Erika Arias, Maureen E Haynes, Neil A Nadkarni, Zoie K Lipfert, William A Muller, Ayush Batra, David P Sullivan
{"title":"EdU tracking of leukocyte recruitment in mouse models of ischemic stroke and sterile lung inflammation.","authors":"Erika Arias, Maureen E Haynes, Neil A Nadkarni, Zoie K Lipfert, William A Muller, Ayush Batra, David P Sullivan","doi":"10.1242/jcs.263835","DOIUrl":"10.1242/jcs.263835","url":null,"abstract":"<p><p>The discovery of copper(I)-catalyzed azide-alkyne cycloaddition (click chemistry) has significantly advanced the detection of proliferating cells by utilizing 5-ethynyl-2'-deoxyuridine (EdU). EdU, a thymidine analogue, is incorporated into DNA during replication and detected by the direct reaction with an azide-conjugated fluorophore. Traditionally, dividing cells are labeled using 5-bromodeoxyuridine (BrdU), another nucleotide analogue. However, BrdU detection is a harsh method that requires substantial sample processing, unlike EdU detection. EdU is classically used to identify proliferating cells; however, we report a streamlined methodology that uses EdU to label and track leukocyte recruitment that is compatible with flow cytometry and microscopy and preserves transgenic fluorophores. EdU labeling was performed in two different models of sterile inflammation: ischemic stroke and hydrochloric acid aspiration. EdU injection was timed to differentially label circulating monocytes, neutrophils and T cells. Tissue analysis showed EdU-positive monocytes and T cells were enriched in both inflammatory models. This suggests that recently divided monocytes and T cells are preferentially recruited to these vascular beds during inflammation and highlights the utility of this labeling approach to track leukocyte subtypes longitudinally during inflammation.</p>","PeriodicalId":15227,"journal":{"name":"Journal of cell science","volume":"138 8","pages":""},"PeriodicalIF":3.3,"publicationDate":"2025-04-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12136169/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143998780","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"Epithelial cell extrusion at a glance.","authors":"Aline Grata, Romain Levayer","doi":"10.1242/jcs.263786","DOIUrl":"https://doi.org/10.1242/jcs.263786","url":null,"abstract":"<p><p>The robustness and plasticity of epithelial tissues rely on the capacity of such tissues to eliminate cells without affecting their sealing. This is achieved by epithelial cell extrusion - a well-orchestrated series of remodelling steps involving the eliminated cell and its neighbours - which ensures the constant maintenance of mechanical and chemical barrier properties while allowing cell expulsion. In this Cell Science at a Glance and the accompanying poster, we describe the remodelling steps that take place within dying or extruding cells, as well as neighbouring cells, outlining the commonalities and variations between tissues and organisms. These steps include reorganization of the cytoskeleton and remodelling of cell-cell junctions that alters their contribution to mechanical coupling and mechanotransduction. We also discuss larger-scale coordination between cells and the contribution of cell extrusion to tissue morphogenesis, epithelial surveillance mechanisms, and pathologies such as cancer and chronic inflammation. Altogether, we outline the complexity and plasticity of this minimalist morphogenetic process.</p>","PeriodicalId":15227,"journal":{"name":"Journal of cell science","volume":"138 8","pages":""},"PeriodicalIF":3.3,"publicationDate":"2025-04-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143997632","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"Med12 cooperates with multiple differentiation signals to facilitate efficient lineage transitions in embryonic stem cells.","authors":"Max Fernkorn, Christian Schröter","doi":"10.1242/jcs.263794","DOIUrl":"10.1242/jcs.263794","url":null,"abstract":"<p><p>Cell differentiation results from coordinated changes in gene transcription in response to combinations of signals. Fibroblast growth factor (FGF), Wnt and mammalian target of rapamycin (mTOR) signals regulate the differentiation of pluripotent mammalian cells towards embryonic and extraembryonic lineages, but how these signals cooperate with general transcriptional regulators is not fully resolved. Here, we report a genome-wide CRISPR screen that reveals both signaling components and general transcriptional regulators for differentiation-associated gene expression in mouse embryonic stem cells (mESCs). Focusing on the Mediator subunit-encoding Med12 gene as one of the strongest hits in the screen, we show that it regulates gene expression in parallel to FGF and mTOR signals. Loss of Med12 is compatible with differentiation along both the embryonic epiblast and the extraembryonic primitive endoderm lineage but impairs pluripotency gene expression and slows down transitions between pluripotency states. These findings suggest that Med12 helps pluripotent cells to efficiently execute transcriptional changes during differentiation, thereby modulating the effects of a broad range of signals.</p>","PeriodicalId":15227,"journal":{"name":"Journal of cell science","volume":"138 8","pages":""},"PeriodicalIF":3.3,"publicationDate":"2025-04-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12079664/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143965817","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"In memoriam - Angelika A. Noegel.","authors":"Annette Müller-Taubenberger, Ludwig Eichinger","doi":"10.1242/jcs.264049","DOIUrl":"https://doi.org/10.1242/jcs.264049","url":null,"abstract":"","PeriodicalId":15227,"journal":{"name":"Journal of cell science","volume":"138 8","pages":""},"PeriodicalIF":3.3,"publicationDate":"2025-04-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144007344","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Ya Gao, Vikas A Tillu, Yeping Wu, James Rae, Thomas E Hall, Kai-En Chen, Saroja Weeratunga, Qian Guo, Emma Livingstone, Wai-Hong Tham, Robert G Parton, Brett M Collins
{"title":"Nanobodies against Cavin1 reveal structural flexibility and regulated interactions of its N-terminal coiled-coil domain.","authors":"Ya Gao, Vikas A Tillu, Yeping Wu, James Rae, Thomas E Hall, Kai-En Chen, Saroja Weeratunga, Qian Guo, Emma Livingstone, Wai-Hong Tham, Robert G Parton, Brett M Collins","doi":"10.1242/jcs.263756","DOIUrl":"10.1242/jcs.263756","url":null,"abstract":"<p><p>Caveolae are abundant plasma membrane structures that regulate signalling, membrane homeostasis and mechanoprotection. Their formation is driven by caveolins and cavins and their coordinated interactions with lipids. Here, we developed nanobodies against the trimeric HR1 coiled-coil domain of Cavin1. We identified specific nanobodies that do not perturb Cavin1 membrane binding and localise to caveolae when expressed in cells. The crystal structure of a nanobody-Cavin 1 HR1 complex reveals a symmetric 3:3 architecture as validated by mutagenesis. In this structure, the C-terminal half of the HR1 domain is disordered, suggesting that the nanobody stabilises an open conformation of Cavin1, which has previously been identified as important for membrane interactions. A phosphomimic mutation in a threonine-serine pair proximal to this region reveals selective regulation of Cavin2 and Cavin3 association. These studies provide new insights into cavin domains required for assembly of multiprotein caveolar assemblies and describe new nanobody tools for structural and functional studies of caveolae.</p>","PeriodicalId":15227,"journal":{"name":"Journal of cell science","volume":"138 8","pages":""},"PeriodicalIF":3.3,"publicationDate":"2025-04-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12079668/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144020206","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Ilka Budde, André Schlichting, David Ing, Sandra Schimmelpfennig, Anna Kuntze, Benedikt Fels, Joelle M-J Romac, Sandip M Swain, Rodger A Liddle, Angela Stevens, Albrecht Schwab, Zoltán Pethő
{"title":"Piezo1-induced durotaxis of pancreatic stellate cells depends on TRPC1 and TRPV4 channels.","authors":"Ilka Budde, André Schlichting, David Ing, Sandra Schimmelpfennig, Anna Kuntze, Benedikt Fels, Joelle M-J Romac, Sandip M Swain, Rodger A Liddle, Angela Stevens, Albrecht Schwab, Zoltán Pethő","doi":"10.1242/jcs.263846","DOIUrl":"10.1242/jcs.263846","url":null,"abstract":"<p><p>Pancreatic stellate cells (PSCs) are primarily responsible for producing the stiff tumor tissue in pancreatic ductal adenocarcinoma (PDAC). Thereby, PSCs generate a stiffness gradient between the healthy pancreas and the tumor. This gradient induces durotaxis, a form of directional cell migration driven by differential stiffness. However, the molecular sensors behind durotaxis are still unclear. To investigate the role of mechanosensitive ion channels in PSC durotaxis, we established a two-dimensional stiffness gradient mimicking PDAC. Using pharmacological and genetic methods, we investigated the contribution of the ion channels Piezo1, TRPC1 and TRPV4 in PSC durotaxis. We found that PSC migration towards a stiffer substrate is diminished by altering Piezo1 activity. Moreover, disrupting TRPC1 along with TRPV4 abolishes PSC durotaxis even when Piezo1 is functional. Our results demonstrate that optimal PSC durotaxis requires an intermediary level of ion channel activity, which we simulated via a numerically discretized mathematical model. These findings suggest that mechanosensitive Piezo1 channels detect the differential stiffness microenvironment. The resulting intracellular signals are amplified by TRPV4 and TRPC1 channels to guide efficient PSC durotaxis.</p>","PeriodicalId":15227,"journal":{"name":"Journal of cell science","volume":" ","pages":""},"PeriodicalIF":3.3,"publicationDate":"2025-04-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12136172/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143523619","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Yuki Ishii, Jessica C Orr, Marie-Belle El Mdawar, Denise R Bairros de Pilger, David R Pearce, Kyren A Lazarus, Rebecca E Graham, Marko Z Nikolić, Robin Ketteler, Neil O Carragher, Sam M Janes, Robert E Hynds
{"title":"Compound screening in human airway basal cells identifies Wnt pathway activators as potential pro-regenerative therapies.","authors":"Yuki Ishii, Jessica C Orr, Marie-Belle El Mdawar, Denise R Bairros de Pilger, David R Pearce, Kyren A Lazarus, Rebecca E Graham, Marko Z Nikolić, Robin Ketteler, Neil O Carragher, Sam M Janes, Robert E Hynds","doi":"10.1242/jcs.263487","DOIUrl":"10.1242/jcs.263487","url":null,"abstract":"<p><p>Regeneration of the airway epithelium restores barrier function and mucociliary clearance following lung injury and infection. The mechanisms regulating the proliferation and differentiation of tissue-resident airway basal stem cells remain incompletely understood. To identify compounds that promote human airway basal cell proliferation, we performed phenotype-based compound screening of 1429 compounds (from the ENZO and Prestwick Chemical libraries) in 384-well format using primary cells transduced with lentiviral luciferase. A total of 17 pro-proliferative compounds were validated in independent donor cell cultures, including the antiretroviral therapy agent abacavir and several Wnt signalling pathway-activating compounds. The effects of compounds on proliferation were further explored in colony formation and 3D organoid assays. Structurally and functionally related compounds that more potently induced Wnt pathway activation were investigated. One such compound, 1-azakenpaullone, induced Wnt target gene activation and basal cell proliferation in mice. Our results demonstrate the pro-proliferative effect of small-molecule Wnt pathway activators on airway basal cells. These findings contribute to the rationale to develop novel approaches to modulate Wnt signalling during airway epithelial repair.</p>","PeriodicalId":15227,"journal":{"name":"Journal of cell science","volume":" ","pages":""},"PeriodicalIF":3.3,"publicationDate":"2025-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12045047/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143597050","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Prashun Acharya, Garima Thapa, Xiayi Liao, Samaneh Matoo, Maura J Graves, Sarah Y Atallah, Ashna K Tipirneni, Tram Nguyen, Niki M Chhabra, Jaden Maschack, Mackenzie R Herod, Favour A Ohaezu, Alder Robison, Ashwini Mudaliyar, Jasvinder Bharaj, Nicole Roeser, Katherine Holmes, Vishwaas Nayak, Rayah Alsayed, Benjamin J Perrin, Scott W Crawley
{"title":"Select autosomal dominant DFNA11 deafness variants activate Myo7A targeting in epithelial cells.","authors":"Prashun Acharya, Garima Thapa, Xiayi Liao, Samaneh Matoo, Maura J Graves, Sarah Y Atallah, Ashna K Tipirneni, Tram Nguyen, Niki M Chhabra, Jaden Maschack, Mackenzie R Herod, Favour A Ohaezu, Alder Robison, Ashwini Mudaliyar, Jasvinder Bharaj, Nicole Roeser, Katherine Holmes, Vishwaas Nayak, Rayah Alsayed, Benjamin J Perrin, Scott W Crawley","doi":"10.1242/jcs.263982","DOIUrl":"10.1242/jcs.263982","url":null,"abstract":"<p><p>Myosin-7A (Myo7A) is a motor protein crucial for the organization and function of stereocilia, specialized actin-rich protrusions on the surface of inner ear hair cells that mediate hearing. Variants in Myo7A cause several forms of genetic hearing loss, including autosomal dominant DFNA11 deafness. Despite its importance, the structural elements that control Myo7A within cells are not well understood. In this study, we used cultured kidney epithelial cells to screen for mutations that activate the motor-dependent targeting of Myo7A to the tips of apical microvilli on these cells. Our findings reveal that the targeting of Myo7A is regulated by specific IQ motifs within its lever arm and that this regulation can function at least partially independent of its tail sequence. Importantly, we demonstrate that many of the DFNA11 deafness variants reported in patients activate Myo7A targeting, providing a potential explanation for the autosomal dominant genetics of this form of deafness.</p>","PeriodicalId":15227,"journal":{"name":"Journal of cell science","volume":" ","pages":""},"PeriodicalIF":3.3,"publicationDate":"2025-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12045598/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143663541","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Mary Fesenko, Daniel J Moore, Peyton Ewbank, Elizabeth Courthold, Stephen J Royle
{"title":"ATG9A vesicles are a subtype of intracellular nanovesicle.","authors":"Mary Fesenko, Daniel J Moore, Peyton Ewbank, Elizabeth Courthold, Stephen J Royle","doi":"10.1242/jcs.263852","DOIUrl":"10.1242/jcs.263852","url":null,"abstract":"<p><p>Cells are filled with thousands of vesicles, which mediate protein transport and ensure homeostasis of the endomembrane system. Distinguishing these vesicles functionally and molecularly represents a major challenge. Intracellular nanovesicles (INVs) are a large class of transport vesicles that likely comprise multiple subtypes. Here, we define the INV proteome and find that it is molecularly heterogeneous and enriched for transmembrane cargo molecules, including integrins, transporters and ATG9A, a lipid scramblase associated with autophagy. ATG9A is known to reside in 'ATG9A vesicles' - small vesicles that contribute to autophagosome formation. Here, using in-cell vesicle capture assays, we found that ATG9A, as well as other ATG9A vesicle cargoes, are in INVs. Quantitative analysis showed that virtually all ATG9A vesicles are INVs, but that only ∼20% of INVs are ATG9A vesicles, suggesting that ATG9A vesicles are in fact a subtype of INV, which we term ATG9A-flavor INVs. Finally, we show that perturbing ATG9A-flavor INVs impairs the autophagy response induced by starvation.</p>","PeriodicalId":15227,"journal":{"name":"Journal of cell science","volume":" ","pages":""},"PeriodicalIF":3.3,"publicationDate":"2025-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12045599/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143605067","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"Announcing the JCS-David Stephens Prize and the 2024 winner Anja Konietzny.","authors":"Michael Way","doi":"10.1242/jcs.263973","DOIUrl":"https://doi.org/10.1242/jcs.263973","url":null,"abstract":"","PeriodicalId":15227,"journal":{"name":"Journal of cell science","volume":"138 7","pages":""},"PeriodicalIF":3.3,"publicationDate":"2025-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143780133","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}