Pub Date : 2024-09-15DOI: 10.1101/2024.09.15.613099
Shae M Milne, Philip T T Edeen, David S Fay
Membrane trafficking is a conserved process required for the movement and distribution of proteins and other macromolecules within cells. The Caenorhabditis elegans NIMA-related kinases NEKL-2 (human NEK8/9) and NEKL-3 (human NEK6/7) are conserved regulators of membrane trafficking and are required for the completion of molting. We used a genetic approach to identify reduction-of-function mutations in tat-1 that suppress nekl-associated molting defects. tat-1 encodes the C. elegans ortholog of mammalian ATP8A1/2, a phosphatidylserine (PS) flippase that promotes the asymmetric distribution of PS to the cytosolic leaflet of lipid membrane bilayers. CHAT-1 (human CDC50), a conserved chaperone, was required for the correct localization of TAT-1, and chat-1 inhibition strongly suppressed nekl defects. Using a PS sensor, we found that TAT-1 was required for the normal localization of PS at apical endosomes and that loss of TAT-1 led to aberrant endosomal morphologies. Consistent with this, TAT-1 localized to early endosomes and to recycling endosomes marked with RME-1, the C. elegans ortholog of the human EPS15 homology (EH) domain-containing protein, EHD1. TAT-1, PS biosynthesis, and the PS-binding protein RFIP-2 (human RAB11-FIP2) were all required for the normal localization of RME-1 to apical endosomes. Consistent with these proteins functioning together, inhibition of RFIP-2 or RME-1 led to the partial suppression of nekl molting defects, as did the inhibition of PS biosynthesis. Using the auxin-inducible degron system, we found that depletion of NEKL-2 or NEKL-3 led to defects in RME-1 localization and that a reduction in TAT-1 function partially restored RME-1 localization in NEKL-3-depleted cells.
{"title":"TAT-1, a phosphatidylserine flippase, affects molting and regulates membrane trafficking in the epidermis of C. elegans","authors":"Shae M Milne, Philip T T Edeen, David S Fay","doi":"10.1101/2024.09.15.613099","DOIUrl":"https://doi.org/10.1101/2024.09.15.613099","url":null,"abstract":"Membrane trafficking is a conserved process required for the movement and distribution of proteins and other macromolecules within cells. The Caenorhabditis elegans NIMA-related kinases NEKL-2 (human NEK8/9) and NEKL-3 (human NEK6/7) are conserved regulators of membrane trafficking and are required for the completion of molting. We used a genetic approach to identify reduction-of-function mutations in tat-1 that suppress nekl-associated molting defects. tat-1 encodes the C. elegans ortholog of mammalian ATP8A1/2, a phosphatidylserine (PS) flippase that promotes the asymmetric distribution of PS to the cytosolic leaflet of lipid membrane bilayers. CHAT-1 (human CDC50), a conserved chaperone, was required for the correct localization of TAT-1, and chat-1 inhibition strongly suppressed nekl defects. Using a PS sensor, we found that TAT-1 was required for the normal localization of PS at apical endosomes and that loss of TAT-1 led to aberrant endosomal morphologies. Consistent with this, TAT-1 localized to early endosomes and to recycling endosomes marked with RME-1, the C. elegans ortholog of the human EPS15 homology (EH) domain-containing protein, EHD1. TAT-1, PS biosynthesis, and the PS-binding protein RFIP-2 (human RAB11-FIP2) were all required for the normal localization of RME-1 to apical endosomes. Consistent with these proteins functioning together, inhibition of RFIP-2 or RME-1 led to the partial suppression of nekl molting defects, as did the inhibition of PS biosynthesis. Using the auxin-inducible degron system, we found that depletion of NEKL-2 or NEKL-3 led to defects in RME-1 localization and that a reduction in TAT-1 function partially restored RME-1 localization in NEKL-3-depleted cells.","PeriodicalId":501590,"journal":{"name":"bioRxiv - Cell Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142265139","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-15DOI: 10.1101/2024.09.15.613115
Yu Deng, Tatsat Banerjee, Dhiman Sankar Pal, Parijat Banerjee, Huiwang David Zhan, Jane Borleis, Pablo A Iglesias, Peter Devreotes
Symmetry breaking, polarity establishment, and spontaneous cell protrusion formation are fundamental but poorly explained cell behaviors. Here, we demonstrate that a biochemical network, where the mutually inhibitory localization of PIP5K and Ras activities plays a central role, governs these processes. First, in resting cells devoid of cytoskeletal activity, PIP5K is uniformly elevated on the plasma membrane, while Ras activity remains minimal. Symmetry is broken by spontaneous local displacements of PIP5K, coupled with simultaneous activations of Ras and downstream signaling events, including PI3K activation. Second, knockout of PIP5K dramatically increases both the incidence and size of Ras-PI3K activation patches, accompanied by branched F-actin assembly. This leads to enhanced cortical wave formation, increased protrusive activity, and a shift in migration mode. Third, high inducible overexpression of PIP5K virtually eliminates Ras-PI3K signaling, cytoskeletal activity, and cell migration, while acute recruitment of cytosolic PIP5K to the membrane induces contraction and blebs in cancer cells. These arrested phenotypes are reversed by reducing myosin II activity, indicating myosin II involvement in the PIP5K-Ras-centered regulatory network. Remarkably, low inducible overexpression of PIP5K unexpectedly facilitates polarity establishment, highlighting PIP5K as a highly sensitive master regulator of these processes. Simulations of a computational model combining an excitable system, cytoskeletal loops, and dynamic partitioning of PIP5K recreates the experimental observations. Taken together, our results reveal that a bistable, mutually exclusive localization of PIP5K and active Ras on the plasma membrane triggers the initial symmetry breaking. Coupled actomyosin reduction and increased actin polymerization lead to intermittently extended protrusions and, with feedback from the cytoskeleton, self-organizing, complementary gradients of PIP5K versus Ras steepen, raising the threshold of the networks at the rear and lowering it at the front to generate polarity for cell migration.
{"title":"PIP5K-Ras bistability initiates plasma membrane symmetry breaking to regulate cell polarity and migration","authors":"Yu Deng, Tatsat Banerjee, Dhiman Sankar Pal, Parijat Banerjee, Huiwang David Zhan, Jane Borleis, Pablo A Iglesias, Peter Devreotes","doi":"10.1101/2024.09.15.613115","DOIUrl":"https://doi.org/10.1101/2024.09.15.613115","url":null,"abstract":"Symmetry breaking, polarity establishment, and spontaneous cell protrusion formation are fundamental but poorly explained cell behaviors. Here, we demonstrate that a biochemical network, where the mutually inhibitory localization of PIP5K and Ras activities plays a central role, governs these processes. First, in resting cells devoid of cytoskeletal activity, PIP5K is uniformly elevated on the plasma membrane, while Ras activity remains minimal. Symmetry is broken by spontaneous local displacements of PIP5K, coupled with simultaneous activations of Ras and downstream signaling events, including PI3K activation. Second, knockout of PIP5K dramatically increases both the incidence and size of Ras-PI3K activation patches, accompanied by branched F-actin assembly. This leads to enhanced cortical wave formation, increased protrusive activity, and a shift in migration mode. Third, high inducible overexpression of PIP5K virtually eliminates Ras-PI3K signaling, cytoskeletal activity, and cell migration, while acute recruitment of cytosolic PIP5K to the membrane induces contraction and blebs in cancer cells. These arrested phenotypes are reversed by reducing myosin II activity, indicating myosin II involvement in the PIP5K-Ras-centered regulatory network. Remarkably, low inducible overexpression of PIP5K unexpectedly facilitates polarity establishment, highlighting PIP5K as a highly sensitive master regulator of these processes. Simulations of a computational model combining an excitable system, cytoskeletal loops, and dynamic partitioning of PIP5K recreates the experimental observations. Taken together, our results reveal that a bistable, mutually exclusive localization of PIP5K and active Ras on the plasma membrane triggers the initial symmetry breaking. Coupled actomyosin reduction and increased actin polymerization lead to intermittently extended protrusions and, with feedback from the cytoskeleton, self-organizing, complementary gradients of PIP5K versus Ras steepen, raising the threshold of the networks at the rear and lowering it at the front to generate polarity for cell migration.","PeriodicalId":501590,"journal":{"name":"bioRxiv - Cell Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142265102","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-15DOI: 10.1101/2024.09.14.613064
Seungwon Yang, Anais Aulas, Paul J Anderson, Pavel Ivanov
Stress granules (SGs) are dynamic cytoplasmic structures assembled in response to various stress stimuli that enhance cell survival under adverse environmental conditions. Here we show that SGs contribute to breast cancer progression by enhancing the survival of cells subjected to anoikis stress. SG assembly is triggered by inhibition of Focal Adhesion Kinase (FAK) or loss of adhesion signals. Combined proteomic analysis and functional studies reveal that SG formation enhances cancer cell proliferation, resistance to metabolic stress, anoikis resistance, and migration. Importantly, inhibiting SG formation promotes the sensitivity of cancer cells to FAK inhibitors being developed as cancer therapeutics. Furthermore, we identify the Rho-ROCK-PERK-eIF2α axis as a critical signaling pathway activated by loss of adhesion signals and inhibition of the FAK-mTOR-eIF4F complex in breast cancer cells. By triggering SG assembly and AKT activation in response to anoikis stress, PERK functions as an oncoprotein in breast cancer cells. Overall, our study highlights the significance of SG formation in breast cancer progression and suggests that therapeutic inhibition of SG assembly may reverse anoikis resistance in treatment-resistant cancers such as triple-negative breast cancer (TNBC).
{"title":"Stress granule formation enables anchorage-independence survival in cancer cells","authors":"Seungwon Yang, Anais Aulas, Paul J Anderson, Pavel Ivanov","doi":"10.1101/2024.09.14.613064","DOIUrl":"https://doi.org/10.1101/2024.09.14.613064","url":null,"abstract":"Stress granules (SGs) are dynamic cytoplasmic structures assembled in response to various stress stimuli that enhance cell survival under adverse environmental conditions. Here we show that SGs contribute to breast cancer progression by enhancing the survival of cells subjected to anoikis stress. SG assembly is triggered by inhibition of Focal Adhesion Kinase (FAK) or loss of adhesion signals. Combined proteomic analysis and functional studies reveal that SG formation enhances cancer cell proliferation, resistance to metabolic stress, anoikis resistance, and migration. Importantly, inhibiting SG formation promotes the sensitivity of cancer cells to FAK inhibitors being developed as cancer therapeutics. Furthermore, we identify the Rho-ROCK-PERK-eIF2α axis as a critical signaling pathway activated by loss of adhesion signals and inhibition of the FAK-mTOR-eIF4F complex in breast cancer cells. By triggering SG assembly and AKT activation in response to anoikis stress, PERK functions as an oncoprotein in breast cancer cells. Overall, our study highlights the significance of SG formation in breast cancer progression and suggests that therapeutic inhibition of SG assembly may reverse anoikis resistance in treatment-resistant cancers such as triple-negative breast cancer (TNBC).","PeriodicalId":501590,"journal":{"name":"bioRxiv - Cell Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142265140","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-14DOI: 10.1101/2024.09.13.612878
Tal Levin, Hector Garcia-Seisdedos, Arseniy Lobov, Matthias Wojtynek, Alexander Alexandrov, Ghil Jona, Dikla Levi, Ohad Medalia, Emmanuel D Levy
The formation of large polymeric structures such as cytoskeletal and enzyme filaments is crucial for normal cellular function. However, such filaments can also form due to mutations that create self-interactions at the surface of symmetric proteins. Often, the proteins forming these structures maintain a folded state and thereby differ from aggregates and amyloids that involve misfolding. We refer to this type of assemblies as agglomerates to mark this difference. While cells have quality control mechanisms to identify, buffer, and eliminate misfolded proteins, it is unclear whether similar mechanisms exist for agglomerates, or whether agglomerates are toxic to cells. Here, we profiled the physiological impact of mutation-induced folded-state protein filamentation in yeast cells. First, we devised a simple strategy to distinguish fluorescently labeled proteins forming agglomerates versus aggregates. We then profiled exogenous protein agglomerates in terms of their recognition by known quality control mechanisms, their effects on specific cellular processes and overall fitness on S. cerevisiae cultures. We found that agglomerates do not colocalize with the proteostasis machinery and do not result in measurable fitness defects. Proteomics profiling of cells expressing the wild type protein, agglomerating or misfolded variants revealed a consistent picture, with only minor, agglomerate-size-dependent changes observed and linked to the cell-wall and plasma-membrane proteins. Overall, our findings indicate that agglomerates form mostly benign structures in cells when compared to aggregates, and thereby offer a promising route for synthetic biology applications.
{"title":"Profiling the physiological impact of aberrant folded-state protein filamentation in cells","authors":"Tal Levin, Hector Garcia-Seisdedos, Arseniy Lobov, Matthias Wojtynek, Alexander Alexandrov, Ghil Jona, Dikla Levi, Ohad Medalia, Emmanuel D Levy","doi":"10.1101/2024.09.13.612878","DOIUrl":"https://doi.org/10.1101/2024.09.13.612878","url":null,"abstract":"The formation of large polymeric structures such as cytoskeletal and enzyme filaments is crucial for normal cellular function. However, such filaments can also form due to mutations that create self-interactions at the surface of symmetric proteins. Often, the proteins forming these structures maintain a folded state and thereby differ from aggregates and amyloids that involve misfolding. We refer to this type of assemblies as agglomerates to mark this difference. While cells have quality control mechanisms to identify, buffer, and eliminate misfolded proteins, it is unclear whether similar mechanisms exist for agglomerates, or whether agglomerates are toxic to cells. Here, we profiled the physiological impact of mutation-induced folded-state protein filamentation in yeast cells. First, we devised a simple strategy to distinguish fluorescently labeled proteins forming agglomerates versus aggregates. We then profiled exogenous protein agglomerates in terms of their recognition by known quality control mechanisms, their effects on specific cellular processes and overall fitness on S. cerevisiae cultures. We found that agglomerates do not colocalize with the proteostasis machinery and do not result in measurable fitness defects. Proteomics profiling of cells expressing the wild type protein, agglomerating or misfolded variants revealed a consistent picture, with only minor, agglomerate-size-dependent changes observed and linked to the cell-wall and plasma-membrane proteins. Overall, our findings indicate that agglomerates form mostly benign structures in cells when compared to aggregates, and thereby offer a promising route for synthetic biology applications.","PeriodicalId":501590,"journal":{"name":"bioRxiv - Cell Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142265144","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-14DOI: 10.1101/2024.09.12.612614
Deepa Ajnar, Ananya Sarkar, Seema Riyaz, Poornima Menon, Dipranil Dutta, Pulkit Asati, Priyanka Sharma, Sai P Pydi, Suresh Kumar
Autophagy Conjugation machinery forms a center piece of autophagy and is essential for sequestration of a broad range of cargo destined for degradation. Apart from its role in canonical autophagy, recent evidence suggests an unconventional role of conjugation machinery. Membrane Atg8ylation is one of the manifestations of autophagy, wherein ATG8 conjugation machinery recruit mammalian ATG8s (mATG8s) to the damaged membranes for repair or removal. Herein, we show that SARS-CoV-2 factor ORF3a induces membrane Atg8ylation and selectively inflicts lysophagy, a cellular response to evade apoptotic cell death. mATG8s and SNARE protein syntaxin 17 (STX17) interact with ORF3a and are required for Atg8ylation induced by ORF3a. ORF3a displaces mTOR from the lysosomes and affects nuclear translocation of TFEB, which is dependent on mATG8s and STX17. Despite mTOR inhibition, its conventional target ULK1 is dispensable for ORF3a induced Atg8ylation. In addition, mATG8s and STX17 protected against the cell death induced by ORF3a. Overall, our findings demonstrate ORF3a induced lysosomal membrane Atg8ylation while identifying the unexpected role of STX17 in Atg8ylation.
{"title":"SARS-CoV-2 protein ORF3a induces Atg8ylation of lysosomal membranes","authors":"Deepa Ajnar, Ananya Sarkar, Seema Riyaz, Poornima Menon, Dipranil Dutta, Pulkit Asati, Priyanka Sharma, Sai P Pydi, Suresh Kumar","doi":"10.1101/2024.09.12.612614","DOIUrl":"https://doi.org/10.1101/2024.09.12.612614","url":null,"abstract":"Autophagy Conjugation machinery forms a center piece of autophagy and is essential for sequestration of a broad range of cargo destined for degradation. Apart from its role in canonical autophagy, recent evidence suggests an unconventional role of conjugation machinery. Membrane Atg8ylation is one of the manifestations of autophagy, wherein ATG8 conjugation machinery recruit mammalian ATG8s (mATG8s) to the damaged membranes for repair or removal. Herein, we show that SARS-CoV-2 factor ORF3a induces membrane Atg8ylation and selectively inflicts lysophagy, a cellular response to evade apoptotic cell death. mATG8s and SNARE protein syntaxin 17 (STX17) interact with ORF3a and are required for Atg8ylation induced by ORF3a. ORF3a displaces mTOR from the lysosomes and affects nuclear translocation of TFEB, which is dependent on mATG8s and STX17. Despite mTOR inhibition, its conventional target ULK1 is dispensable for ORF3a induced Atg8ylation. In addition, mATG8s and STX17 protected against the cell death induced by ORF3a. Overall, our findings demonstrate ORF3a induced lysosomal membrane Atg8ylation while identifying the unexpected role of STX17 in Atg8ylation.","PeriodicalId":501590,"journal":{"name":"bioRxiv - Cell Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142265141","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-14DOI: 10.1101/2024.09.13.612932
Jinsha Koroth, Ismael Y Karkache, Elizabeth K Vu, Kim C Mansky, Elizabeth W Bradley
Disruptions in the bone remodeling cycle that occur with increasing age lead to degeneration of the skeleton and increased risk of fragility fractures. Our understanding of how the bone remodeling process within cortical bone is controlled and altered with age in males and females is limited. Here, we generated bone marrow chimeric mice to understand the impacts of age and sex on the bone remodeling process. We demonstrate that transplantation of aged male or female bone marrow into young lethally irradiated male hosts unexpectedly enhances cortical bone mass without an impacting cancellous bone. Our single cell RNA-sequencing data show that mice reconstituted with aged bone marrow exhibited subsets of cells marked by CD11B/CD36 expression that demonstrate enhanced production of anabolic cytokines as compared to young counterparts, and that these myeloid subsets exist under conditions of normal physiology in aged mice. Importantly, CD11B+CD36+ cells do not differentiate into osteoclasts in vitro, and CD36 does not mark TRAP+ cells in vivo. Instead, CD36+ cells localize to resorption sites, including within cortical bone defects, suggesting their involvement in cortical bone remodeling and healing. CD11B+CD36+ cells also express elevated levels of bone anabolic WNT ligands, especially Wnt6. In functional assays, we demonstrate that soluble factors produced by CD11B+CD36+ cells enhance osteoblast progenitor commitment, mineralization, and activation of WNT signaling in vitro. Moreover, CD11B/CD36 exquisitely mark a subset of anabolic myeloid cells within human bone marrow. In conclusion, our studies identified a novel population of aged macrophages that limit cortical bone loss.
{"title":"CD11B+CD36+ cells are bone anabolic macrophages that limit age-associated bone loss","authors":"Jinsha Koroth, Ismael Y Karkache, Elizabeth K Vu, Kim C Mansky, Elizabeth W Bradley","doi":"10.1101/2024.09.13.612932","DOIUrl":"https://doi.org/10.1101/2024.09.13.612932","url":null,"abstract":"Disruptions in the bone remodeling cycle that occur with increasing age lead to degeneration of the skeleton and increased risk of fragility fractures. Our understanding of how the bone remodeling process within cortical bone is controlled and altered with age in males and females is limited. Here, we generated bone marrow chimeric mice to understand the impacts of age and sex on the bone remodeling process. We demonstrate that transplantation of aged male or female bone marrow into young lethally irradiated male hosts unexpectedly enhances cortical bone mass without an impacting cancellous bone. Our single cell RNA-sequencing data show that mice reconstituted with aged bone marrow exhibited subsets of cells marked by CD11B/CD36 expression that demonstrate enhanced production of anabolic cytokines as compared to young counterparts, and that these myeloid subsets exist under conditions of normal physiology in aged mice. Importantly, CD11B+CD36+ cells do not differentiate into osteoclasts in vitro, and CD36 does not mark TRAP+ cells in vivo. Instead, CD36+ cells localize to resorption sites, including within cortical bone defects, suggesting their involvement in cortical bone remodeling and healing. CD11B+CD36+ cells also express elevated levels of bone anabolic WNT ligands, especially Wnt6. In functional assays, we demonstrate that soluble factors produced by CD11B+CD36+ cells enhance osteoblast progenitor commitment, mineralization, and activation of WNT signaling in vitro. Moreover, CD11B/CD36 exquisitely mark a subset of anabolic myeloid cells within human bone marrow. In conclusion, our studies identified a novel population of aged macrophages that limit cortical bone loss.","PeriodicalId":501590,"journal":{"name":"bioRxiv - Cell Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142265142","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-14DOI: 10.1101/2024.09.13.612962
Daniel T. Schneider, Eduard I. Dedkov
Introduction: Myocardial CD34+ stromal cells/telocytes (SC/TCs) have been recently recognized as a novel resident cell which may play an important role in the repair process following acute myocardial infarction (MI). This study aims to determine the spatiotemporal dynamics of CD34+ SC/TCs within the left ventricular (LV) wall during the late inflammatory and proliferative phases of post-MI scar formation. Methods: A large transmural MI was induced in middle-aged, Sprague-Dawley rats by permanent ligation of the left anterior descending coronary artery. To recognize proliferating cells, rats were infused with 5-bromo-2'-deoxyuridine (BrdU) in a dose of 12.5 mg/kg/day for 72 hours via intraperitoneal osmotic minipumps on day 0, 4, or 11 after surgery. The rats were euthanized on day 3, 7 and 14 after MI, and their hearts were processed for histology and immunostaining. Results: Three days after MI, CD34+ SC/TCs were absent within the necrotic myocardial tissue but were visible around the surviving cardiac myocytes (CMs) bordering the infarcted region, including those remaining in subepicardial and subendocardial regions, and in the adventitia of residual coronary vessels. Seven days after MI, many of the CD34+ SC/TCs located at the periphery of the developing scar appeared enlarged and contained the BrdU labeling, indicating the cell proliferation. At the same time, elongated CD34+ SC/TCs, which lacked BrdU labeling, were noticed closer to the necrotic zone residing in the interstitial areas between the intact basement membranes left from resorbed CMs, suggesting their migratory activity. Fourteen days after MI, CD34+ SC/TCs were distributed throughout the entire post-infarcted region except for the areas occupied by necrotic tissue, myofibroblast-rich granulation tissue, and the fibroelastic thickenings of the endocardium affected by an MI. Furthermore, accumulated clusters of flattened CD34+ SC/TCs cells were apparent in the areas where the edges of surviving CMs extend into the fibrotic portion of the scar. Conclusion: These findings, for the first time, demonstrate that a population of myocardial CD34+ SC/TCs follow a dynamic pattern of spatiotemporal distribution within the healing myocardium suggesting their direct involvement in post-MI repair process and scar formation.
{"title":"CD34+ Stromal Cell/Telocytes Demonstrate a Dynamic Pattern of Distribution During Healing of Post-Infarcted Myocardium in Middle-Aged Sprague-Dawley Rats","authors":"Daniel T. Schneider, Eduard I. Dedkov","doi":"10.1101/2024.09.13.612962","DOIUrl":"https://doi.org/10.1101/2024.09.13.612962","url":null,"abstract":"<strong>Introduction:</strong> Myocardial CD34+ stromal cells/telocytes (SC/TCs) have been recently recognized as a novel resident cell which may play an important role in the repair process following acute myocardial infarction (MI). This study aims to determine the spatiotemporal dynamics of CD34+ SC/TCs within the left ventricular (LV) wall during the late inflammatory and proliferative phases of post-MI scar formation. <strong>Methods:</strong> A large transmural MI was induced in middle-aged, Sprague-Dawley rats by permanent ligation of the left anterior descending coronary artery. To recognize proliferating cells, rats were infused with 5-bromo-2'-deoxyuridine (BrdU) in a dose of 12.5 mg/kg/day for 72 hours via intraperitoneal osmotic minipumps on day 0, 4, or 11 after surgery. The rats were euthanized on day 3, 7 and 14 after MI, and their hearts were processed for histology and immunostaining. <strong>Results:</strong> Three days after MI, CD34+ SC/TCs were absent within the necrotic myocardial tissue but were visible around the surviving cardiac myocytes (CMs) bordering the infarcted region, including those remaining in subepicardial and subendocardial regions, and in the adventitia of residual coronary vessels. Seven days after MI, many of the CD34+ SC/TCs located at the periphery of the developing scar appeared enlarged and contained the BrdU labeling, indicating the cell proliferation. At the same time, elongated CD34+ SC/TCs, which lacked BrdU labeling, were noticed closer to the necrotic zone residing in the interstitial areas between the intact basement membranes left from resorbed CMs, suggesting their migratory activity. Fourteen days after MI, CD34+ SC/TCs were distributed throughout the entire post-infarcted region except for the areas occupied by necrotic tissue, myofibroblast-rich granulation tissue, and the fibroelastic thickenings of the endocardium affected by an MI. Furthermore, accumulated clusters of flattened CD34+ SC/TCs cells were apparent in the areas where the edges of surviving CMs extend into the fibrotic portion of the scar. <strong>Conclusion:</strong> These findings, for the first time, demonstrate that a population of myocardial CD34+ SC/TCs follow a dynamic pattern of spatiotemporal distribution within the healing myocardium suggesting their direct involvement in post-MI repair process and scar formation.","PeriodicalId":501590,"journal":{"name":"bioRxiv - Cell Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142265143","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-13DOI: 10.1101/2024.09.12.612747
Mari Minekawa, Atsushi Suzuki
The dynamic regulation of neuronal polarity is essential for establishing neural networks during brain development. The primary culture of rodent neurons recapitulates several aspects of this polarity regulation and thus provides powerful tools for revealing the cellular and molecular mechanisms underlying axon specification and neuronal migration. However, little is known about how preexisting bipolarity breaks to form multipolar dendrites. Here, we demonstrated that the Golgi-associated, microtubule (MT) cross-linking protein MTCL2 plays an essential role in this type of polarity change observed in the differentiation of cerebellar granule neurons (CGNs). MTCL2 is highly expressed in CGNs and exhibited gradual accumulation in dendrites in parallel to their polarity development. MTCL2 depletion resulted in the generation of longer and fewer dendrites by suppressing the bipolar-to-multipolar transition of dendrite extension observed in the normal polarization process. During this process, the Golgi apparatus changes its localization from the base of the preexisting bipolar neurites to the lateral or apical side of the nucleus, where it associates closely with the MT cage wrapping the nucleus. The resulting upward extension of the Golgi apparatus is tightly coupled with randomization of its position in x-y plane. Knockdown/rescue experiments demonstrated that MTCL2 promotes these changes in Golgi position in an MT- and Golgi-binding activity-dependent manner. These results suggest that MTCL2 promotes the development of multipolar short dendrites by sequestering the Golgi apparatus from the base of preexisting neurites, enabling its random movements around nuclei.
{"title":"The Golgi-associated microtubule cross-linking protein MTCL2 promotes the multipolar extension of dendrites in cerebellar granule neurons.","authors":"Mari Minekawa, Atsushi Suzuki","doi":"10.1101/2024.09.12.612747","DOIUrl":"https://doi.org/10.1101/2024.09.12.612747","url":null,"abstract":"The dynamic regulation of neuronal polarity is essential for establishing neural networks during brain development. The primary culture of rodent neurons recapitulates several aspects of this polarity regulation and thus provides powerful tools for revealing the cellular and molecular mechanisms underlying axon specification and neuronal migration. However, little is known about how preexisting bipolarity breaks to form multipolar dendrites. Here, we demonstrated that the Golgi-associated, microtubule (MT) cross-linking protein MTCL2 plays an essential role in this type of polarity change observed in the differentiation of cerebellar granule neurons (CGNs). MTCL2 is highly expressed in CGNs and exhibited gradual accumulation in dendrites in parallel to their polarity development. MTCL2 depletion resulted in the generation of longer and fewer dendrites by suppressing the bipolar-to-multipolar transition of dendrite extension observed in the normal polarization process. During this process, the Golgi apparatus changes its localization from the base of the preexisting bipolar neurites to the lateral or apical side of the nucleus, where it associates closely with the MT cage wrapping the nucleus. The resulting upward extension of the Golgi apparatus is tightly coupled with randomization of its position in x-y plane. Knockdown/rescue experiments demonstrated that MTCL2 promotes these changes in Golgi position in an MT- and Golgi-binding activity-dependent manner. These results suggest that MTCL2 promotes the development of multipolar short dendrites by sequestering the Golgi apparatus from the base of preexisting neurites, enabling its random movements around nuclei.","PeriodicalId":501590,"journal":{"name":"bioRxiv - Cell Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142189932","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-13DOI: 10.1101/2024.09.12.612751
James W Swann, Ruiyuan Zhang, Evgenia V Verovskaya, Fernando J Calero-Nieto, Xiaonan Wang, Melissa A Proven, Peter T Shyu, Edward Guo, Berthold Gottgens, Emmanuelle Passegue
Hematopoietic stem and progenitor cells (HSPC) are regulated by interactions with stromal cells in the bone marrow (BM) cavity, which can be segregated into two spatially defined central marrow (CM) and endosteal (Endo) compartments. However, the importance of this spatial compartmentalization for BM responses to inflammation and neoplasia remains largely unknown. Here, we extensively validate a combination of scRNA-seq profiling and matching flow cytometry isolation that reproducibly identifies 7 key CM and Endo populations across mouse strains and accurately surveys both niche locations. We demonstrate that different perturbations exert specific effects on different compartments, with type I interferon responses causing CM mesenchymal stromal cells to adopt an inflammatory phenotype associated with overproduction of chemokines modulating local monocyte dynamics in the surrounding microenvironment. Our results provide a comprehensive method for molecular and functional stromal characterization and highlight the importance of altered stomal cell activity in regulating hematopoietic responses to inflammatory challenges.
造血干细胞和祖细胞（HSPC）通过与骨髓（BM）腔中的基质细胞相互作用而受到调控，骨髓（BM）腔在空间上可分为骨髓中央（CM）和骨膜内（Endo）两个区室。然而，这种空间分区对于骨髓对炎症和肿瘤反应的重要性在很大程度上仍不为人所知。在这里，我们广泛验证了 scRNA-seq 图谱分析和匹配流式细胞术分离的组合，它能在不同小鼠品系中重复鉴定 7 个关键的 CM 和内膜群体，并准确调查这两个壁龛位置。我们证明，不同的扰动会对不同的分区产生特定的影响，I型干扰素反应会导致CM间充质基质细胞出现炎症表型，与此同时，趋化因子的过度分泌会调节周围微环境中局部单核细胞的动态变化。我们的研究结果为基质的分子和功能特征描述提供了一种全面的方法，并强调了改变的气孔细胞活性在调节造血对炎症挑战的反应中的重要性。
{"title":"Inflammation perturbs hematopoiesis by remodeling specific compartments of the bone marrow niche","authors":"James W Swann, Ruiyuan Zhang, Evgenia V Verovskaya, Fernando J Calero-Nieto, Xiaonan Wang, Melissa A Proven, Peter T Shyu, Edward Guo, Berthold Gottgens, Emmanuelle Passegue","doi":"10.1101/2024.09.12.612751","DOIUrl":"https://doi.org/10.1101/2024.09.12.612751","url":null,"abstract":"Hematopoietic stem and progenitor cells (HSPC) are regulated by interactions with stromal cells in the bone marrow (BM) cavity, which can be segregated into two spatially defined central marrow (CM) and endosteal (Endo) compartments. However, the importance of this spatial compartmentalization for BM responses to inflammation and neoplasia remains largely unknown. Here, we extensively validate a combination of scRNA-seq profiling and matching flow cytometry isolation that reproducibly identifies 7 key CM and Endo populations across mouse strains and accurately surveys both niche locations. We demonstrate that different perturbations exert specific effects on different compartments, with type I interferon responses causing CM mesenchymal stromal cells to adopt an inflammatory phenotype associated with overproduction of chemokines modulating local monocyte dynamics in the surrounding microenvironment. Our results provide a comprehensive method for molecular and functional stromal characterization and highlight the importance of altered stomal cell activity in regulating hematopoietic responses to inflammatory challenges.","PeriodicalId":501590,"journal":{"name":"bioRxiv - Cell Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142189928","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-13DOI: 10.1101/2024.09.11.612435
Michele Weiss-Gayet, Gaetan Juban, Emmeran Le Moal, Antonio Moretta, Camilla Farnetari, Christelle Gobet, Jules Guillemaud, Marie-Catherine Le Bihan, Oded Shoseyov, Annie Adrait, Katharina Ternka, Odile Boespflug-Tanguy, Matthias Kettwig, Yohann Coute, Remi Mounier, Francesco Acquati, Robert Knight, Benedicte Chazaud
Muscle stem cells (MuSCs) fuse to form myofibers to repair skeletal muscle after injury. Within the regenerative MuSC niche, restorative macrophages stimulate MuSC fusion, although the molecular mechanisms involved are largely unknown. Here, we show that restorative macrophages secrete ribonuclease T2 (RNAseT2) to stimulate MuSC fusion. RNAseT2 entered MuSCs via the mannose receptor and induced the formation of actin bundles in MuSCs, enabling cell/cell fusion. Mechanistically, RNAseT2 bound to Ste20-like kinase (SLK), which itself triggered the phosphorylation-mediated activation of N-WASP, through Paxillin phosphorylation, allowing actin bundling necessary for MuSC fusion. In vivo, overexpressing RNAseT2 in regenerating muscle increased fusion in newly formed myofibers in mouse and zebrafish while macrophages deficient for RNAseT2 gene led to fusion defect and smaller myofibers. This study reveals a new function for the highly conserved RNAseT2 and provides a new molecular mechanism by which restorative macrophages support MuSC fusion during muscle repair.
{"title":"Macrophage-derived RNAseT2 stimulates muscle stem cell fusion via SLK/N-WASP/actin bundling","authors":"Michele Weiss-Gayet, Gaetan Juban, Emmeran Le Moal, Antonio Moretta, Camilla Farnetari, Christelle Gobet, Jules Guillemaud, Marie-Catherine Le Bihan, Oded Shoseyov, Annie Adrait, Katharina Ternka, Odile Boespflug-Tanguy, Matthias Kettwig, Yohann Coute, Remi Mounier, Francesco Acquati, Robert Knight, Benedicte Chazaud","doi":"10.1101/2024.09.11.612435","DOIUrl":"https://doi.org/10.1101/2024.09.11.612435","url":null,"abstract":"Muscle stem cells (MuSCs) fuse to form myofibers to repair skeletal muscle after injury. Within the regenerative MuSC niche, restorative macrophages stimulate MuSC fusion, although the molecular mechanisms involved are largely unknown. Here, we show that restorative macrophages secrete ribonuclease T2 (RNAseT2) to stimulate MuSC fusion. RNAseT2 entered MuSCs via the mannose receptor and induced the formation of actin bundles in MuSCs, enabling cell/cell fusion. Mechanistically, RNAseT2 bound to Ste20-like kinase (SLK), which itself triggered the phosphorylation-mediated activation of N-WASP, through Paxillin phosphorylation, allowing actin bundling necessary for MuSC fusion. In vivo, overexpressing RNAseT2 in regenerating muscle increased fusion in newly formed myofibers in mouse and zebrafish while macrophages deficient for RNAseT2 gene led to fusion defect and smaller myofibers. This study reveals a new function for the highly conserved RNAseT2 and provides a new molecular mechanism by which restorative macrophages support MuSC fusion during muscle repair.","PeriodicalId":501590,"journal":{"name":"bioRxiv - Cell Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142189927","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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