According to our knowledges, this is the first time that it is shown that undifferentiated spermatogonia in apoptosis are cleaned by peritubular macrophages.
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Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.13.511998v1?rss=1
Authors: Zeng, Y., Staley, J. P.
Abstract: To promote fidelity in nuclear pre-mRNA splicing, the spliceosome rejects and discards suboptimal splicing substrates after they have engaged the spliceosome. Although nuclear quality control mechanisms have been proposed to retain immature mRNPs, evidence indicates that discarded splicing substrates, including lariat intermediates, do export to the cytoplasm, as indicated by their translation and degradation by cytoplasmic nucleases. However, the mechanism for exporting these species has remained unknown. By single molecule (sm) RNA FISH in budding yeast, we have directly observed the nuclear export of lariat intermediates. Further, by crosslinking, export reporter assays, and smRNA FISH, we have demonstrated that the export of lariat intermediates requires the general mRNA export receptor Mex67p and three of its mRNA export adapter proteins, Nab2p, Yra1p, and Nlp3, establishing that both mRNAs and lariat intermediates share the same export machinery. Unexpectedly, the export of lariat intermediates, but not mRNA, requires an interaction between Nab2p and Mlp1p, a nuclear basket component implicated in retaining immature mRNPs, including unspliced pre-mRNA, in the nucleus of budding yeast. Finally, the export of lariat intermediates, like mRNA, relies on the E3 ubiquitin ligase Tom1p and its target sites in Yra1p. Overall, our data indicate that the nuclear basket can promote, rather than antagonize, the export of an immature mRNP. Further, our data imply that the export of discarded lariat intermediates requires both Mlp1p-dependent docking onto the nuclear basket and subsequent Tom1p-mediated undocking, a mechanism our data suggests generalizes to the export of mRNA but in a manner obscured by redundant pathways.
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Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.13.511617v1?rss=1
Authors: Ok, M. T., Liu, J., Bliton, R. J., Hinesley, C. M., San Pedro, E. E. T., Breau, K. A., Gomez-Martinez, I., Burclaff, J., Magness, S. T.
Abstract: Background & Aims: Clostridioides difficile (C. difficile) toxins A (TcdA) and B (TcdB) cause antibiotic-associated colitis and increase morbidity and mortality. Accurate in vitro models are necessary to detect early toxicity kinetics, investigate disease etiology, and develop pre-clinical models for new therapies. Properties of cancer cell lines and 3D organoids inherently limit these efforts. Here, we develop adult stem cell-derived monolayers of differentiated human colonic epithelium (hCE) with barrier function, investigate the impact of toxin application to apical/basal aspects of monolayers, and evaluate whether a leaky epithelial barrier enhances toxicity. Methods: Single-cell RNA-sequencing (scRNAseq) mapped C. difficile-relevant genes to cell lineages across the human gut. Transcriptomics informed timing of stem cell differentiation to achieve in vitro colonocyte maturation like that observed in vivo. Transepithelial electrical resistance (TEER) and fluorescent dextran permeability assays measured cytotoxicity as barrier loss post-toxin exposure. Leaky epithelial barriers were induced with diclofenac (DCF). Results: scRNAseq demonstrated broad and variable toxin receptor expression across the human gut lineages. Absorptive colonocytes displayed generally enhanced toxin receptor, Rho GTPase, and cell junction expression. 21-day differentiated Caco-2 cells remained immature whereas hCE monolayers were similar to mature colonocytes. hCE monolayers exhibited high barrier function after 1-day differentiation. Basal TcdA/B application to monolayers caused more toxicity and apoptosis than apical exposure. DCF induced leaky hCE monolayers and enhanced toxicity of TcdB exposure. Conclusions: hCE monolayers represent a physiologically relevant and sensitive culture system to evaluate impact of microbial toxins on gut epithelium, demonstrate uncoupled onset and magnitude of apical/basal toxicities with delayed apical toxicity, and highlight that leaky paracellular junctions enhance toxicity of apical TcdB exposure.
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Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.13.512077v1?rss=1
Authors: Kim, J. Y., Atanassov, I., Dethloff, F., Kroczek, L., Langer, T.
Abstract: Mitochondrial dysfunction and cellular senescence are hallmarks of aging. However, the relationship between these two phenomena remains incompletely understood. In this study, we investigated the rewiring of mitochondria upon development of the senescent state in human IMR90 fibroblasts. Determining the bioenergetic activities and abundance of mitochondria, we demonstrate that senescent cells accumulate mitochondria with reduced OXPHOS activity, resulting in an overall increase of mitochondrial activities in senescent cells. Time-resolved proteomic analyses revealed extensive reprogramming of the mitochondrial proteome upon senescence development and allowed the identification of metabolic pathways that are rewired with different kinetics upon establishment of the senescent state. Among the early-responding pathways, the degradation of branched-chain amino acid (BCAA) was increased, while the one carbon-folate metabolism was decreased. Late-responding pathways include lipid metabolism and mitochondrial translation. These signatures were confirmed by metabolic tracing experiments, highlighting metabolic rewiring as a central feature of mitochondria in cellular senescence. Together, our data provide an unprecedentedly comprehensive view on the metabolic status of mitochondria in senescent cells and reveal how the mitochondrial proteome adapts to the induction of senescence.
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Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.14.512051v1?rss=1
Authors: Stier, A., Gilberto, S., Mohamed, W. I., Helenius, J., Mikicic, I., Sajic, T., Beli, P., Mueller, D. J., Peter, M.
Abstract: The cullin-4 paralogs CUL4A and CUL4B assemble E3 ubiquitin ligase complexes regulating multiple chromatin-associated cellular functions. Although they are structurally similar, we found that the unique N-terminal extension of CUL4B is heavily phosphorylated during mitosis, and the phosphorylation pattern is perturbed in the CUL4B-P50L mutation causing X-linked intellectual disability (XLID). Phenotypic characterization and mutational analysis revealed that CUL4B phosphorylation is required for efficient progression through mitosis, controlling spindle positioning and cortical tension. Interestingly, while CUL4B phosphorylation triggers chromatin exclusion, it critically promotes binding to actin regulators and two previously unrecognized, CUL4B-specific DCAFs, LIS1 and WDR1. Indeed, co-immunoprecipitation experiments and biochemical analysis revealed that LIS1 and WDR1 interact with DDB1, but their binding requires the phosphorylated N-terminal domain of CUL4B. Together, our study uncovers previously unrecognized DCAFs relevant for mitosis and brain development that specifically bind CUL4B, but not the CUL4B-P50L patient mutant, by a phosphorylation-dependent mechanism.
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Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.14.512102v1?rss=1
Authors: Martinez-Heredia, V., Blackwell, D., Sebastian, S., Pearson, T., Mok, G. F., Mincarelli, L., Utting, C., Folkes, L., Poschl, E., Macaulay, I. C., Mayer, U. C., Munsterberg, A. E.
Abstract: Skeletal muscle stem cells (MuSC) are crucial for tissue homeostasis and repair after injury. Following activation, they proliferate to generate differentiating myoblasts. A proportion of cells self-renew, re-enter the MuSC niche under the basal lamina outside the myofiber and become quiescent. Quiescent MuSC have a primary cilium, which is disassembled upon cell cycle entry. Ex vivo experiments suggest cilia are important for MuSC self-renewal, however, their role in muscle regeneration in vivo remains poorly understood. Talpid3 (TA3) is essential for primary cilia formation and Hedgehog (Hh) signalling. Here we use tamoxifen-inducible conditional deletion of TA3 in MuSC (iSC-KO) and show that regeneration is impaired in response to cytotoxic injury. Repeat injury exacerbates the regeneration phenotype in TA3iSC-KO mice, indicating depletion of MuSCs. Single cell transcriptomics of MuSC progeny isolated from myofibers identifies components of several signalling pathways, which are deregulated in absence of TA3, including Hh and Wnt. Pharmacological activation of Wnt restores muscle regeneration, while purmorphamine, an activator of the Smoothened (Smo) co-receptor in the Hh pathway, has no effect. Together, our data suggest that TA3 and primary cilia are important for MuSC self-renewal, and that pharmacological treatment can efficiently restore muscle regeneration.
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Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.12.511926v1?rss=1
Authors: Xin, Z., Qing, Z., Yong, L. D., Meng, L. Y., Qing, X., Hong, L. X., Wen, L., Min, Z., Li, L., Lu, Y., Cheng, J., Chen, Y.
Abstract: Necroptosis and pyroptosis are lytic and inflammatory types of programmed cell death that require the membrane destruction predominantly driven by the mixed lineage kinase domain-like (MLKL) and gasdermin D (GSDMD) proteins. However, the crosstalk between them remains largely unknown. Here, our research discloses that endoplasmic reticulumn transmembrane protein inositol-requiring enzyme-1 (IRE-1) is a potential modulator of both necroptosis and pyroptosis, paricularly in liver pathology. Interestingly, enhanced expression of IRE-1 triggers hepatic pyroptosis, while defective IRE-1 level activates hepatic necroptosis, and both processes are closed related to the activity of GSDMD. To elucidate unknown crosstalk, by using pharmacological and genetic methods, we first demonstrated that IRE-1 suppresses necroptosis by promoting the expression of GSDMD and cleaves caspase-8 and by inhibiting the expression of receptor-interacting serine/threonine-protein kinase 1 (RIPK1), RIPK3 and MLKL. Unexpectedly, excess IRE-1 initiates pyroptosis by increasing GSDMD and NLRP3 levels. Our work clearly provides insight into the modulation of IRE-1 to dominate necroptosis and pyroptosis and suggests that IRE-1 may be a promising therapeutic target for drug discovery in both types of tissue injuries.
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Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.12.511969v1?rss=1
Authors: Bogucka-Janczi, K., Harms, G., May-Coissieux, M., Bentires-Alj, M., Thiede, B., Rajalingam, K.
Abstract: The actin cytoskeleton is tightly controlled by RhoGTPases, actin binding proteins and nucleation-promoting factors to perform fundamental cellular functions. Here, we show that ERK3, an atypical MAPK, directly acts as a guanine nucleotide exchange factor for Cdc42 and phosphorylates the ARP3 subunit of the ARP2/3 complex at S418 to promote filopodia formation and actin polymerization, respectively. Consistently, depletion of ERK3 prevented both basal and EGF-dependent Rac1 and Cdc42 activation, maintenance of F-actin content, filopodia formation and epithelial cell migration. Further, ERK3 protein binds directly to the purified ARP2/3 complex and augments polymerization of actin in vitro. ERK3 kinase activity is required for the formation of actin-rich protrusions in mammalian cells. These findings unveil a fundamentally unique pathway employed by cells to control actin-dependent cellular functions.
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Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.12.511145v1?rss=1
Authors: Goudswaard, L. J., Williams, C. M., Khalil, J., Burley, K. L., Hamilton, F., Arnold, D., Milne, A., Lewis, P. A., Heesom, K. J., Mundell, S. J., Davidson, A. D., Poole, A. W., Hers, I.
Abstract: BackgroundPatients with coronavirus disease-19 (COVID-19) are at increased risk of thrombosis, which is associated with altered platelet function and coagulopathy, contributing to excess mortality.
ObjectivesWe aimed to characterise the mechanism of altered platelet function in COVID-19 patients.
MethodsThe platelet proteome, platelet functional responses and platelet-neutrophil aggregates were compared between patients hospitalised with COVID-19 and healthy control subjects using Tandem Mass Tag (TMT) proteomic analysis, Western blotting and flow cytometry.
ResultsCOVID-19 patients showed a different profile of platelet protein expression (858 altered out of 5773 quantified). Levels of COVID-19 plasma markers were enhanced in COVID-19 platelets. Gene ontology (GO) pathway analysis demonstrated that levels of granule secretory proteins were raised, whereas some platelet activation proteins, such as the thrombopoietin receptor and PKC, were lowered. Basally, COVID-19 platelets showed enhanced phosphatidylserine (PS) exposure, with unaltered integrin IIb{beta}3 activation and P-selectin expression. Agonist-stimulated integrin IIb{beta}3 activation and PS exposure, but not P-selectin expression, were significantly decreased in COVID-19 patients. COVID-19 patients had high levels of platelet-neutrophil aggregates, even under basal conditions, compared to controls. This interaction was disrupted by blocking P-selectin, demonstrating that platelet P-selectin is critical for the interaction.
ConclusionsOverall, our data suggests the presence of two platelet populations in patients with COVID-19: one with circulating platelets with an altered proteome and reduced functional responses and another with P-selectin expressing neutrophil-associated platelets. Platelet driven thromboinflammation may therefore be one of the key factors enhancing the risk of thrombosis in COVID-19 patients.
Essentials- COVID-19 patient platelet function and platelet proteins were compared with healthy controls - Proteomic analysis of platelets indicated that COVID-19 decreased platelet activation proteins - Agonist induced PS exposure and integrin IIb{beta}3 activation were impaired in COVID-19 - COVID-19 led to maximal levels of P-selectin dependent platelet-neutrophil aggregates
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Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.12.511902v1?rss=1
Authors: De-Souza, E. A., Thompson, M. A., Taylor, R. C.
Abstract: Animals rely on chemosensory cues to survive in pathogen-rich environments. In C. elegans, pathogenic bacteria are known to trigger aversive behaviors through neuronal perception, and to activate molecular defenses throughout the animal. This suggests that neurons may be able to coordinate the activation of organism-wide defensive responses upon pathogen perception. We find that exposure to volatile pathogen-associated compounds induces cell non-autonomous activation of the endoplasmic reticulum unfolded protein response (UPRER) in peripheral tissues following xbp-1 splicing in neurons. This odorant-induced UPRER activation is dependent upon transforming growth factor beta (TGF-{beta}) signaling and leads to extended lifespan and enhanced clearance of toxic proteins. Our data suggest that the cell non-autonomous UPRER rewires organismal proteostasis in response to pathogen detection, pre-empting the arrival of proteotoxic stress. Thus, chemosensation of particular odors may be a novel way to manipulate stress responses and longevity.
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Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.12.511987v1?rss=1
Authors: Matsuyama, S., Matsuyama, M., Ortega, J., Fedorov, Y., Scott-McKean, J., Muller-Greven, J., Buck, M., Adams, D., Jastrzebska, B., Greenlee, W.
Abstract: We identified cyto-protective small molecules (CSMs) by a cell-based high-throughput screening of Bax inhibitors. Through a medicinal chemistry program, M109S was developed, which is orally bioactive and penetrates the blood-brain/retina barriers. M109S protected retinal cells in ocular disease mouse models. M109S directly interacted with Bax and inhibited the conformational change and mitochondrial translocation of Bax. M109S inhibited ABT-737-induced apoptosis both in Bax-only and Bak-only mouse embryonic fibroblasts. M109S also inhibited apoptosis induced by staurosporine, etoposide, and obatoclax. M109S decreased maximal mitochondrial oxygen consumption rate and reactive oxygen species production whereas it increased glycolysis. These effects on cellular metabolism may contribute to the cytoprotective activity of M109S. M109S is a novel small molecule protecting cells from mitochondria-dependent apoptosis both in vitro and in vivo. M109S has the potential to become a new research tool for cell death mechanisms and to develop therapeutics targeting mitochondria-dependent cell death pathway. (146 words)
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Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.12.511914v1?rss=1
Authors: Di, A., Huang, L. S., Zhou, B., Toth, P. T., Krishnan, Y., Malik, A. B.
Abstract: Potassium efflux via the two pore K+ channel TWIK2 is a requisite step for the activation of the NLRP3 inflammasome, however it is unclear how the efflux is activated in response to cues. Here we report that during homeostasis, TWIK2 resides in endosomal compartments. TWIK2 is transported by endosomal fusion to the plasmalemma in response to increased extracellular ATP resulting in extrusion of K+ ATP-induced endosomal TWIK2 plasmalemma translocation is regulated by Rab11a. Deleting Rab11a or ATP ligated purinergic receptor P2X7 prevented endosomal fusion with the plasmalemma and K+ efflux and NLRP3 inflammasome activation in macrophages. Adoptive transfer of Rab11a-deleted macrophages into mouse lungs prevented NLRP3 inflammasome activation and inflammatory lung injury. Rab11a-mediated endosomal trafficking in macrophages thus regulates TWIK2 abundance and activity on the cell surface and downstream activation of the NLRP3 inflammasome. Endosomal trafficking of TWIK2 to the plasmalemma is therefore a potential therapy target in acute or chronic inflammatory states.
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Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.12.511867v1?rss=1
Authors: Pagan, J., Nguyen-Dien, G., Kozul, K., Cui, Y., Townsend, B., Ooi, S. S., Pagano, M., Lazarou, M., Taylor, R., Collins, B. M., Parton, R. G., Kulkarni, P., Carrodus, N., Zuryn, S., Millard, S., Marzio, A., Jones, M.
Abstract: Cells selectively remove damaged or excessive mitochondria through mitophagy, a specialized form of autophagy, to maintain mitochondrial quality and quantity. Mitophagy is induced in response to diverse conditions, including hypoxia, cellular differentiation, and mitochondrial damage. However, the mechanisms by which cells remove specific dysfunctional mitochondria under steady-state conditions to fine-tune mitochondrial content are not well understood. Here, we report that SCFFBXL4, an SKP1/CUL1/F-box protein ubiquitin ligase complex, localizes to the mitochondrial outer membrane in unstressed cells and mediates the constitutive ubiquitylation and degradation of the mitophagy receptors NIX and BNIP3 to suppress basal levels of mitophagy. We demonstrate that, unlike wild-type FBXL4, pathogenic variants of FBXL4 that cause encephalopathic mtDNA depletion syndrome (MTDPS13), do not efficiently interact with the core SCF ubiquitin ligase machinery or mediate the degradation of NIX and BNIP3. Thus, we reveal a molecular mechanism that actively suppresses mitophagy via preventing NIX and BNIP3 accumulation and propose that excessive basal mitophagy in the FBXL4-associated mtDNA depletion syndrome is caused by dysregulation of NIX and BNIP3 turnover.
O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=195 SRC="FIGDIR/small/511867v1_ufig1.gif" ALT="Figure 1" greater than View larger version (60K): org.highwire.dtl.DTLVardef@102ff7dorg.highwire.dtl.DTLVardef@1d39846org.highwire.dtl.DTLVardef@b23b60org.highwire.dtl.DTLVardef@1582329_HPS_FORMAT_FIGEXP M_FIG C_FIG
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Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.12.511955v1?rss=1
Authors: Newman, L. E., Tadepalle, N., Novak, S. W., Schiavon, C. R., Rojas, G. R., Chevez, J. A., Lemersal, I., Medina, M., Rocha, S., Towers, C. G., Grotjahn, D. A., Manor, U., Shadel, G. S.
Abstract: Maternally inherited mitochondrial DNA (mtDNA) encodes essential subunits of the mitochondrial oxidative phosphorylation system, but is also a major damage-associated molecular pattern (DAMP) that engages innate immune sensors when released into the cytoplasm, outside of cells or into circulation1. This function of mtDNA contributes to antiviral resistance, but unfortunately also causes pathogenic inflammation in many disease contexts2. Cells experiencing mtDNA stress due to depletion of the mtDNA-packaging protein, Transcription Factor A, Mitochondrial (TFAM), or HSV-1 infection exhibit elongated mitochondria, mtDNA depletion, enlargement of nucleoids (mtDNA-protein complexes), and activation of cGAS/STING innate immune signaling via mtDNA released into the cytoplasm3. However, the relationships between altered mitochondrial dynamics and mtDNA-mediated activation of the cGAS-STING pathway remain unclear. Here, we show that entire enlarged nucleoids are released from mitochondria that remain bound to TFAM and colocalize with cGAS. These nucleoids arise at sites of mtDNA replication due to a block in mitochondrial fission at a stage when endoplasmic reticulum (ER) actin polymerization would normally commence, which we propose is a fission checkpoint to ensure that mtDNA has completed replication and is competent for segregation into daughter mitochondria. Released nucleoids also colocalize with the early endosomal marker RAB5 as well as the late endosomal marker RAB7 in TFAM-deficient cells and in response to mtDNA stress caused by the HSV-1 UL12.5 protein. Loss of RAB7 increases interferon stimulated gene (ISG) expression. Thus, we propose that defects in mtDNA replication and/or segregation enact a late mitochondrial fission checkpoint that, if persistent, leads to selective removal of dysfunctional nucleoids by a mitochondrial-endosomal pathway. Early steps in this pathway are prone to mtDNA release and cGAS-STING activation, but the immunostimulatory mtDNA is ultimately disposed of through a mechanism involving RAB7-containing late endosomes to prevent excessive innate immune signaling. This mtDNA quality control pathway might represent a therapeutic target to prevent mtDNA-mediated inflammation and associated pathology.
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Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.12.511771v1?rss=1
Authors: Castellano-Pozo, M., Sioutas, G., Barroso, C., Lopez-Jimenez, P., Jaso-Tamame, A. L., Crawley, O., Shao, N., Page, J., Martinez-Perez, E.
Abstract: The cohesin complex plays essential roles in chromosome segregation, 3D genome organisation, and DNA damage repair through its ability to modify DNA topology. In higher eukaryotes, meiotic chromosome function, and therefore fertility, requires cohesin complexes containing meiosis-specific kleisin subunits: REC8 and RAD21L in mammals and REC-8 and COH-3/4 in C. elegans. How these complexes perform the multiple functions of cohesin during meiosis and whether this involves different modes of DNA binding or dynamic association with chromosomes is poorly understood. Combining time-resolved methods of protein removal with live imaging and exploiting the temporospatial organisation of the C. elegans germline, we show that REC-8 complexes provide sister chromatid cohesion (SCC) and DNA repair, while COH-3/4 complexes control higher-order chromosome structure. High-abundance COH-3/4 complexes associate dynamically with individual chromatids in a manner dependent on cohesin loading (SCC-2) and removal (WAPL-1) factors. In contrast, low-abundance REC-8 complexes associate stably with chromosomes, tethering sister chromatids from S-phase until the meiotic divisions. Our results reveal that kleisin identity determines the function of meiotic cohesin by controlling the mode and regulation of cohesin-DNA association, and are consistent with a model in which SCC and DNA looping are performed by variant cohesin complexes that coexist on chromosomes.
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Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.11.511646v1?rss=1
Authors: Novy, B., Adoff, H., De Maria, M., Kampmann, M., Tsvetanova, N., Von Zastrow, M., Lobingier, B.
Abstract: G protein-coupled receptors (GPCRs) are the largest family of membrane-bound signaling molecules. Activity of these receptors is critically regulated by their trafficking through the endo-lysosomal pathway. Identifying the genes involved in GPCR trafficking is challenging due the complexity of sorting operations and low affinity protein-protein interactions. Here we present a chemical biology fluorescence-based technique to interrogate GPCR trafficking. We show that the engineered enzyme APEX2 is a highly sensitive biosensor for GPCR trafficking to the lysosome, and this trafficking can be monitored through APEX-based activation of fluorogenic substrates such as Amplex UltraRed (AUR). We used this approach to perform a genome-wide CRISPR interference screen focused on the delta type opioid receptor (DOR), a GPCR which modulates anxiety, depression, and pain. The screen identified 492 genes including known- and novel-regulators of DOR expression and trafficking. We demonstrate that one of the novel genes, RME-8, localizes to early endosomes and plays a critical role in regulating DOR trafficking to the lysosome. Together, our data demonstrate that GPCR-APEX2/AUR is a flexible and highly sensitive chemical biology platform for genetic interrogation of receptor trafficking.
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Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.10.511652v1?rss=1
Authors: Liu, B., Paudel, S., Flowers, W. L., Piedrahita, J. A., Wang, X.
Abstract: Adrenomedullin (ADM) as a highly conserved peptide hormone has been reported to increase significantly in the uterine lumen during the peri-implantation period of pregnancy in pigs, but its functional roles in growth and development of porcine conceptus (embryonic/fetus and its extra-embryonic membranes) as well as underlying mechanisms remain largely unknown. Therefore, we conducted in vitro experiments using our established porcine trophectoderm cell line (pTr1) isolated from Day-12 porcine conceptuses to test the hypothesis that porcine ADM stimulates cell proliferation, migration and adhesion via AKT-TSC2-MTOR cell signaling pathway in pTr1 cells. Porcine ADM at 10-7 M stimulated (P less than 0.05) pTr1 cell proliferation, migration and adhesion by 1.4-, 1.5- and 1.2-folds, respectively. These ADM-induced effects were abrogated (P less than 0.05) by siRNA-mediated knockdown of ADM (siADM) and its shared receptor component calcitonin-receptor-like receptor (CALCRL; siCALCRL), as well as by rapamycin, the inhibitor of mechanistic target of rapamycin (MTOR). Using siRNA mediated knockdown of CALCRL coupled with Western blot analyses, ADM signaling transduction was determined in which ADM binds to CALCRL to increase phosphorylation of MTOR, its downstream effectors (4EBP1, P70S6K, and S6), and upstream regulators (AKT and TSC2). Collectively, these results suggest that porcine ADM in histotroph act on its receptor component CALCRL to activate AKT-TSC2-MTOR, particularly MTORC1 signaling cascade, leading to elongation,migration and attachment of conceptuses.
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Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.10.511666v1?rss=1
Authors: Karasmanis, E. P., Reimer, J. M., Kendrick, A. A., Rodriguez, J. A., Truong, J. B., Lahiri, I., Reck-Peterson, S. L., Leschziner, A. E.
Abstract: Cytoplasmic dynein-1 transports many intracellular cargos towards microtubule minus ends. Dynein is autoinhibited and undergoes conformational changes to form an active complex, consisting of one or two dynein dimers, the dynactin complex and activating adaptor(s)1,2. The Lissencephaly 1 gene, LIS1, is genetically linked to the dynein pathway from fungi to mammals and is mutated in patients with the neurodevelopmental disease lissencephaly3-5. Lis1 is required for active dynein complexes to form6-10, but how it does so is unclear. Here, we present a structure of two dynein motor domains with two Lis1 dimers wedged in-between. The contact sites between dynein and Lis1 in this structure, termed "Chi", are required for Lis1s regulation of dynein in Saccharomyces cerevisiae in vivo and the formation of active human dynein-dynactin- activating adaptor complexes in vitro. We propose that this structure represents an intermediate in dyneins activation pathway, revealing how Lis1 relieves dyneins autoinhibited state.
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Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.11.511735v1?rss=1
Authors: Elcocks, H., Brazel, A. J., McCarron, K. R., Kaulich, M., Husnjak, K., Mortiboys, H. J., Clague, M. J., Urbe, S.
Abstract: The selective autophagy of mitochondria is linked to mitochondrial quality control and is critical to a healthy organism. We have conducted a CRISPR/Cas9 screen of human E3 ubiquitin ligases for influence on mitophagy under both basal cell culture conditions and following acute mitochondrial depolarisation. We identify two Cullin RING ligases, VHL and FBXL4 as the most profound negative regulators of basal mitophagy. We show that these converge through control of the mitophagy adaptors BNIP3 and BNIP3L/NIX through different mechanisms. FBXL4 suppression of BNIP3 and NIX levels is mediated via direct interaction and protein destabilisation rather than suppression of HIF1-mediated transcription. Depletion of NIX but not BNIP3 is sufficient to restore mitophagy levels. Our study enables a full understanding of the aetiology of early onset mitochondrial encephalomyopathy that is supported by analysis of a disease associated mutation. We further show that the compound MLN4924, which globally interferes with Cullin RING ligase activity, is a strong inducer of mitophagy providing a research tool in this context and a candidate therapeutic agent for conditions linked to mitochondrial dysfunction.
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