{"messages":[{"status":"ok","category":"cell biology","interval":"2025-03-21:2025-03-28","funder":"all","cursor":0,"count":30,"count_new_papers":"57","total":"77"}], "collection":[{"title":"Coro1A and TRIM67 collaborate in netrin-dependent neuronal morphogenesis","authors":"Ho, C. T.; Evans, E. B.; Lukasik, K.; O'Shaughnessy, E. C.; Shah, A.; Hsu, C.-H.; Temple, B.; Bear, J. E.; Gupton, S. L.","author_corresponding":"Stephanie L Gupton","author_corresponding_institution":"University of North Carolina at Chapel Hill","doi":"10.1101\/2025.03.20.644333","date":"2025-03-21","version":"1","type":"new results","license":"cc_no","category":"cell biology","jatsxml":"https:\/\/www.biorxiv.org\/content\/early\/2025\/03\/21\/2025.03.20.644333.source.xml","abstract":"Neuronal morphogenesis depends on extracellular guidance cues accurately instructing intracellular cytoskeletal remodeling. Here, we describe a novel role for the actin binding protein Coronin 1A (Coro1A) in neuronal morphogenesis, where it mediates responses to the axon guidance cue netrin-1. We found that Coro1A localizes to growth cones and filopodial structures and is required for netrindependent axon turning, branching, and corpus callosum development. We previously discovered that Coro1A interacts with TRIM67, a brain enriched E3 ubiquitin ligase that interacts with a netrin receptor and is also required for netrin-mediated neuronal morphogenesis. Loss of Coro1A and loss of TRIM67 shared similar phenotypes, suggesting that they may function together in the same netrin pathway. A Coro1A mutant deficient in binding TRIM67 was not able to rescue loss of Coro1A phenotypes, indicating that the interaction between Coro1A and TRIM67 is required for netrin responses. Together, our findings reveal that Coro1A is required for proper neuronal morphogenesis, where it collaborates with TRIM67 downstream of netrin.","funder":"NA","published":"10.1083\/jcb.202503068","server":"bioRxiv"},{"title":"Inhibition of Renin Release, a Crucial Event in Homeostasis, is Mediated by Coordinated Calcium Oscillations within Juxtaglomerular Cell Clusters.","authors":"Yamaguchi, H.; Guagliardo, N. A.; Bell, L. A.; Yamaguchi, M.; Matsuoka, D.; Xu, F.; Smith, J. P.; Diagne, M.; Almeida, L. F.; Medrano, S.; Barrett, P. Q.; Nieh, E. H.; Gomez, A. R.; Sequeira-Lopez, M. L. S.","author_corresponding":"Maria Luisa S Sequeira-Lopez","author_corresponding_institution":"Department of Pediatrics, Child Health Research Center, University of Virginia School of Medicine","doi":"10.1101\/2024.12.23.629519","date":"2025-03-21","version":"2","type":"new results","license":"cc_by_nc_nd","category":"cell biology","jatsxml":"https:\/\/www.biorxiv.org\/content\/early\/2025\/03\/21\/2024.12.23.629519.source.xml","abstract":"BACKGROUNDJuxtaglomerular (JG) cells are sensors that control blood pressure (BP) and fluid-electrolyte homeostasis. They are arranged as clusters at the tip of each afferent arteriole. In response to a decrease in BP or extracellular fluid volume, JG cells secrete renin, initiating an enzymatic cascade that culminates in the production of angiotensin II (AngII), a potent vasoconstrictor that restores BP and fluid-electrolyte homeostasis. In turn, AngII exerts negative feedback on renin release concomitantly with increased intracellular Ca2+, preventing excessive circulating renin and hypertension. However, within their native structural organization, the intricacies of intracellular Ca2+ signaling dynamics and their sources remain uncharacterized.\n\nMETHODSWe generated mice expressing the JG cell-specific genetically encoded Ca2+ indicator (GCaMP6f) to investigate Ca2+ dynamics within JG cell clusters ex vivo and in vivo. For ex vivo Ca2+ imaging, acutely prepared kidney slices were perfused continuously with a buffer containing variable Ca2+ and AngII concentrations {+\/-} Ca2+ channel inhibitors. For in vivo Ca2+ image capture, native mouse kidneys were imaged in situ using multi-photon microscopy with and without AngII administration. ELISA measurements of renin concentrations determined acute renin secretion ex vivo and in vivo, respectively.\n\nRESULTSEx vivo Ca2+ imaging revealed that JG cells exhibit robust and coordinated intracellular oscillatory signals with cell-cell propagation following AngII stimulation. AngII dose-dependently induced stereotypical burst patterns characterized by consecutive Ca2+ spikes, which inversely correlated with renin secretion. Pharmacological channel inhibition identified key sources of these oscillations: endoplasmic reticulum Ca2+ storage and release, extracellular Ca2+ uptake via ORAI channels, and intercellular communication through gap junctions. Blocking ORAI channels and gap junctions reduced AngII inhibitory effect on renin secretion. In vivo Ca2+ imaging demonstrated robust intracellular and intercellular Ca2+ oscillations within JG cell clusters under physiological conditions, exhibiting spike patterns consistent with those measured in ex vivo preparations. Administration of AngII enhanced the Ca2+ oscillatory signals and suppressed acute renin secretion in vivo.\n\nCONCLUSIONAngII elicits coordinated intracellular and intercellular Ca2+ oscillations within JG cell clusters, ex vivo and in vivo. The effect is driven by endoplasmic reticulum-derived Ca2+ release, ORAI channels, and gap junctions, leading to suppressed renin secretion.","funder":"NA","published":"NA","server":"bioRxiv"},{"title":"Human Satellite 3 DNA encodes megabase-scale transcription factor binding platforms","authors":"Franklin, J. M.; Dubocanin, D.; Chittenden, C.; Barillas, A. G.; Lee, R. J.; Ghosh, R. P.; Gerton, J. L.; Guan, K.-L.; Altemose, N.","author_corresponding":"Nicolas Altemose","author_corresponding_institution":"Stanford University","doi":"10.1101\/2024.10.22.616524","date":"2025-03-21","version":"2","type":"new results","license":"cc_by_nc","category":"cell biology","jatsxml":"https:\/\/www.biorxiv.org\/content\/early\/2024\/10\/22\/2024.10.22.616524.source.xml","abstract":"Eukaryotic genomes frequently contain large arrays of tandem repeats, called satellite DNA. While some satellite DNAs participate in centromere function, others do not. For example, Human Satellite 3 (HSat3) forms the largest satellite DNA arrays in the human genome, but these multi-megabase regions were almost fully excluded from genome assemblies until recently, and their potential functions remain understudied and largely unknown. To address this, we performed a systematic screen for HSat3 binding proteins. Our work revealed that HSat3 contains millions of copies of transcription factor (TF) motifs bound by over a dozen TFs from various signaling pathways, including the growth-regulating transcription effector family TEAD1-4 from the Hippo pathway. Imaging experiments show that TEAD recruits the co-activator YAP to HSat3 regions in a cell-state specific manner. Using synthetic reporter assays, targeted repression of HSat3, inducible degradation of YAP, and super-resolution microscopy, we show that HSat3 arrays can localize YAP\/TEAD inside the nucleolus, enhancing RNA Polymerase I activity. Beyond discovering a direct relationship between the Hippo pathway and ribosomal DNA regulation, this work demonstrates that satellite DNA can encode multiple transcription factor binding motifs, defining an important functional role for these enormous genomic elements.","funder":"NA","published":"NA","server":"bioRxiv"},{"title":"Exosomes Released from Senescent Cells and Circulatory Exosomes Isolated from Human Plasma Reveal Aging-associated Proteomic and Lipid Signatures","authors":"Patel, S. K.; Bons, J.; Rose, J. P.; Chappel, J. R.; Beres, R. L.; Watson, M.; Webster, C.; Burton, J. B.; Bruderer, R.; Desprez, P. Y.; Reiter, L.; Campisi, J.; Baker, E.; Schilling, B.","author_corresponding":"Birgit Schilling","author_corresponding_institution":"Buck Institute for Research on Aging","doi":"10.1101\/2024.06.22.600215","date":"2025-03-22","version":"2","type":"new results","license":"cc_no","category":"cell biology","jatsxml":"https:\/\/www.biorxiv.org\/content\/early\/2025\/03\/22\/2024.06.22.600215.source.xml","abstract":"Senescence emerged as significant mechanism of aging and age-related diseases, offering an attractive target for clinical interventions. Senescent cells release a senescence-associated secretory phenotype (SASP), including exosomes that may act as signal transducers between distal tissues, propagating secondary or bystander senescence and signaling throughout the body. However, the composition of exosome SASP remains underexplored, presenting an opportunity for novel unbiased discovery. We present a detailed proteomic and lipidomic analysis of exosome SASP using mass spectrometry from human plasma from young and older individuals and from tissue culture of senescent primary human lung fibroblasts. We identified [~]1,300 exosome proteins released by senescent cells induced by three different senescence inducers. In parallel, a human plasma cohort from young (20-26 years) and old (65-74 years) individuals revealed over 1,350 exosome proteins and 171 plasma exosome proteins were regulated when comparing old vs young individuals. Of the age-regulated plasma exosome proteins, we observed 52 exosome SASP factors that were also regulated in exosomes from the senescent fibroblasts, including serine protease inhibitors (SERPINs), Prothrombin, Coagulation factor V, Plasminogen, and Reelin. 247 lipids were identified in exosome samples. Following senescence induction, identified phosphatidylcholines, phosphatidylethanolamines, and sphingomyelins increased significantly indicating cellular membrane changes. Interestingly, significantly changed proteins were related to extracellular matrix remodeling and inflammation, both potentially detrimental pathways that can damage surrounding tissues and even induce secondary senescence. Our findings reveal mechanistic insights and potential senescence biomarkers, enabling a better approach to surveilling the senescence burden in the aging population and offering therapeutic targets for interventions.","funder":"NA","published":"10.18632\/aging.206292","server":"bioRxiv"},{"title":"A niche-dependent redox rheostat regulates epithelial stem cell fate in the distal colon","authors":"Chen, X.; Raghunathan, K.; Bao, B.; Ngwa, E.; Kwong, A.; Wu, Z.; Babcock, S.; Baek, C.; Ye, G.; Muppirala, A.; Peng, Q.; Rutlin, M.; Bhaumik, M.; Yang, D.; Kotlarz, D.; Jadhav, U.; Rao, M.; Weerapana, E.; Zhou, X.; Ordovas-Montanes, J.; Snapper, S. B.; Thiagarajah, J. R.","author_corresponding":"Jay R Thiagarajah","author_corresponding_institution":"Division of Gastroenterology, Hepatology and Nutrition, Boston Childrens Hospital, Harvard Medical School, Boston, MA 02115, USA; Congenital Enteropathy Program","doi":"10.1101\/2025.01.26.634856","date":"2025-03-22","version":"2","type":"new results","license":"cc_by_nc_nd","category":"cell biology","jatsxml":"https:\/\/www.biorxiv.org\/content\/early\/2025\/03\/22\/2025.01.26.634856.source.xml","abstract":"The niche environment surrounding intestinal stem cells (ISCs) varies along the length of intestine and provides key cues that regulate stem cell fate. Here, we investigated the role of cellular redox balance in colonic ISC function. We show that hypoxia and Wnt signaling synergize to restrict the reactive oxygen species (ROS) generating enzyme NADPH oxidase 1 (NOX1) to the crypt base in the distal colon. NOX1 function maintains a more oxidative cell state that licenses cell cycle entry, altering the balance of asymmetric stem cell self-renewal and directing lineage commitment. Mechanistically, cell redox state directs a self-reinforcing circuit that connects hypoxia inducible factor 1 (HIF1)-dependent signaling with regulation of the metabolic enzyme isocitrate dehydrogenase 1 (IDH1). Our studies show that cellular redox balance is a central and niche-dependent regulator of epithelial homeostasis and regeneration and provide a basis for understanding disease propensity in the distal large intestine.\n\nGRAPHICAL ABSTRACT\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=176 SRC=\"FIGDIR\/small\/634856v2_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (55K):\norg.highwire.dtl.DTLVardef@107798aorg.highwire.dtl.DTLVardef@1bc9013org.highwire.dtl.DTLVardef@96d56aorg.highwire.dtl.DTLVardef@95268d_HPS_FORMAT_FIGEXP  M_FIG C_FIG HIGHLIGHTSO_LIThe balance of cycling intestinal stem cells (ISCs) versus committed epithelial cells in the uniquely hypoxic niche of the distal colon is regulated by NADPH oxidase 1 (NOX1) dependent H2O2 both at homeostasis and during regeneration.\nC_LIO_LIPhysiological increase in cellular H2O2 favors maintenance of glycolysis in ISCs for self-renewal through regulation of isocitrate dehydrogenase 1 activity.\nC_LIO_LIMaintenance of the increased cellular oxidative state stabilizes HIF1 through a re-enforcing metabolic circuit.\nC_LIO_LIA shift from a relatively oxidative to a reductive cell environment in distal colonic ISCs leads to decreased progression through the cell cycle and altered cell fate determination.\nC_LI","funder":"NA","published":"10.1038\/s41467-025-66636-5","server":"bioRxiv"},{"title":"GMCL1 Controls 53BP1 Stability and Modulates Paclitaxel Sensitivity in Cancer","authors":"Kito, Y.; Gonzalez-Robles, T. J.; Kaisari, S.; Pae, J.; Garcia, S. F.; Ortiz-Pacheco, J.; Ueberheide, B.; Lehmann, R.; Marzio, A.; Rona, G.; Pagano, M.","author_corresponding":"Michele Pagano","author_corresponding_institution":"NYU GROSSMAN SCHOOL OF MEDICINE HHMI","doi":"10.1101\/2025.03.18.643855","date":"2025-03-22","version":"2","type":"new results","license":"cc_by","category":"cell biology","jatsxml":"https:\/\/www.biorxiv.org\/content\/early\/2025\/03\/22\/2025.03.18.643855.source.xml","abstract":"Mitotic surveillance pathways monitor the duration of mitosis (M phase) in the cell cycle. Prolonged M phase, caused by spindle attachment defects or microtubule-targeting drugs triggers formation of the ternary \"mitotic stopwatch pathway\" complex (MSP) consisting of 53BP1, USP28, and p53. This complex stabilizes p53, leading to cell cycle arrest or apoptosis in daughter cells. In cancers that are resistant to paclitaxel, a microtubule-targeting agent, cells bypass mitotic surveillance activation, allowing unchecked proliferation, although the underlying mechanisms remain poorly understood. Here, we identify GMCL1 as a key negative regulator of MSP signaling. We show that 53BP1 physically interacts with GMCL1, but not its paralog GMCL2, and we map their interaction domains. CRL3GMCL1 functions as a ubiquitin ligase that targets 53BP1 for degradation during the M phase, thereby reducing p53 accumulation in daughter cells. Depletion of GMCL1 inhibits cell cycle progression upon release from prolonged mitotic arrest, a defect that is rescued by co-silencing 53BP1 or USP28. Moreover, GMCL1 depletion sensitizes cancer cells to paclitaxel in a p53-dependent manner. Together, our findings support a model in which dysregulated CRL3GMCL1-mediated degradation of 53BP1 prevents proper MSP function, leading to p53 degradation and continued proliferation. Targeting GMCL1 may therefore represent one possible avenue for addressing paclitaxel resistance in cancer cells with functional p53.","funder":[{"name":"National Institute of General Medical Sciences","id":"https:\/\/ror.org\/04q48ey07","id-type":"ROR","award":""}],"published":"10.7554\/eLife.106730.3","server":"bioRxiv"},{"title":"Discovery of unique mitotic mechanisms in Paradiplonema papillatum","authors":"Akiyoshi, B.; Faktorova, D.; Lukes, J.","author_corresponding":"Bungo Akiyoshi","author_corresponding_institution":"University of Edinburgh","doi":"10.1101\/2025.03.21.644664","date":"2025-03-24","version":"1","type":"new results","license":"cc_by","category":"cell biology","jatsxml":"https:\/\/www.biorxiv.org\/content\/early\/2025\/03\/24\/2025.03.21.644664.source.xml","abstract":"Diplonemids are highly diverse and abundant marine plankton with significant ecological importance. However, little is known about their biology, even in the model diplonemid Paradiplonema papillatum whose genome sequence is available. Examining the subcellular localization of proteins using fluorescence microscopy is a powerful approach to infer their putative function. Here we report a plasmid-based method that enables YFP-tagging of a gene at the endogenous locus. By examining the localization of proteins whose homologs are involved in chromosome segregation in other eukaryotes, we discovered several interesting features in mitotically dividing P. papillatum cells. Cohesin is enriched on condensed interphase chromatin. During mitosis, chromosomes organize into two rings (termed metaphase rings herein) that surround the elongating nucleolus. Homologs of chromosomal passenger complex components (INCENP, two Aurora kinases, and KIN-A), a CLK1 kinase, spindle checkpoint protein Mad1, and microtubule regulator XMAP215 localize in between the two metaphase rings, suggesting that kinetochores may assemble in between them. We also found that a homolog of the meiotic chromosome axis protein SYCP2L1 is enriched in between metaphase rings during mitosis. These features have some resemblance to the bivalent bridge (also known as the modified synaptonemal complex), which is thought to mediate the linkage between homologous chromosomes using axis components in Bombyx mori female meiotic metaphase I. By representing the first molecular characterization of mitotic mechanisms in P. papillatum and raising a number of questions, this study forms the foundation for dissecting mitotic mechanisms in diplonemids.","funder":"NA","published":"10.1098\/rsob.250096","server":"bioRxiv"},{"title":"Munc13-4 mediates tumor immune evasion by regulating the sorting and secretion of PD-L1 via exosomes","authors":"Liu, C.; Liu, D.; Zheng, X.; Guan, J.; Zhou, X.; Zhang, H.; Wang, S.; Li, Q.; Gan, L.; He, J.; Ma, C.","author_corresponding":"Cong Ma","author_corresponding_institution":"Huazhong University of Science and Technology","doi":"10.1101\/2025.03.22.644518","date":"2025-03-24","version":"1","type":"new results","license":"cc_by_nc","category":"cell biology","jatsxml":"https:\/\/www.biorxiv.org\/content\/early\/2025\/03\/24\/2025.03.22.644518.source.xml","abstract":"Tumor-derived extracellular vesicles primarily carry PD-L1 via exosomes, which interact with PD-1 receptors on T cells, impacting immune responses in the tumor microenvironment and beyond, leading to a more extensive immunosuppressive landscape. However, the mechanisms governing exosomal PD-L1 sorting and secretion remain elusive. In this study, we identified Munc13-4 as a crucial regulator of exosomal PD-L1 sorting and secretion. Deletion of Munc13-4 in breast tumors enhances T cell-mediated anti-tumor immunity, suppresses tumor growth, and improves the efficacy of immune checkpoint inhibitors. Our results illustrate how Munc13-4 collaborates with HRS, Rab27, and SNAREs to facilitate PD-L1 sorting and secretion via exosomes. The cryo-EM structure of the Munc13-4-Rab27a complex provide new insights into its role in exosome secretion. Importantly, we discovered that Munc13-4 has a novel role in sorting PD-L1 onto exosomes, which relies on the formation of a ternary complex with PD-L1 and HRS. In addition, IFN{gamma} stimulation modifies Munc13-4 and HRS, establishing a dynamic regulatory mechanism that enables tumor cells to adapt to immune pressure by modulating PD-L1 sorting. Using a specially designed peptide to disrupt the Munc13-4-PD-L1 interaction and impede PD-L1 sorting significantly enhances anti-tumor immunity and slows tumor growth in vivo. These results highlight the potential of targeting the Munc13-4-PD-L1 axis to suppress tumor immune evasion.","funder":"NA","published":"NA","server":"bioRxiv"},{"title":"Hyperactivation of TAK1 causes skeletal muscle pathology reminiscent of inflammatory myopathies","authors":"Tomaz da Silva, M.; Roy, A.; Kumar, A.","author_corresponding":"Ashok Kumar","author_corresponding_institution":"University of Houston","doi":"10.1101\/2025.03.21.644671","date":"2025-03-24","version":"1","type":"new results","license":"cc_by_nc","category":"cell biology","jatsxml":"https:\/\/www.biorxiv.org\/content\/early\/2025\/03\/24\/2025.03.21.644671.source.xml","abstract":"Loss of skeletal muscle mass and strength is a debilitating consequence of various chronic diseases, inflammatory myopathies, and neuromuscular disorders. Inflammation plays a major role in the perpetuation of myopathy in degenerative muscle diseases. TAK1 is a major signaling protein that mediates the activation of multiple signaling pathways in response to inflammatory cytokines and microbial products. Recent studies have demonstrated that TAK1 is essential for the growth and maintenance of skeletal muscle mass in adult mice. However, the effects of overstimulation of TAK1 activity in the regulation of skeletal muscle mass remain unknown. In the present study, using AAV vectors, we investigated the effect of varying levels of TAK1 activation on skeletal muscle in adult mice. Our results demonstrate that while low levels of TAK1 activation improve skeletal muscle mass, sustained hyperactivation of TAK1 causes myopathy in adult mice. Excessive stimulation of TAK1 manifests pathological features, such as myofiber degeneration and regeneration, cellular infiltration, increased expression of proinflammatory molecules, and interstitial fibrosis. Hyperactivation of TAK1 also upregulates proteolytic systems and various catabolic signaling pathways in skeletal muscle of adult mice. Altogether, our study demonstrates that physiological levels of activation of TAK1 lead to myofiber hypertrophy, whereas its hyperactivation results in myofiber damage and other pathological features resembling inflammatory myopathies.","funder":"NA","published":"10.1016\/j.ajpath.2025.07.005","server":"bioRxiv"},{"title":"Glioblastoma stem cell morphotypes convey distinct cell states and clinically relevant functions","authors":"Barelli, C.; Bonfanti, M.; Mirabella, F.; Ricca, D.; Alizadehmohajer, N.; Sokolova, V.; Campione, A.; Sicuri, G. M.; Peano, C.; Faletti, S.; Stefini, R.; Kalebic, N.","author_corresponding":"Nereo Kalebic","author_corresponding_institution":"Human Technopole","doi":"10.1101\/2025.03.24.644884","date":"2025-03-24","version":"1","type":"new results","license":"cc_by_nd","category":"cell biology","jatsxml":"https:\/\/www.biorxiv.org\/content\/early\/2025\/03\/24\/2025.03.24.644884.source.xml","abstract":"Glioblastoma (GBM) is an aggressive brain tumor and an unmet clinical need due to its invasiveness and therapy-resistance. These features are driven by glioblastoma stem cells (GSCs), which exhibit remarkable functional heterogeneity. However, GSC transcriptional profiling alone cannot predict clinically relevant behaviors. Here, we developed CellShape-seq, a spatial transcriptomics platform that integrates cell morphology with transcriptome. This identified three GSC morphoclasses corresponding to distinct transcriptomic states and functions: (1) nonpolar cells show differentiation and therapy sensitivity, (2) elongated cells are invasive, and (3) multipolar cells form intercellular networks. Importantly, chemoresistance is morphoclass-specific: elongated GSCs depend on YAP\/TEAD1 signaling, while multipolar GSCs rely on gap junction-mediated networks. Targeting these vulnerabilities with specific inhibitors sensitized resistant GSC morphoclasses to temozolomide (TMZ) in patient-derived organoids. Our findings demonstrate that morphology provides critical insights into GSC behavior and establish a rationale for morphology-informed therapies to overcome resistance and improve outcomes in GBM.\n\nGraphical abstract\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=85 SRC=\"FIGDIR\/small\/644884v2_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (15K):\norg.highwire.dtl.DTLVardef@141e698org.highwire.dtl.DTLVardef@18a238corg.highwire.dtl.DTLVardef@23ef62org.highwire.dtl.DTLVardef@1390c07_HPS_FORMAT_FIGEXP  M_FIG C_FIG","funder":"NA","published":"NA","server":"bioRxiv"},{"title":"Sox9-dependent plasticity of esophageal progenitors is fine-tuned by cues from the microenvironment","authors":"Descampe, L.; Dassy, B.; Charara, F.; Craciun, L.; Verset, L.; Pirard, S.; Verheye, Q.; Leprovost, M.; Garcia, M.-I.; Vercauteren Drubbel, A.; Beck, B.","author_corresponding":"Benjamin Beck","author_corresponding_institution":"IRIBHM J.E. Dumont, Faculty of medicine, Universite Libre de Bruxelles (ULB), 808 route de Lennik, 1070 Brussels.","doi":"10.1101\/2025.03.21.644516","date":"2025-03-25","version":"1","type":"new results","license":"cc_no","category":"cell biology","jatsxml":"https:\/\/www.biorxiv.org\/content\/early\/2025\/03\/25\/2025.03.21.644516.source.xml","abstract":"Cell plasticity governs tissue regeneration but can also drive metaplasia, the replacement of one cell type with another. This process increases the risk of cancer development in several tissues, including esophagus. Esophageal metaplasia development partly depends on keratinocyte plasticity, making regulation of esophageal progenitor fate critical.\n\nWe previously identified Sox9 as instrumental in regulating esophageal cell plasticity following Hedgehog pathway activation. Our current study reveals that Hedgehog indirectly regulates Sox9 by modifying epithelial-stromal communication. This activates TGF-{beta} and BMP pathways in epithelial cells, which synergistically regulate Sox9 and stimulate a transcriptomic program resembling squamo-columnar junction progenitors, which are prone to initiate metaplasia. Importantly, we demonstrate pharmacological modulation of this plasticity in vivo. Indeed, Ibuprofen inhibits Hedgehog-induced Sox9 expression by directly targeting epithelial cells, providing proof of concept for pharmacological intervention in cell plasticity with implications for regenerative medicine and metaplasia treatment.","funder":"NA","published":"NA","server":"bioRxiv"},{"title":"Using lightsheet microscopy to investigate the initial lymphatic network in the murine knee joints","authors":"Lin, X.; Xing, L.","author_corresponding":"Xi Lin","author_corresponding_institution":"University of Rochester Medical Center","doi":"10.1101\/2025.03.21.644620","date":"2025-03-25","version":"1","type":"new results","license":"cc_no","category":"cell biology","jatsxml":"https:\/\/www.biorxiv.org\/content\/early\/2025\/03\/25\/2025.03.21.644620.source.xml","abstract":"Introduction. Lymphatic vasculature in mouse joints is difficult to study due to its vast anatomical variation. We reported increased lymphatic capillaries in post-traumatic OA (PTOA) murine joint based on 2D immunostaining. However, a gap of knowledge with this approach is that histology highly subjected to histological sampling, which might not be appropriate for a highly variable system such as the lymphatic vasculature. We hypothesize that using lightsheet microscopy would reveal the detailed structure of the initial lymphatic network.\n\nMethods. We established lightsheet imaging protocol and four quantifiable outcome parameters for visualizing and analyzing the initial lymphatic network in young C57\/B6 wild type mouse knees. Lymphatic vessels were identified by staining samples with lymphatic endothelial marker LYVE1. We validated our lightsheet imaging protocol in mouse knee joints following PTOA surgery and knee joints received sham operation.\n\nResults. We used the histological landmarks of growth plates and menisci shape for consistent data collection for the 3D reconstruction of initial lymphatic network. Comparing to sham joints, PTOA joints have less mean branches length (58.91{+\/-} 6.50 vs. 146.29 {+\/-} 2.48 m in sham, p<0.0001), and mean branch diameter (12.25{+\/-} 0.80] vs. 28.51 {+\/-} 4.99 m in sham, p=0.0051), but higher total branching points (378{+\/-} 239 vs. 69 {+\/-} 17 m in sham, p=0.020), and total initial lymphatic network volume (51247{+\/-} 14239 vs. 1019289 {+\/-} 544458 m3 in sham, p=0.037). Conclusion. Lightsheet imaging of murine knee joints is a powerful and promising tool to study the joint lymphatic system and arthritis. Similar to previous report, the PTOA joint has increased branching with smaller and shorter branches.","funder":"NA","published":"NA","server":"bioRxiv"},{"title":"Patient-derived liver biopsy organoids enable precision alcohol-associated liver disease modeling","authors":"Arino, S.; Zannatto, L.; Martinez-Garcia de la Torre, R. A.; Ferrer-Lorente, R.; Cratacos-Gines, J.; Belen Rubio, A.; Perez, M.; Aguilar-Bravo, B.; Serrano, G.; Atkinson, S.; Xu, Z.; Cantallops-Vila, P.; Sererols-Vinas, L.; Ruiz-Blazquez, P.; Rill, A.; Lozano, J. J.; Coll, M.; Ochoa, I.; Affo, S.; Moles, A.; Mereu, E.; Bataller, R.; Pose, E.; Sancho-Bru, P.","author_corresponding":"Pau Sancho-Bru","author_corresponding_institution":"Institut d'Investigacions Biom\u00e8diques August Pi i Sunyer (IDIBAPS), Barcelona, Spain.","doi":"10.1101\/2025.03.22.644563","date":"2025-03-25","version":"1","type":"new results","license":"cc_no","category":"cell biology","jatsxml":"https:\/\/www.biorxiv.org\/content\/early\/2025\/03\/25\/2025.03.22.644563.source.xml","abstract":"Background & AimsAlcohol-associated liver disease (ALD) is a major cause of liver disease worldwide with scarce therapeutic options. Animal models poorly recapitulate advanced ALD precluding the development of new treatments. Organoids have emerged as a powerful human-based preclinical tool. However, current patient-derived liver organoids fail to recapitulate the epithelial heterogeneity and its generation requires liver surgical resections, thus limiting personalized disease modeling. Here, we report the development of organoids from liver needle biopsies (b-Orgs) from patients with ALD.\n\nMethodsb-Orgs were generated from tru-cut biopsies from patients at early (n=28) and advanced (n=34) stages of ALD. b-Orgs were characterized by immunofluorescence, bulk and single cell RNA-sequencing and compared to parental tissues. b-Orgs were used to model ALD progression, identify pathogenic drivers, induce alcohol-associated hepatitis (AH) and evaluate response to prednisolone.\n\nResultsPhenotypic and functional analysis of b-Orgs showed hepatocyte- enriched features. Single-cell RNA-sequencing revealed a heterogeneous cell composition comprising hepatocyte, biliary and progenitor populations, mirroring the epithelial landscape found in patients with advanced ALD. Moreover, b-Orgs preserved disease-stage features and allowed to identify the association of ELF3 with cell plasticity and disease progression. Finally, stimulation of b-Orgs with drivers of ALD induced pathophysiological features of alcohol-associated hepatitis, including ROS production, lipid accumulation, inflammation and decreased cell proliferation, which were mitigated in response to prednisolone.\n\nConclusions\n\nOverall, we provide a human-based model that recapitulates epithelial complexity and patient specific features, allowing to identify drivers of cell plasticity and expanding organoid-based liver disease modeling for personalized medicine.\n\nImpact and implications\n\nHere, we describe the generation of biopsy-derived organoids (b-Orgs) from patients with liver disease. b-Orgs reproduce the liver epithelial cell composition found in patients liver tissue and are efficiently generated from different stages of the disease, providing a platform for patient- tailored disease modeling and drug testing.","funder":"NA","published":"NA","server":"bioRxiv"},{"title":"TTC7A organizes glandular lumen formation in the intestine through a Class II phosphatidylinositol 3-kinase","authors":"Bugda Gwilt, K.; Wijayratne, D.; Emberling, G.; Gillette, L.; La, J.; Zheng, H. B.; Wendel, D.; Karam, L. B.; Muise, A. M.; Snapper, S. B.; Lencer, W. I.; Raghunathan, K.; Thiagarajah, J. R.","author_corresponding":"Jay R Thiagarajah","author_corresponding_institution":"Division of Gastroenterology, Hepatology and Nutrition, Boston Children's Hospital","doi":"10.1101\/2025.03.22.644724","date":"2025-03-25","version":"1","type":"new results","license":"cc_by_nc_nd","category":"cell biology","jatsxml":"https:\/\/www.biorxiv.org\/content\/early\/2025\/03\/25\/2025.03.22.644724.source.xml","abstract":"The formation of a single central lumen is a critical step for glandular morphogenesis. Patients with loss of function variants in the chaperone protein TTC7A have multiple lumen formation in colonic crypt glands. We show that trafficking and localization of TTC7A to the plasma membrane is required for directionally specifying the apical membrane with TTC7A patient loss-of-function variants leading to mislocalized membrane and lumen formation. Our experiments show that TTC7A, in early stages of apical membrane development, functions as a molecular chaperone for the Class II phosphatidylinositol 3-kinase, PIK3C2A and is trafficked in Rab11a positive vesicles to generate phosphatidylinositol 3,4-bisphosphate (PI(3,4)P2). We show that the apical specification process is dependent on PIK3C2A dependent generation of PI(3,4)P2 in intestinal epithelia and that defective lumen formation can be rescued by exogenous PI(3,4)P2 or small molecules that modulate phosphoinositide homeostasis.","funder":"NA","published":"NA","server":"bioRxiv"},{"title":"A novel function for endothelial protease-activated receptors in modulating insulin receptor activity with implications for diabetes","authors":"Rajala, R.; Griffin, C. T.","author_corresponding":"Courtney T. Griffin","author_corresponding_institution":"Oklahoma Medical Research Foundation","doi":"10.1101\/2025.03.21.644607","date":"2025-03-25","version":"1","type":"new results","license":"cc_by_nc_nd","category":"cell biology","jatsxml":"https:\/\/www.biorxiv.org\/content\/early\/2025\/03\/25\/2025.03.21.644607.source.xml","abstract":"Thrombin, a serine protease with increased activity in diabetics, signals through protease-activated receptors 1 and 4 (PAR1\/PAR4) on endothelial cells (ECs). While studying the roles of endothelial PAR1\/4 in diabetic pathology, we found that mice with inducible deletion of both receptors on ECs (Par1\/4iECko) displayed increased insulin sensitivity and were protected against streptozotocin (STZ)-induced diabetes. Concordantly, we found that cultured primary ECs with PAR1\/4 deficiency had increased basal activity\/phosphorylation of the insulin receptor (IR) and insulin transcytosis. This elevated IR activity correlated with reduced activity of protein tyrosine phosphatase 1B (PTP1B), which is a negative regulator of IR activity. Lastly, Par1\/4iECkomice with additional deletion of one allele of the IR gene demonstrated restoration of diabetic phenotypes after STZ treatment, indicating that these phenotypes are driven by heightened IR activity. These findings establish a novel link between endothelial PAR signaling and IR regulation, underscoring the critical role of ECs in metabolic homeostasis and identifying a potential therapeutic target for diabetes.\n\nGRAPHICAL ABSTRACT\n\nO_FIG O_LINKSMALLFIG WIDTH=199 HEIGHT=200 SRC=\"FIGDIR\/small\/644607v1_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (62K):\norg.highwire.dtl.DTLVardef@4a5d4forg.highwire.dtl.DTLVardef@34012corg.highwire.dtl.DTLVardef@391efeorg.highwire.dtl.DTLVardef@21e3f8_HPS_FORMAT_FIGEXP  M_FIG C_FIG","funder":"NA","published":"10.1161\/ATVBAHA.125.323140","server":"bioRxiv"},{"title":"The Hitchdock domain in Kinesin-2 Tail Enables Adaptor Assembly and Cargo Binding","authors":"Jiang, X.; Danev, R.; Niu, B.; Ohtsuki, S.; Yanagisawa, H.; Hirokawa, N.; Kikkawa, M.","author_corresponding":"Masahide Kikkawa","author_corresponding_institution":"The University of Tokyo","doi":"10.1101\/2025.03.21.644525","date":"2025-03-25","version":"1","type":"new results","license":"cc_by_nc_nd","category":"cell biology","jatsxml":"https:\/\/www.biorxiv.org\/content\/early\/2025\/03\/25\/2025.03.21.644525.source.xml","abstract":"Intracellular transport relies on motor proteins like kinesins to deliver essential cargoes along microtubules, yet the mechanisms of cargo recognition remain unclear. Here, we present high-resolution cryo-electron microscopy structures of the heterotrimeric kinesin-2 complex (KIF3A\/KIF3B\/KAP3) bound to the adenomatous polyposis coli (APC) cargo. Our findings reveal a previously uncharacterized KIF3 tail motif, termed the \"Hitchdock domain,\" which plays a pivotal role in mediating interactions with both the KAP3 adaptor and the APC cargo. In this domain, the KIF3A helical regions facilitate specific cargo binding, while the {beta}-hairpin region and KIF3B provide structural support. Mutagenesis and molecular dynamics simulations confirm the domains functional importance. Interestingly, the Hitchdock\/KAP3 structure suggests a conserved structural basis for cargo recognition across molecular motors, including kinesin-1 and dynein, which utilize similar hook-like architectures, highlighting the potential universality of this mechanism. Furthermore, our findings provide insights into kinesin-2 cargo specificity and offer a molecular framework for understanding related diseases.","funder":[{"name":"Japan Science and Technology Agency","id":"https:\/\/ror.org\/00097mb19","id-type":"ROR","award":"JPMJER2202;"},{"name":"Japan Society for the Promotion of Science","id":"https:\/\/ror.org\/00hhkn466","id-type":"ROR","award":"JP23000013;JP16H06372;JP21H05247;JP24K18106;JP22H02554;JP24KF0141;"},{"name":"Japan Agency for Medical Research and Development","id":"https:\/\/ror.org\/004rtk039","id-type":"ROR","award":"JP23ama121002;JP23ama121018;"}],"published":"NA","server":"bioRxiv"},{"title":"Circulating extracellular vesicles can transport stress signals to the male germline","authors":"Alshanbayeva, A.; Steg, L.; Othman, A.; Manuella, F.; Arzate-Mejia, R. G.; Zamboni, N.; Mansuy, I. M.","author_corresponding":"Isabelle M Mansuy","author_corresponding_institution":"University\/ETH Zurich","doi":"10.1101\/2025.03.21.644551","date":"2025-03-25","version":"1","type":"new results","license":"cc_by_nc_nd","category":"cell biology","jatsxml":"https:\/\/www.biorxiv.org\/content\/early\/2025\/03\/25\/2025.03.21.644551.source.xml","abstract":"Extracellular vesicles (EVs) play a key role in cell-cell communication by transporting bioactive molecules from donor to recipient cells across the body. While their involvement in somatic cells communication is well described, somatic-to-germ cells communication remains understudied. We show that small EVs (sEVs) can vehicle signals of early life stress (ELS) from circulation to sperm consequentially for the offspring. In mice, ELS persistently modifies RNA, lipids and metabolites in plasma sEVs. Chronic injection of plasma sEVs from ELS-exposed males alters the sperm transcriptome and is reflected in the transcriptome of embryos. This leads to metabolic dysfunctions in the adult offspring. These findings highlight a role of circulating sEVs in soma-to-germline communication relevant for the intergenerational transmission of ELS effects.","funder":"NA","published":"NA","server":"bioRxiv"},{"title":"The ganglioside GD3 and its Synthase (ST8SIA1) as novel senescence markers associated with osteoarthritis","authors":"Fissoun, C.; Maroun, G.; Silva, R.; Milano, M.; Guibert, B.; Dagneaux, L.; Ferreira-Lopez, R.; Commes, T.; Gilson, E.; Jorgensen, C.; Cherfils-Vicini, J.; Pers, Y.-M.; Brondello, J.-M.","author_corresponding":"Jean-Marc Brondello","author_corresponding_institution":"Institute of Regenerative Medicine and Biotherapies (IRMB), INSERM U1183, University of Montpellier, Montpellier, France","doi":"10.1101\/2025.03.22.640206","date":"2025-03-25","version":"1","type":"new results","license":"cc_no","category":"cell biology","jatsxml":"https:\/\/www.biorxiv.org\/content\/early\/2025\/03\/25\/2025.03.22.640206.source.xml","abstract":"Osteoarthritis (OA), is the most common age-induced degenerative joint disease. It is associated with synovial inflammation, subchondral bone remodeling and cartilage degradation. One of the significant emerging causes of OA progression is senescent cell accumulation within the joint compartment during lifespan. Currently, there are no therapeutic approaches nor stratification tools that rely on the senescence burden in OA. In this study, we identified the b-series ganglioside 3 (GD3) as new senescent cell surface marker associated with OA. Joint RNA sequencing analysis revealed an increase expression of the GD3 synthase, ST8SIA1 in cartilage, synovial tissue, and subchondral bone marrow from OA patients compared to healthy donors. Moreover, we revealed a strong correlative association between the expression of ST8SIA1 and GD3 production with senescence hallmarks in an in vitro-induced 3D organotypic OA cartilage model but also with cartilage histological grading scores in human and preclinical murine OA joints. Anti-GD3 cell sorting showed that GD3-positive human OA chondrocytes or human OA synoviocytes are enriched in senescence and SASP markers compared to GD3-negative counterparts confirming that GD3 is a cell surface marker linked to the senescence stage. Intra-articular anti-GD3 antibody delivery in experimental OA model, reduced local expression of senescence and OA markers in association with a protection against OA-induced subchondral bone remodeling. Our research demonstrates a compelling linkage between ST8SIA1 gene, GD3 and senescence in OA pathology, revealing knowledge and perspectives for a better understanding and anti-senescence treatment of OA pathogenesis.","funder":"NA","published":"10.1007\/s11357-025-01903-3","server":"bioRxiv"},{"title":"An Assessment of the Functional State of Endothelial Colony Forming Cells from Patients with Diabetes Mellitus and Chronic Limb Threatening Ischemia","authors":"Lyons, C. J.; Creane, M.; Soomro, N.; Sanz-Nogues, C.; Shafik, L.; Straszewicz, A.; Griffin, T. P.; Stitt, A.; O'Brien, T.","author_corresponding":"Timothy O'Brien","author_corresponding_institution":"University of Galway","doi":"10.1101\/2025.03.24.644359","date":"2025-03-25","version":"1","type":"new results","license":"cc_by_nc_nd","category":"cell biology","jatsxml":"https:\/\/www.biorxiv.org\/content\/early\/2025\/03\/25\/2025.03.24.644359.source.xml","abstract":"Chronic limb threatening ischemia (CLTI) is the most severe form of peripheral vascular disease which can lead to amputation with a high associated mortality rate. Endothelial colony forming cells (ECFCs) show potential as a cell therapy to revascularize the limbs of individuals with CLTI. However, autologous ECFCs from patient peripheral blood (PB) have been reported to have a dysfunctional phenotype. We investigated this disease phenotype in individuals with CLTI, with and without diabetes mellitus (DM), to determine ECFC suitability as an autologous cell therapy.\n\nPB-ECFCs were isolated from age-matched controls, individuals with DM, and individuals with CLTI, with and without DM. The frequency of isolating ECFCs from this donor cohort was calculated. Furthermore, in vitro characterization assays were performed (growth kinetics, angiogenic properties, and reactive oxygen species (ROS) levels) and compared between donor groups.\n\nWe report a significantly increased frequency of ECFCs from individuals with CLTI, with and without DM. Furthermore, our results demonstrate no significant disease related effect on the in vitro functional properties of ECFCs between cohorts. However, there is a significantly higher in vitro angiogenic capacity in individuals with DM vs age-matched controls.\n\nOur results demonstrate that ECFCs can be isolated in individuals with CLTI, with and without DM, and that ECFC functionality is similar between cohorts. Therefore, if the 70% isolation efficiency from CLTI cohorts is overcome, then autologous PB-ECFCs may be a suitable therapeutic for CLTI. Further analysis is needed to determine the critical quality attributes of ECFCs from this patient population.\n\nSignificance StatementTo the authors knowledge, this paper shows for the first time that endothelial colony forming cells can be isolated from individuals with chronic limb threatening ischemia, with and without diabetes. Additionally, we show a significantly higher frequency of endothelial colony forming cells isolated from chronic limb threatening ischemia patient cohorts. There is no significant difference in endothelial colony forming cells between age-matched controls and chronic limb threatening ischemia patient with and without diabetes mellitus in vitro, potentially suggesting an autologous approach may be a viable therapeutic option in the future.","funder":"NA","published":"10.1093\/stcltm\/szaf054","server":"bioRxiv"},{"title":"T-World: A highly general computational model of a human ventricular myocyte","authors":"Tomek, J.; Zhou, X.; Martinez-Navarro, H.; Holmes, M.; Bury, T.; Arantes Berg, L.; Tomkova, M.; Jo, E.; Nagy, N.; Bertrand, A.; Bueno-Orovio, A.; Colman, M. A.; Rodriguez, B.; Bers, D. M.; Heijman, J.","author_corresponding":"Jakub Tomek","author_corresponding_institution":"University of Oxford","doi":"10.1101\/2025.03.24.645031","date":"2025-03-25","version":"1","type":"new results","license":"cc_by_nc","category":"cell biology","jatsxml":"https:\/\/www.biorxiv.org\/content\/early\/2025\/03\/25\/2025.03.24.645031.source.xml","abstract":"Cardiovascular disease is the leading cause of death, demanding new tools to improve mechanistic understanding and overcome limitations of stem cell and animal-based research. We introduce T-World, a highly general virtual model of human ventricular cardiomyocyte suitable for multiscale studies. T-World shows comprehensive agreement with human physiology, from electrical activation to contraction, and is the first to replicate all key cellular mechanisms driving life-threatening arrhythmias. Extensively validated on unseen data, it demonstrates strong predictivity across applications and scales. Using T-World we revealed a likely sex-specific arrhythmia risk in females related to restitution properties, identified arrhythmia drivers in type 2 diabetes, and describe unexpected pro-arrhythmic role of NaV1.8 in heart failure. T-World demonstrates strong performance in predicting drug-induced arrhythmia risk and opens new opportunities for predicting and explaining drug efficacy, demonstrated by unpicking effects of mexiletine in Long QT syndrome 2. T-World is available as open-source code and an online app.","funder":"NA","published":"NA","server":"bioRxiv"},{"title":"The differential mechanisms of eIF4A1-mediated translational activation instructed by distinct RNA features","authors":"Schmidt, T.; Turnbull, A. P.; Gupta, A.; Waldron, J. A.; Pollock, K.; Munro, J.; Nepravishta, R.; Herviou, P.; Dabrowska, A.; Hodge, K.; Voelker, A. M.; Pang, L.; Pasquali, C.; Kanellos, G.; Zanivan, S.; Sansom, O. J.; Beaudoin, J.-D.; Bushell, M.","author_corresponding":"Martin Bushell","author_corresponding_institution":"Cancer Research UK Scotland Institute, Garscube Estate, Switchback Road, Glasgow, G61 1BD, UK","doi":"10.1101\/2025.03.24.644895","date":"2025-03-25","version":"1","type":"new results","license":"cc_by_nc_nd","category":"cell biology","jatsxml":"https:\/\/www.biorxiv.org\/content\/early\/2025\/03\/25\/2025.03.24.644895.source.xml","abstract":"All mRNAs require eukaryotic translation initiation factor (eIF) 4A1 for translation through its different functions: loading of the pre-initiation complex onto mRNAs and unwinding of RNA structure. eIF4A1 is the catalytic subunit of the cap- binding eIF4F complex and presumed to select the mRNAs for translation through these activities, instructed by signalling pathways driving cell fate. The mechanisms underlying activation of the distinct eIF4A1 functions and establish translational selectivity are unknown.\n\nHere, we have unravelled the complexity of mRNA selection by eIF4A1. We have mechanistically characterised the biological and atomic basis of inhibition by gain- and loss-of-function eIF4A1-inhibitors eFT226 and hippuristanol, and used machine learning to model the mRNA features associated with specific inhibition. This uncovered the eIF4A1 function - mRNA sequence relationship: 5UTRs containing C\/CG-rich require efficient mRNA loading by eIF4F which is specifically targeted by hippuristanol, while 5UTRs harbouring alternatives starts sites together with AG-rich motifs utilise eIF4A1 for start site selection, specifically perturbed by eFT226. Our model is validated through a massively parallel reporter assay using 5UTRs from a distinct evolutionary origin. This prompted us to examine the conservation between mRNA sequence and eIF4A1 function, and revealed their co- development.\n\nOur findings highlight opportunities for novel therapeutic strategies targeting eIF4A1 and for improved design of mRNA-based therapeutics.","funder":"NA","published":"NA","server":"bioRxiv"},{"title":"Mechanisms of mitochondrial reactive oxygen species action in bone mesenchymal cells","authors":"Ali, M. M.; Nookaew, I.; Resende-Coelho, A.; Marques-Carvalho, A.; Aaron Warren, A.; Fu, Q.; Kim, H.-N.; OBrien, C. A.; Almeida, M.","author_corresponding":"Maria Almeida","author_corresponding_institution":"University of Arkansas for Medical Sciences","doi":"10.1101\/2025.03.24.643319","date":"2025-03-25","version":"1","type":"new results","license":"cc_by_nc","category":"cell biology","jatsxml":"https:\/\/www.biorxiv.org\/content\/early\/2025\/03\/25\/2025.03.24.643319.source.xml","abstract":"Mitochondrial reactive oxygen species (mtROS), insufficient NAD+, and cellular senescence all contribute to the decrease in bone formation with aging. ROS can cause senescence and decrease NAD+, but it remains unknown whether these mechanisms mediate the effects of ROS in vivo. Here, we generated mice with deletion of the mitochondrial antioxidant enzyme Sod2 in Osx1-Cre (Sp7-tTA, tetO-EGFP\/cre) targeted cells designated Sod2{Delta}Osx1 mice. We showed that Sod2 deletion caused low bone mass. Osteoblastic cells from these mice had impaired mitochondrial respiration and attenuated NAD+ levels. Administration of an NAD+ precursor improved mitochondrial function in vitro but failed to rescue the low bone mass of Sod2{Delta}Osx1 mice. Single-cell RNA-sequencing of bone mesenchymal cells indicated that ROS had no significant effects on markers of senescence but disrupted parathyroid hormone signaling, iron metabolism, and proteostasis. Our data support the rationale that treatment combinations aimed at decreasing mtROS and senescent cells and increasing NAD+ should confer additive effects in delaying age-associated osteoporosis.","funder":[{"name":"National Institutes of Health","id":"https:\/\/ror.org\/01cwqze88","id-type":"ROR","award":"R01AG068449;R01AR56679;"},{"name":"Center for Musculoskeletal Disease Research COBRE","id":"","id-type":"ROR","award":"P20GM125503;"}],"published":"10.1016\/j.jbc.2025.110551","server":"bioRxiv"},{"title":"ATG2A engages Rab1a and ARFGAP1 positive membranes during autophagosome biogenesis","authors":"Fuller, D. M.; Wu, Y.; Schueder, F.; Rasool, B.; Nag, S.; Korfhage, J. L.; Garcia-Milian, R.; Melnyk, K. D.; Bewersdorf, J.; De Camilli, P.; Melia, T.","author_corresponding":"Thomas Melia","author_corresponding_institution":"Yale School of Medicine","doi":"10.1101\/2025.03.24.645038","date":"2025-03-25","version":"1","type":"new results","license":"cc_by","category":"cell biology","jatsxml":"https:\/\/www.biorxiv.org\/content\/early\/2025\/03\/25\/2025.03.24.645038.source.xml","abstract":"Autophagosomes form from seed membranes that expand through bulk-lipid transport via the bridge-like lipid transporter ATG2. The origins of the seed membranes and their relationship to the lipid transport machinery are poorly understood. Using proximity labeling and a variety of fluorescence microscopy techniques, we show that ATG2A localizes to extra-Golgi ARFGAP1 puncta during autophagosome biogenesis. ARFGAP1 itself is dispensable during macroautophagy, but among other proteins associating to these membranes, we find that RAB1 is essential. ATG2A co-immunoprecipitates strongly, albeit indirectly, with RAB1A, and siRNA-mediated depletion of RAB1A\/B blocks autophagy downstream of LC3B lipidation, similar to ATG2A depletion. Further, when either autophagosome formation or the early secretory pathway is perturbed, ARFGAP1 and RAB1A accumulate at ectopic locations with autophagic machinery. Our results indicate that ATG2A engages a RAB1A complex on select early secretory membranes in support of autophagosome biogenesis.\n\nSignificance StatementThis study expounds upon the role of early secretory membranes in autophagosome biogenesis. The authors demonstrate that RAB1\/ARFGAP1 positive membranes are essential to autophagy and are recruited to the phagophore assembly site at an early step of autophagosome biogenesis. These membranes interact with the bridge-like lipid transport protein ATG2A and are positive for LC3B and WIPI2, suggesting that RAB1 membranes are a direct source for autophagosome formation.","funder":[{"name":"National Institutes of Health","id":"https:\/\/ror.org\/01cwqze88","id-type":"ROR","award":"R01 GM100930;R35 GM153482;R01 GM151829;DA018343;F31 AG079606;F31 DK136246;"},{"name":"Aligning Science Across Parkinson's","id":"https:\/\/ror.org\/03zj4c476","id-type":"ROR","award":"ASAP-025173;"},{"name":"Human Frontier Science Program","id":"","id-type":"ROR","award":"LT000056\/2020-C;"},{"name":"Wellcome Leap Foundation","id":"","id-type":"ROR","award":""},{"name":"HHMI","id":"","id-type":"ROR","award":""},{"name":"nih","id":"","id-type":"ROR","award":"R01 GM100930;R35 GM153482;R01 GM151829;DA018343;F31 AG079606;F31 DK136246;"}],"published":"NA","server":"bioRxiv"},{"title":"Membrane-associated polymerases deliver most of the actin subunits to a lamellipodial network","authors":"Skruber, K.; Sept, D.; Mullins, D.","author_corresponding":"Dyche Mullins","author_corresponding_institution":"UCSF School of Medicine","doi":"10.1101\/2025.03.24.645090","date":"2025-03-25","version":"1","type":"new results","license":"cc_by","category":"cell biology","jatsxml":"https:\/\/www.biorxiv.org\/content\/early\/2025\/03\/25\/2025.03.24.645090.source.xml","abstract":"Actin filaments are two-stranded protein polymers that form the basic structural unit of the eukaryotic actin cytoskeleton. While filaments assembled from purified actin in vitro elongate when soluble monomers bind to free filament ends, in cells the mechanism of filament elongation is less clear. Most monomeric actin in the cytoplasm is bound to the accessory protein profilin, and many regulators of filament assembly recruit actin-profilin complexes to membrane surfaces where they locally accelerate filament elongation. Employing quantitative live-cell imaging of actin-profilin fusion proteins and biochemically defined mutants of the branched actin regulator, WAVE1, we find that only [~]25% of the actin in leading-edge lamellipodial networks enters directly from solution, while the majority enters via membrane-associated polymerases.","funder":"NA","published":"NA","server":"bioRxiv"},{"title":"In situ cryo-ET visualization of mitochondrial depolarization and mitophagic engulfment","authors":"Rose, K.; Herrmann, E.; Kakudji, E. V.; Lizarrondo, J.; Celebi, A. Y.; Wilfling, F.; Lewis, S. C.; Hurley, J. H.","author_corresponding":"James H Hurley","author_corresponding_institution":"UC Berkeley","doi":"10.1101\/2025.03.24.645001","date":"2025-03-25","version":"1","type":"new results","license":"cc_by","category":"cell biology","jatsxml":"https:\/\/www.biorxiv.org\/content\/early\/2025\/03\/25\/2025.03.24.645001.source.xml","abstract":"Defective mitochondrial quality control in response to loss of mitochondrial membrane polarization is implicated in Parkinsons disease by mutations in PINK1 and PRKN. Application of in situ cryo-electron tomography (cryo-ET) made it possible to visualize the consequences of mitochondrial depolarization at higher resolution than heretofore attainable. Parkin-expressing U2OS cells were treated with the depolarizing agents oligomycin and antimycin A (OA), subjected to cryo-FIB milling, and mitochondrial structure was characterized by in situ cryo-ET. Phagophores were visualized in association with mitochondrial fragments. Bridge-like lipid transporter (BLTP) densities potentially corresponding to ATG2A were seen connected to mitophagic phagophores. Mitochondria in OA-treated cells were fragmented and devoid of matrix calcium phosphate crystals. The intermembrane gap of cristae was narrowed and the intermembrane volume reduced, and some fragments were devoid of cristae. A subpopulation of ATP synthases re-localized from cristae to the inner boundary membrane (IBM) apposed to the outer membrane (OMM). The structure of the dome-shaped prohibitin complex, a dodecamer of PHB1-PHB2 dimers, was determined in situ by sub-tomogram averaging in untreated and treated cells and found to exist in open and closed conformations, with the closed conformation is enriched by OA treatment. These findings provide a set of native snapshots of the manifold nano-structural consequences of mitochondrial depolarization and provide a baseline for future in situ dissection of Parkin-dependent mitophagy.","funder":"NA","published":"10.1073\/pnas.2511890122","server":"bioRxiv"},{"title":"CRISPR-Cas9 Screening Reveals Microproteins Regulating Adipocyte Proliferation and Lipid Metabolism","authors":"Pai, V. J.; Shan, H.; Donaldson, C.; Vaughan, J.; O'Connor, C.; Liem, M.; Pinto, A.; Diedrich, J. K.; Saghatelian, A.","author_corresponding":"Alan Saghatelian","author_corresponding_institution":"Salk Institute for Biological Studies","doi":"10.1101\/2025.03.21.644636","date":"2025-03-25","version":"1","type":"new results","license":"cc_no","category":"cell biology","jatsxml":"https:\/\/www.biorxiv.org\/content\/early\/2025\/03\/25\/2025.03.21.644636.source.xml","abstract":"Small open reading frames (smORFs) encode microproteins that play crucial roles in various biological processes, yet their functions in adipocyte biology remain largely unexplored. In a previous study, we identified thousands of smORFs in white and brown adipocytes derived from the stromal vascular fraction (SVF) of mice using ribosome profiling (Ribo-Seq). Here, we expand on this work by identifying additional smORFs related to adipocytes using the in vitro 3T3-L1 preadipocyte model. To systematically investigate the functional relevance of these smORFs, we designed a custom CRISPR\/Cas9 guide RNA (sgRNA) library and screened for smORFs influencing adipocyte proliferation and differentiation. Through a dropout screen and fluorescence-assisted cell sorting (FACS) of lipid droplets, we identified dozens of smORFs that regulate either cell proliferation or lipid accumulation. Among these, we validated a novel microprotein as a key regulator of adipocyte differentiation. These findings highlight the potential of CRISPR\/Cas9-based screening to uncover functional smORFs and provide a framework for further exploration of microproteins in adipocyte biology and metabolic regulation.\n\nSignificanceObesity and its associated metabolic disorders pose significant public health challenges, yet the molecular mechanisms regulating adipocyte function remain incompletely understood. Small open reading frames (smORFs) and their encoded microproteins represent an emerging class of regulatory elements with potential roles in metabolism. Here, we leveraged CRISPR\/Cas9 screening to functionally characterize smORFs in adipocytes, identifying novel regulators of cell proliferation and lipid metabolism. Our findings demonstrate that conservation is not a prerequisite for smORF function, as we validated a mouse-specific microprotein that modulates adipocyte differentiation. This work establishes a robust pipeline for unbiased smORF discovery and highlights the potential for species-specific microproteins to regulate adipose biology. Future studies in human adipocytes may uncover additional microproteins with therapeutic relevance for obesity and metabolic disease.","funder":"NA","published":"NA","server":"bioRxiv"},{"title":"Ser14-phosphorylated Rpn6 Limits Proteostasis Impairment and Pathology in Both Brain and Heart of Tauopathy Mice","authors":"Ejaz, S.; Sternburg, J. O.; Rezvani, K.; Ahammed, M. S.; Giri, S.; Liu, J.; Wang, H.; Wang, X.","author_corresponding":"Xuejun Wang","author_corresponding_institution":"University of South Dakota","doi":"10.1101\/2025.03.24.645024","date":"2025-03-25","version":"1","type":"new results","license":"cc_no","category":"cell biology","jatsxml":"https:\/\/www.biorxiv.org\/content\/early\/2025\/03\/25\/2025.03.24.645024.source.xml","abstract":"Alzheimers disease (AD) patients often display neurobehavioral and cardiac impairments, but the underlying factors remain unclear. Ser14 phosphorylation in RPN6 (p-S14-RPN6) mediates the activation of 26S proteasomes by protein kinase A (PKA). Proteasome priming is implicated in protection by cAMP-PKA against AD, but this remains to be established. Hence, this study was conducted to interrogate homeostatic p-S14-RPN6 in AD. The recently validated Rpn6S14A knock-in (S14A) mice were crossbred with the PS19 tauopathy mice (RRID: IMSR_JAX:008169). The resultant wild type (WT), PS19, and PS19::S14A littermates were compared. Expedited declines in cognitive and motor functions as indicated respectively by significant decreases in object recognition and discrimination indexes and rotarod time were observed in PS19::S14A mice vs. PS19 mice, which is associated with more pronounced synaptic losses, microglial activation, and gliosis in the hippocampus. Compared with WT and PS19 mice, PS19::S14A mice showed exacerbated cardiac malfunction, cardiac hypertrophic responses and fibrosis, and greater increases of total and hyperphosphorylated tau proteins and ubiquitin conjugates in both hippocampi and hearts. These findings demonstrate that genetic blockade of p-S14-RPN6 exacerbates tauopathy in both the brain and heart, which for the first time establishes that homeostatic p-S14-RPN6 promotes proteostasis and protects against pathogenesis in AD.","funder":"NA","published":"NA","server":"bioRxiv"},{"title":"Depletion of Polypyrimidine tract binding protein 1 (ptbp1) activates Muller glia-derived proliferation during zebrafish retina regeneration via modulation of the senescence secretome","authors":"Konar, G. J.; Lingan, A. L.; Vallone, K. T.; Nguyen, T. D.; Flickinger, Z. R.; Patton, J. G.","author_corresponding":"James G Patton","author_corresponding_institution":"Vanderbilt University","doi":"10.1101\/2025.03.24.645057","date":"2025-03-25","version":"1","type":"new results","license":"cc_by","category":"cell biology","jatsxml":"https:\/\/www.biorxiv.org\/content\/early\/2025\/03\/25\/2025.03.24.645057.source.xml","abstract":"Polypyrimidine Tract Binding protein 1 (PTB) is an alternative splicing factor linked to neuronal induction and maturation. Previously, knockdown experiments supported a model in which PTB can function as a potent reprogramming factor, able to elicit direct glia-to-neuron conversion in vivo, in both the brain and retina. However, later lineage tracing and genetic knockouts of PTB did not support direct neuronal reprogramming. Nevertheless, consistent with the PTB depletion experiments, we show that antisense knockdown of PTB (ptbp1a) in the zebrafish retina can activate Muller glia-derived proliferation and that depletion of PTB can further enhance proliferation when combined with acute NMDA damage. The effects of PTB are consistent with a role in controlling key senescence and pro-inflammatory genes that are part of the senescence secretome that initiates retina regeneration.","funder":"NA","published":"10.1016\/j.exer.2025.110420","server":"bioRxiv"},{"title":"Armed with PRICKLE(3)s: Stabilizing WNT\/PCP complexes against RNF43-mediated ubiquitination","authors":"Radaszkiewicz, K.; Radaszkiewicz, T.; Kolarova, P.; Paclikova, P.; Gomoryova, K.; Barta, T.; Hanakova, K.; Zdrahal, Z.; Harnos, J.","author_corresponding":"Jakub Harnos","author_corresponding_institution":"Masaryk University","doi":"10.1101\/2025.03.24.644882","date":"2025-03-25","version":"1","type":"new results","license":"cc_by_nc_nd","category":"cell biology","jatsxml":"https:\/\/www.biorxiv.org\/content\/early\/2025\/03\/25\/2025.03.24.644882.source.xml","abstract":"The human Prickle protein family, consisting of PRICKLE1, PRICKLE2, PRICKLE3, and PRICKLE4, is an integral component of the WNT\/planar cell polarity (WNT\/PCP) pathway and is essential for various cellular and developmental processes. Despite their significance, the detailed roles and involvement in molecular mechanisms of these proteins in cells remain not fully understood. In this study, we used enhanced proximity biotinylation (miniTurboID) combined with mass spectrometry to characterize the microenvironment of PRICKLE1-3. Our results reveal that PRICKLE3 is directly linked to the WNT\/PCP pathway, primarily localizing at the plasma membrane and forming complexes with VANGL proteins. This observation prompted us to examine its role in the non-canonical WNT signalling pathway in more detail. Using an inducible expression system to achieve protein levels closer to physiological conditions, we found that PRICKLE3 enhances the stability of VANGL1 and VANGL2 by shielding them from Casein kinase 1 {varepsilon}-mediated phosphorylation. Furthermore, our results indicate that PRICKLE3 modulates WNT receptor complexes by negatively regulating the interaction between Casein kinase 1 {varepsilon} and ubiquitin ligase RNF43, resulting in decreased ubiquitination and increased stabilization of VANGL1\/2 at the plasma membrane. Notably, these effects were specific to PRICKLE3, with PRICKLE1 showing no comparable activity. Contrary to previous findings based mainly on standard overexpression studies, neither PRICKLE3 nor PRICKLE1 influenced the levels or phosphorylation status of WNT proteins DISHEVELLED2 and DISHEVELLED3, which are the PRICKLE proteins binding partners. In summary, we have identified a key mechanism specific to PRICKLE3 that positively regulates WNT\/PCP complexes by suppressing RNF43. Additionally, we present a comprehensive interactome and new tools for the functional specification of Prickle isoforms to support further research.","funder":"NA","published":"NA","server":"bioRxiv"},{"title":"Respiratory Airway Secretory Cells act as Immune Sentinels in Human Distal Airways","authors":"Sun, J.; Sun, H.; Jiang, S.; Xie, X.; Wang, D.; Yao, H.; Guan, W.; Zhao, J.; Zhang, W. K.; Xu, T.; Liu, H.","author_corresponding":"Jincun Zhao","author_corresponding_institution":"The First Affiliated Hospital of Guangzhou Medical University","doi":"10.1101\/2025.03.24.644887","date":"2025-03-25","version":"1","type":"new results","license":"cc_no","category":"cell biology","jatsxml":"https:\/\/www.biorxiv.org\/content\/early\/2025\/03\/25\/2025.03.24.644887.source.xml","abstract":"Pulmonary immunity in the human distal respiratory airways is essential for lung function but remains less explored. Here we report that respiratory airway secretory (RAS) cells, a newly identified lung epithelial population unique to humans and large mammals, act as immune sentinels in the distal airways by safeguarding against infection and injury. Leveraging human pluripotent stem cell-derived lung organoids, animal models, and clinical specimens, we elucidate that RAS cells arise from distal lung progenitors and exhibit context-dependent immune competence. Upon viral or bacterial challenge, RAS cells display interferon-mediated or TLR5-dependent defense responses, respectively. In chronic obstructive pulmonary disease, they produce elevated adaptive immune responses. Notably, complement C3 is upregulated under these conditions and is suppressed by a selective TLR5 inhibitor. Our findings reveal RAS cells as previously unrecognized sensors and effectors of mucosal immunity at human distal airways, highlighting the TLR5-complement axis as a potential therapeutic target in lung disease.","funder":[{"name":"National Natural Science Foundation of China","id":"https:\/\/ror.org\/01h0zpd94","id-type":"ROR","award":"82171419;"},{"name":"Major Project of Guangzhou National Laboratory","id":"","id-type":"ROR","award":"MP-GZNL2025C03007;"},{"name":"National Key Research and Development Program of China","id":"","id-type":"ROR","award":"2021YFA1101304;2020YFA0908200;"}],"published":"NA","server":"bioRxiv"}]}