Pub Date : 2024-09-13DOI: 10.1101/2024.09.13.612812
Syeda Kubra, Michelle Sun, William Dion, Ahmet Catak, Hannah Luong, Haokun Wang, Yinghong Pan, Jia-Jun Liu, Aishwarya Ponna, Ian Sipula, Michael J Jurczak, Silvia Liu, Bokai Zhu
Proteostasis is vital for cellular health, with disruptions leading to pathologies including aging, neurodegeneration and metabolic disorders. Traditionally, proteotoxic stress responses were studied as acute reactions to various noxious factors; however, recent evidence reveals that many proteostasis stress-response genes exhibit ~12-hour ultradian rhythms under physiological conditions in mammals. These rhythms, driven by an XBP1s-dependent 12h oscillator, are crucial for managing proteostasis. By exploring the chromatin landscape of the murine 12h hepatic oscillator, we identified RBBP5, a key subunit of the COMPASS complex writing H3K4me3, as an essential epigenetic regulator of proteostasis. RBBP5 is indispensable for regulating both the hepatic 12h oscillator and transcriptional response to acute proteotoxic stress, acting as a co-activator for proteostasis transcription factor XBP1s. RBBP5 ablation leads to increased sensitivity to proteotoxic stress, chronic inflammation, and hepatic steatosis in mice, along with impaired autophagy and reduced cell survival in vitro. In humans, lower RBBP5 expression is associated with reduced adaptive stress-response gene expression and hepatic steatosis. Our findings establish RBBP5 as a central regulator of proteostasis, essential for maintaining mammalian organismal health.
{"title":"Epigenetic regulation of global proteostasis dynamics by RBBP5 ensures mammalian organismal health","authors":"Syeda Kubra, Michelle Sun, William Dion, Ahmet Catak, Hannah Luong, Haokun Wang, Yinghong Pan, Jia-Jun Liu, Aishwarya Ponna, Ian Sipula, Michael J Jurczak, Silvia Liu, Bokai Zhu","doi":"10.1101/2024.09.13.612812","DOIUrl":"https://doi.org/10.1101/2024.09.13.612812","url":null,"abstract":"Proteostasis is vital for cellular health, with disruptions leading to pathologies including aging, neurodegeneration and metabolic disorders. Traditionally, proteotoxic stress responses were studied as acute reactions to various noxious factors; however, recent evidence reveals that many proteostasis stress-response genes exhibit ~12-hour ultradian rhythms under physiological conditions in mammals. These rhythms, driven by an XBP1s-dependent 12h oscillator, are crucial for managing proteostasis. By exploring the chromatin landscape of the murine 12h hepatic oscillator, we identified RBBP5, a key subunit of the COMPASS complex writing H3K4me3, as an essential epigenetic regulator of proteostasis. RBBP5 is indispensable for regulating both the hepatic 12h oscillator and transcriptional response to acute proteotoxic stress, acting as a co-activator for proteostasis transcription factor XBP1s. RBBP5 ablation leads to increased sensitivity to proteotoxic stress, chronic inflammation, and hepatic steatosis in mice, along with impaired autophagy and reduced cell survival in vitro. In humans, lower RBBP5 expression is associated with reduced adaptive stress-response gene expression and hepatic steatosis. Our findings establish RBBP5 as a central regulator of proteostasis, essential for maintaining mammalian organismal health.","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":"142189923","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: 10.1101/2024.09.13.612863
Corbin J Renken, Susie Kim, Youjun Wu, Marc Hammarlund, Shaul Yogev
Neurons rely on local protein synthesis to rapidly modify the proteome of neurites distant from the cell body. A prerequisite for local protein synthesis is the presence of ribosomes in the neurite, but the mechanisms of ribosome transport in neurons remain poorly defined. Here, we find that ribosomes hitchhike on mitochondria for their delivery to the dendrite of a sensory neuron in C. elegans. Ribosomes co-transport with dendritic mitochondria, and their association requires the atypical Rho GTPase MIRO-1. Disrupting mitochondrial transport prevents ribosomes from reaching the dendrite, whereas ectopic re-localization of mitochondria results in a concomitant re-localization of ribosomes, demonstrating that mitochondria are required and sufficient for instructing ribosome distribution in dendrites. Endolysosomal organelles that are involved in mRNA transport and translation can associate with mitochondria and ribosomes but do not play a significant role in ribosome transport. These results reveal a mechanism for dendritic ribosome delivery, which is a critical upstream requirement for local protein synthesis.
{"title":"Cytoplasmic ribosomes hitchhike on mitochondria to dendrites","authors":"Corbin J Renken, Susie Kim, Youjun Wu, Marc Hammarlund, Shaul Yogev","doi":"10.1101/2024.09.13.612863","DOIUrl":"https://doi.org/10.1101/2024.09.13.612863","url":null,"abstract":"Neurons rely on local protein synthesis to rapidly modify the proteome of neurites distant from the cell body. A prerequisite for local protein synthesis is the presence of ribosomes in the neurite, but the mechanisms of ribosome transport in neurons remain poorly defined. Here, we find that ribosomes hitchhike on mitochondria for their delivery to the dendrite of a sensory neuron in C. elegans. Ribosomes co-transport with dendritic mitochondria, and their association requires the atypical Rho GTPase MIRO-1. Disrupting mitochondrial transport prevents ribosomes from reaching the dendrite, whereas ectopic re-localization of mitochondria results in a concomitant re-localization of ribosomes, demonstrating that mitochondria are required and sufficient for instructing ribosome distribution in dendrites. Endolysosomal organelles that are involved in mRNA transport and translation can associate with mitochondria and ribosomes but do not play a significant role in ribosome transport. These results reveal a mechanism for dendritic ribosome delivery, which is a critical upstream requirement for local protein synthesis.","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":"142189926","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 : 2024-09-12DOI: 10.1101/2024.09.05.611401
William Toubiana, Zoe Dumas, Marie Delattre, Tanja Schwander
In eukaryotes, the cellular processes contributing to gamete formation form the building blocks of genetic inheritance across generations. While traditionally viewed as conserved across model organisms, emerging studies reveal significant variation among eukaryotes in meiotic and post-meiotic processes. Extending our knowledge to non-model organisms is therefore critical to improve our understanding of the evolutionary origin, significance and subsequent modifications associated with gamete formation. We describe the mechanisms underlying recombination, chromosome segregation, and meiotic sex chromosome inactivation in the stick insect group Timema. Our results reveal that in males, 1) recombination initiates before synapsis, 2) the mechanisms of X inactivation are variable among species and the X remains silenced despite two waves of transcriptional activation in autosomes during spermatogenesis, 3) chromosome segregation is preceded by the recruitment of centromere proteins that either bind locally or longitudinally along the chromosomes. Together, our observations help understand the evolutionary significance of key cellular processes related to spermatogenesis and shed light on the diversity of their underlying mechanisms among species, including Timema.
在真核生物中，配子形成的细胞过程构成了跨代遗传的基石。虽然传统上认为模式生物之间的配子形成过程是保守的，但新的研究显示，真核生物之间的减数分裂和减数分裂后过程存在显著差异。因此，将我们的知识扩展到非模式生物，对于提高我们对配子形成的进化起源、意义和后续修饰的理解至关重要。我们描述了粘虫类群蒂玛虫的重组、染色体分离和减数分裂性染色体失活的基本机制。我们的研究结果表明，在雄性个体中，1）重组在突触之前就开始了；2）X 失活的机制在不同物种之间存在差异，尽管在精子形成过程中常染色体发生了两波转录激活，但 X 仍然保持沉默；3）染色体分离之前需要招募中心粒蛋白，这些蛋白或沿染色体局部结合，或沿染色体纵向结合。我们的观察结果有助于理解与精子发生有关的关键细胞过程的进化意义，并揭示了包括蒂默马在内的不同物种之间精子发生机制的多样性。
{"title":"Dynamics of recombination, X inactivation and centromere proteins during stick insect spermatogenesis","authors":"William Toubiana, Zoe Dumas, Marie Delattre, Tanja Schwander","doi":"10.1101/2024.09.05.611401","DOIUrl":"https://doi.org/10.1101/2024.09.05.611401","url":null,"abstract":"In eukaryotes, the cellular processes contributing to gamete formation form the building blocks of genetic inheritance across generations. While traditionally viewed as conserved across model organisms, emerging studies reveal significant variation among eukaryotes in meiotic and post-meiotic processes. Extending our knowledge to non-model organisms is therefore critical to improve our understanding of the evolutionary origin, significance and subsequent modifications associated with gamete formation. We describe the mechanisms underlying recombination, chromosome segregation, and meiotic sex chromosome inactivation in the stick insect group Timema. Our results reveal that in males, 1) recombination initiates before synapsis, 2) the mechanisms of X inactivation are variable among species and the X remains silenced despite two waves of transcriptional activation in autosomes during spermatogenesis, 3) chromosome segregation is preceded by the recruitment of centromere proteins that either bind locally or longitudinally along the chromosomes. Together, our observations help understand the evolutionary significance of key cellular processes related to spermatogenesis and shed light on the diversity of their underlying mechanisms among species, including Timema.","PeriodicalId":501590,"journal":{"name":"bioRxiv - Cell Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142189934","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 : 2024-09-12DOI: 10.1101/2024.09.03.610974
Qianman Peng, Kulandaisamy Arulsamy, Yao Wei Lu, Hao Wu, Bo Zhu, Bandana Singh, Kui Cui, Jill Wylie-Sears, Kathryn S. Li, Scott Wong, Douglas B. Cowan, Masanori Aikawa, Da-Zhi Wang, Joyce Bischoff, Kaifu Chen, Hong Chen
Background: Protein-tyrosine-phosphatase CD45 is exclusively expressed in all nucleated cells of the hematopoietic system but is rarely expressed in endothelial cells. Interestingly, our recent study indicated that activation of the endogenous CD45 promoter in human endothelial colony forming cells (ECFCs) induced expression of multiple EndoMT marker genes. However, the detailed molecular mechanisms underlying CD45 that drive EndoMT and the therapeutic potential of manipulation of CD45 expression in atherosclerosis are entirely unknown. Method: We generated a tamoxifen-inducible EC-specific CD45 deficient mouse strain (EC-iCD45KO) in an ApoE-deficient (ApoE-/-) background and fed with a Western diet (C57BL/6) for atherosclerosis and molecular analyses. We isolated and enriched mouse aortic endothelial cells with CD31 beads to perform single-cell RNA sequencing. Biomedical, cellular, and molecular approaches were utilized to investigate the role of endothelial CD45-specific deletion in the prevention of EndoMT in ApoE-/- model of atherosclerosis. Results: Single-cell RNA sequencing revealed that loss of endothelial CD45 inhibits EndoMT marker expression and transforming growth factor-β signaling in atherosclerotic mice. which is associated with the reductions of lesions in the ApoE-/- mouse model. Mechanistically, the loss of endothelial cell CD45 results in increased KLF2 expression, which inhibits transforming growth factor-β signaling and EndoMT. Consistently, endothelial CD45 deficient mice showed reduced lesion development, plaque macrophages, and expression of cell adhesion molecules when compared to ApoE-/- controls. Conclusions: These findings demonstrate that the loss of endothelial CD45 protects against EndoMT-driven atherosclerosis, promoting KLF2 expression while inhibiting TGFβ signaling and EndoMT markers. Thus, targeting endothelial CD45 may be a novel therapeutic strategy for EndoMT and atherosclerosis.
{"title":"Novel Role of Endothelial CD45 in Regulating Endothelial-to-Mesenchymal Transition in Atherosclerosis","authors":"Qianman Peng, Kulandaisamy Arulsamy, Yao Wei Lu, Hao Wu, Bo Zhu, Bandana Singh, Kui Cui, Jill Wylie-Sears, Kathryn S. Li, Scott Wong, Douglas B. Cowan, Masanori Aikawa, Da-Zhi Wang, Joyce Bischoff, Kaifu Chen, Hong Chen","doi":"10.1101/2024.09.03.610974","DOIUrl":"https://doi.org/10.1101/2024.09.03.610974","url":null,"abstract":"Background: Protein-tyrosine-phosphatase CD45 is exclusively expressed in all nucleated cells of the hematopoietic system but is rarely expressed in endothelial cells. Interestingly, our recent study indicated that activation of the endogenous CD45 promoter in human endothelial colony forming cells (ECFCs) induced expression of multiple EndoMT marker genes. However, the detailed molecular mechanisms underlying CD45 that drive EndoMT and the therapeutic potential of manipulation of CD45 expression in atherosclerosis are entirely unknown.\u0000Method: We generated a tamoxifen-inducible EC-specific CD45 deficient mouse strain (EC-iCD45KO) in an ApoE-deficient (ApoE-/-) background and fed with a Western diet (C57BL/6) for atherosclerosis and molecular analyses. We isolated and enriched mouse aortic endothelial cells with CD31 beads to perform single-cell RNA sequencing. Biomedical, cellular, and molecular approaches were utilized to investigate the role of endothelial CD45-specific deletion in the prevention of EndoMT in ApoE-/- model of atherosclerosis.\u0000Results: Single-cell RNA sequencing revealed that loss of endothelial CD45 inhibits EndoMT marker expression and transforming growth factor-β signaling in atherosclerotic mice. which is associated with the reductions of lesions in the ApoE-/- mouse model. Mechanistically, the loss of endothelial cell CD45 results in increased KLF2 expression, which inhibits transforming growth factor-β signaling and EndoMT. Consistently, endothelial CD45 deficient mice showed reduced lesion development, plaque macrophages, and expression of cell adhesion molecules when compared to ApoE-/- controls.\u0000Conclusions: These findings demonstrate that the loss of endothelial CD45 protects against EndoMT-driven atherosclerosis, promoting KLF2 expression while inhibiting TGFβ signaling and EndoMT markers. Thus, targeting endothelial CD45 may be a novel therapeutic strategy for EndoMT and atherosclerosis.","PeriodicalId":501590,"journal":{"name":"bioRxiv - Cell Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142189931","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 : 2024-09-12DOI: 10.1101/2024.09.11.611976
Christopher Lambert, Marius Karger, Anika Steffen, Yubo Tang, Hermann Doering, Theresia E.B. Stradal, Pekka Lappalainen, Jan Faix, Peter Bieling, Klemens Rottner
Dynamic actin filament remodeling is crucial for a plethora of fundamental cell biological processes, ranging from cell division and migration to cell communication, intracellular trafficking or tissue development. Cytochalasin B and -D are fungal secondary metabolites frequently used for interference with such processes. Although generally assumed to block actin filament polymerization at their rapidly growing barbed ends and compete with regulators at these sites, our molecular understanding of their precise effects in dynamic actin structures is scarce. Here we combine live cell imaging and analysis of fluorescent actin-binding protein dynamics with acute treatment of lamellipodia in migrating cells with cytochalasin B. Our results show that in spite of an abrupt halt of lamellipodium protrusion, cytochalasin B affects various actin filament barbed end-binding proteins in a differential fashion. Cytochalasin B enhances instead of diminishes the accumulation of prominent barbed end-binding factors such as Ena/VASP family proteins and heterodimeric capping protein (CP) in the lamellipodium. Similar results were obtained with cytochalasin D. All these effects are highly specific, as cytochalasin-induced VASP accumulation requires the presence of CP, but not vice versa, and coincides with abrogation of both actin and VASP turnover. Cytochalasin B can also increase apparent barbed end interactions with the actin-binding β-tentacle of CP and partially mimic its Arp2/3 complex-promoting activity in the lamellipodium. In conclusion, our results reveal a new spectrum of cytochalasin activities on barbed end-binding factors, with important implications for the interpretation of their effects on dynamic actin structures.
动态肌动蛋白丝重塑对从细胞分裂和迁移到细胞通讯、细胞内贩运或组织发育等大量基本细胞生物学过程至关重要。Cytochalasin B 和 -D 是真菌的次级代谢产物，经常用于干扰此类过程。虽然人们普遍认为细胞松弛素 B 和-D 会在其快速增长的倒钩末端阻断肌动蛋白丝的聚合，并在这些部位与调节剂竞争，但我们对它们在动态肌动蛋白结构中的精确作用的分子认识却很匮乏。在这里，我们将活细胞成像和荧光肌动蛋白结合蛋白动态分析与用细胞松弛素 B 对迁移细胞中的片层突起进行急性处理相结合。细胞松弛素 B 会增强而不是减少薄壁基质中主要的带刺末端结合因子（如 Ena/VASP 家族蛋白和异源二聚体盖层蛋白 (CP)）的积累。所有这些效应都具有高度的特异性，因为细胞松素诱导的 VASP 积累需要 CP 的存在，反之亦然，并且与肌动蛋白和 VASP 的周转减弱同时发生。细胞松弛素 B 还能增加与 CP 的肌动蛋白结合 β 触角的明显倒钩末端相互作用，并部分模拟其在瓣膜中的 Arp2/3 复合物促进活性。总之，我们的研究结果揭示了细胞松弛素对带刺末端结合因子的新的活性谱系，对解释它们对动态肌动蛋白结构的影响具有重要意义。
{"title":"Differential interference with actin-binding protein function by acute Cytochalasin B","authors":"Christopher Lambert, Marius Karger, Anika Steffen, Yubo Tang, Hermann Doering, Theresia E.B. Stradal, Pekka Lappalainen, Jan Faix, Peter Bieling, Klemens Rottner","doi":"10.1101/2024.09.11.611976","DOIUrl":"https://doi.org/10.1101/2024.09.11.611976","url":null,"abstract":"Dynamic actin filament remodeling is crucial for a plethora of fundamental cell biological processes, ranging from cell division and migration to cell communication, intracellular trafficking or tissue development. Cytochalasin B and -D are fungal secondary metabolites frequently used for interference with such processes. Although generally assumed to block actin filament polymerization at their rapidly growing barbed ends and compete with regulators at these sites, our molecular understanding of their precise effects in dynamic actin structures is scarce. Here we combine live cell imaging and analysis of fluorescent actin-binding protein dynamics with acute treatment of lamellipodia in migrating cells with cytochalasin B. Our results show that in spite of an abrupt halt of lamellipodium protrusion, cytochalasin B affects various actin filament barbed end-binding proteins in a differential fashion. Cytochalasin B enhances instead of diminishes the accumulation of prominent barbed end-binding factors such as Ena/VASP family proteins and heterodimeric capping protein (CP) in the lamellipodium. Similar results were obtained with cytochalasin D. All these effects are highly specific, as cytochalasin-induced VASP accumulation requires the presence of CP, but not vice versa, and coincides with abrogation of both actin and VASP turnover. Cytochalasin B can also increase apparent barbed end interactions with the actin-binding β-tentacle of CP and partially mimic its Arp2/3 complex-promoting activity in the lamellipodium. In conclusion, our results reveal a new spectrum of cytochalasin activities on barbed end-binding factors, with important implications for the interpretation of their effects on dynamic actin structures.","PeriodicalId":501590,"journal":{"name":"bioRxiv - Cell Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142189936","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 : 2024-09-12DOI: 10.1101/2024.09.12.612721
Wen Lu, Brad S. Lee, Helen Deng, Margot Lakonishok, Enrique Martin-Blanco, Vladimir I Gelfand
During neuronal development, neurons undergo significant microtubule reorganization to shape axons and dendrites, establishing the framework for efficient wiring of the nervous system. Previous studies from our laboratory demonstrated the key role of kinesin-1 in driving microtubule-microtubule sliding, which provides the mechanical forces necessary for early axon outgrowth and regeneration in Drosophila melanogaster. In this study, we reveal the critical role of kinesin-5, a mitotic motor, in modulating the development of postmitotic neurons. Kinesin-5, a conserved homotetrameric motor, typically functions in mitosis by sliding antiparallel microtubules apart in the spindle. Here, we demonstrate that the Drosophila kinesin-5 homolog, Klp61F, is expressed in larval brain neurons, with high levels in ventral nerve cord (VNC) neurons. Knockdown of Klp61F using a pan-neuronal driver leads to severe locomotion defects and complete lethality in adult flies, mainly due to the absence of kinesin-5 in VNC motor neurons during early larval development. Klp61F depletion results in significant axon growth defects, both in cultured and in vivo neurons. By imaging individual microtubules, we observe a significant increase in microtubule motility, and excessive penetration of microtubules into the axon growth cone in Klp61F-depleted neurons. Adult lethality and axon growth defects are fully rescued by a chimeric human-Drosophila kinesin-5 motor, which accumulates at the axon tips, suggesting a conserved role of kinesin-5 in neuronal development. Altogether, our findings show that at the growth cone, kinesin-5 acts as a brake on kinesin-1-driven microtubule sliding, preventing premature microtubule entry into the growth cone. This regulatory role of kinesin-5 is essential for precise axon pathfinding during nervous system development.
{"title":"'Mitotic' kinesin-5 is a dynamic brake for axonal growth","authors":"Wen Lu, Brad S. Lee, Helen Deng, Margot Lakonishok, Enrique Martin-Blanco, Vladimir I Gelfand","doi":"10.1101/2024.09.12.612721","DOIUrl":"https://doi.org/10.1101/2024.09.12.612721","url":null,"abstract":"During neuronal development, neurons undergo significant microtubule reorganization to shape axons and dendrites, establishing the framework for efficient wiring of the nervous system. Previous studies from our laboratory demonstrated the key role of kinesin-1 in driving microtubule-microtubule sliding, which provides the mechanical forces necessary for early axon outgrowth and regeneration in Drosophila melanogaster. In this study, we reveal the critical role of kinesin-5, a mitotic motor, in modulating the development of postmitotic neurons.\u0000Kinesin-5, a conserved homotetrameric motor, typically functions in mitosis by sliding antiparallel microtubules apart in the spindle. Here, we demonstrate that the Drosophila kinesin-5 homolog, Klp61F, is expressed in larval brain neurons, with high levels in ventral nerve cord (VNC) neurons. Knockdown of Klp61F using a pan-neuronal driver leads to severe locomotion defects and complete lethality in adult flies, mainly due to the absence of kinesin-5 in VNC motor neurons during early larval development. Klp61F depletion results in significant axon growth defects, both in cultured and in vivo neurons. By imaging individual microtubules, we observe a significant increase in microtubule motility, and excessive penetration of microtubules into the axon growth cone in Klp61F-depleted neurons. Adult lethality and axon growth defects are fully rescued by a chimeric human-Drosophila kinesin-5 motor, which accumulates at the axon tips, suggesting a conserved role of kinesin-5 in neuronal development.\u0000Altogether, our findings show that at the growth cone, kinesin-5 acts as a brake on kinesin-1-driven microtubule sliding, preventing premature microtubule entry into the growth cone. This regulatory role of kinesin-5 is essential for precise axon pathfinding during nervous system development.","PeriodicalId":501590,"journal":{"name":"bioRxiv - Cell Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142189948","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 : 2024-09-12DOI: 10.1101/2024.09.11.612195
Sandy Peltier, Mickael Marin, Monika Dzieciatkowska, Michael Dussiot, Micaela Roy, Johanna Bruce, Louise Leblanc, Youcef Hadjou, Sonia Georgeault, Aurelie Fricot, Camille Roussel, Daniel Stephenson, Madeleine Casimir, Abdoulaye Sissoko, Francois Paye, Safi Dokmak, Papa Alioune Ndour, Philippe Roingeard, Emilie-Fleur Gautier, Steven Spitalnik, Olivier Hermine, Pierre A Buffet, Angelo D'Alessandro, Pascal Amireault
Although refrigerated storage slows the metabolism of volunteer donor RBCs, cellular aging still occurs throughout this in vitro process, which is essential in transfusion medicine. Storage-induced microerythrocytes (SMEs) are morphologically-altered senescent RBCs that accumulate during storage and which are cleared from circulation following transfusion. However, the molecular and cellular alterations that trigger clearance of this RBC subset remain to be identified. Using a staining protocol that sorts long-stored SMEs (i.e., CFSEhigh) and morphologically-normal RBCs (CFSElow), these in vitro aged cells were characterized. Metabolomics analysis identified depletion of energy, lipid-repair, and antioxidant metabolites in CFSEhigh RBCs. By redox proteomics, irreversible protein oxidation primarily affected CFSEhigh RBCs. By proteomics, 96 proteins, mostly in the proteostasis family, had relocated to CFSEhigh RBC membranes. CFSEhigh RBCs exhibited decreased proteasome activity and deformability; increased phosphatidylserine exposure, osmotic fragility, and endothelial cell adherence; and were cleared from the circulation during human spleen ex vivo perfusion. Conversely, molecular, cellular, and circulatory properties of long-stored CFSElow RBCs resembled those of short-stored RBCs. CFSEhigh RBCs are morphologically and metabolically altered, have irreversibly oxidized and membrane-relocated proteins, and exhibit decreased proteasome activity. In vitro aging during storage selectively alters metabolism and proteostasis in SMEs, targeting these senescent cells for clearance.
{"title":"Proteostasis and metabolic dysfunction in a distinct subset of storage-induced senescent erythrocytes targeted for clearance","authors":"Sandy Peltier, Mickael Marin, Monika Dzieciatkowska, Michael Dussiot, Micaela Roy, Johanna Bruce, Louise Leblanc, Youcef Hadjou, Sonia Georgeault, Aurelie Fricot, Camille Roussel, Daniel Stephenson, Madeleine Casimir, Abdoulaye Sissoko, Francois Paye, Safi Dokmak, Papa Alioune Ndour, Philippe Roingeard, Emilie-Fleur Gautier, Steven Spitalnik, Olivier Hermine, Pierre A Buffet, Angelo D'Alessandro, Pascal Amireault","doi":"10.1101/2024.09.11.612195","DOIUrl":"https://doi.org/10.1101/2024.09.11.612195","url":null,"abstract":"Although refrigerated storage slows the metabolism of volunteer donor RBCs, cellular aging still occurs throughout this in vitro process, which is essential in transfusion medicine. Storage-induced microerythrocytes (SMEs) are morphologically-altered senescent RBCs that accumulate during storage and which are cleared from circulation following transfusion. However, the molecular and cellular alterations that trigger clearance of this RBC subset remain to be identified. Using a staining protocol that sorts long-stored SMEs (i.e., CFSEhigh) and morphologically-normal RBCs (CFSElow), these in vitro aged cells were characterized.\u0000Metabolomics analysis identified depletion of energy, lipid-repair, and antioxidant metabolites in CFSEhigh RBCs. By redox proteomics, irreversible protein oxidation primarily affected CFSEhigh RBCs. By proteomics, 96 proteins, mostly in the proteostasis family, had relocated to CFSEhigh RBC membranes. CFSEhigh RBCs exhibited decreased proteasome activity and deformability; increased phosphatidylserine exposure, osmotic fragility, and endothelial cell adherence; and were cleared from the circulation during human spleen ex vivo perfusion. Conversely, molecular, cellular, and circulatory properties of long-stored CFSElow RBCs resembled those of short-stored RBCs. CFSEhigh RBCs are morphologically and metabolically altered, have irreversibly oxidized and membrane-relocated proteins, and exhibit decreased proteasome activity. In vitro aging during storage selectively alters metabolism and proteostasis in SMEs, targeting these senescent cells for clearance.","PeriodicalId":501590,"journal":{"name":"bioRxiv - Cell Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142189949","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 : 2024-09-12DOI: 10.1101/2024.09.12.611851
Josie A Christopher, Lisa M Breckels, Oliver M Crook, Mercedes Vazquez-Chantada, Derek Barratt, Kathryn Susan Lilley
Cells have many protective mechanisms against background levels of ionizing radiation (IR) orchestrated by molecular changes in expression, post-translation modifications and subcellular localization. Radiotherapeutic treatment in oncology attempts to overwhelm such mechanisms, but radio-resistance is an ongoing challenge. Here, global subcellular proteomics combined with Bayesian modelling identified 544 differentially localized proteins in A549 cells upon 6 Gy x-ray exposure, revealing subcellular-specific changes of proteins involved in ferroptosis, an iron-dependent cell death, suggestive of potential radio-resistance mechanisms. These observations were independent of expression changes, emphasizing the utility of global subcellular proteomics and the promising prospect of ferroptosis-inducing therapies for combatting radioresistance.
细胞通过表达、翻译后修饰和亚细胞定位的分子变化，对本底水平的电离辐射（IR）具有多种保护机制。肿瘤学中的放射治疗试图压制这些机制，但放射抗性是一个持续的挑战。在这里，全局亚细胞蛋白质组学与贝叶斯建模相结合，确定了 A549 细胞在 6 Gy X 射线照射下 544 个不同定位的蛋白质，揭示了参与铁凋亡（一种依赖铁的细胞死亡）的蛋白质的亚细胞特异性变化，提示了潜在的放射抗性机制。这些观察结果与表达变化无关，强调了全局亚细胞蛋白质组学的实用性，以及诱导铁蛋白沉积以对抗放射抗性的疗法的广阔前景。
{"title":"Global proteomics indicates subcellular-specific anti-ferroptotic responses to ionizing radiation","authors":"Josie A Christopher, Lisa M Breckels, Oliver M Crook, Mercedes Vazquez-Chantada, Derek Barratt, Kathryn Susan Lilley","doi":"10.1101/2024.09.12.611851","DOIUrl":"https://doi.org/10.1101/2024.09.12.611851","url":null,"abstract":"Cells have many protective mechanisms against background levels of ionizing radiation (IR) orchestrated by molecular changes in expression, post-translation modifications and subcellular localization. Radiotherapeutic treatment in oncology attempts to overwhelm such mechanisms, but radio-resistance is an ongoing challenge. Here, global subcellular proteomics combined with Bayesian modelling identified 544 differentially localized proteins in A549 cells upon 6 Gy x-ray exposure, revealing subcellular-specific changes of proteins involved in ferroptosis, an iron-dependent cell death, suggestive of potential radio-resistance mechanisms. These observations were independent of expression changes, emphasizing the utility of global subcellular proteomics and the promising prospect of ferroptosis-inducing therapies for combatting radioresistance.","PeriodicalId":501590,"journal":{"name":"bioRxiv - Cell Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142224503","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 : 2024-09-12DOI: 10.1101/2024.09.12.612654
Karen KL Yee, Nobukatsu Morooka, Takashi Sato
In acute inflammation, macrophages polarises its phenotype in order to participate effectively in the inflammatory, anti-inflammatory and resolving phases. Particularly, the resolving phase is vital for homeostatic recovery. The in vivo murine peritonitis model had identified various subtypes of resolving macrophages. However, the in vivo model has limitations in deciphering the molecular mechanisms required for resolving macrophage polarisation. Therefore the aim of this study is to establish an in vitro model that could simplify the reproduction of resolving macrophage polarisation. This model will be a useful tool to screen for molecular mechanisms essential for triggering resolution. Our in vitro model showed Raw 264.7 cells exhibited classical inflammatory-like (M1-like) phenotype between 2-24 h with increased interleukin-1β expression and tumour necrosis factor-α secretion. Concurrently, at 22-24 h there was an increase in Raw 264.7 cells polarising to anti-inflammatory like (M2-like) phenotype. These M2-like macrophages were increased in arginase activity and interleukin-10 expression. By 48 h, Raw 264.7 cells were polarised to resolving-like (Mres-like/CD11b low) phenotype. These macrophages were characterised by high efferocytic index and a decrease in inflammatory cytokine expression, low arginase activity and low CD11b expression. In summary, this in vitro resolution model showed resolving-like polarisation in a macrophage cell line.
{"title":"An in vitro modelling of resolving macrophage with Raw 264.7 macrophage cell line","authors":"Karen KL Yee, Nobukatsu Morooka, Takashi Sato","doi":"10.1101/2024.09.12.612654","DOIUrl":"https://doi.org/10.1101/2024.09.12.612654","url":null,"abstract":"In acute inflammation, macrophages polarises its phenotype in order to participate effectively in the inflammatory, anti-inflammatory and resolving phases. Particularly, the resolving phase is vital for homeostatic recovery. The in vivo murine peritonitis model had identified various subtypes of resolving macrophages. However, the in vivo model has limitations in deciphering the molecular mechanisms required for resolving macrophage polarisation. Therefore the aim of this study is to establish an in vitro model that could simplify the reproduction of resolving macrophage polarisation. This model will be a useful tool to screen for molecular mechanisms essential for triggering resolution. Our in vitro model showed Raw 264.7 cells exhibited classical inflammatory-like (M1-like) phenotype between 2-24 h with increased interleukin-1β expression and tumour necrosis factor-α secretion. Concurrently, at 22-24 h there was an increase in Raw 264.7 cells polarising to anti-inflammatory like (M2-like) phenotype. These M2-like macrophages were increased in arginase activity and interleukin-10 expression. By 48 h, Raw 264.7 cells were polarised to resolving-like (Mres-like/CD11b low) phenotype. These macrophages were characterised by high efferocytic index and a decrease in inflammatory cytokine expression, low arginase activity and low CD11b expression. In summary, this in vitro resolution model showed resolving-like polarisation in a macrophage cell line.","PeriodicalId":501590,"journal":{"name":"bioRxiv - Cell Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142189929","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 : 2024-09-12DOI: 10.1101/2024.09.11.612431
Korbinian Traeuble, Matthias Munz, Jessica Pauli, Nadja Sachs, Eshan Vafadarnejad, Tania Carrillo-Roa, Lars Maegdefessel, Peter Kastner, Matthias Heinig
Atherosclerosis, a major cause of cardiovascular diseases, is characterized by the buildup of lipids and chronic inflammation in the arteries, leading to plaque formation and potential rupture. The underlying causal immune mechanisms and alterations in structural cell composition and plasticity driving plaque progression remain incompletely defined. Recent advances in single-cell transcriptomics (scRNA-seq) have provided deeper insights into the roles of immune and non-immune cells in atherosclerosis. However, existing public scRNA-seq datasets often lack comprehensive cell type coverage and consistent annotations, posing challenges for downstream analyses. In this study, we present an integrated single-cell atlas of human atherosclerotic plaques, encompassing 261,747 high-quality annotated cells from carotid, coronary, and femoral arteries. By benchmarking and applying the best-performing data integration method, scPoli, we achieved robust cell type annotations validated by expert consensus and surface protein measurements. This comprehensive atlas enables accurate automatic cell type annotation of new datasets, optimal experimental design, and deconvolution of existing as well as novel bulk RNA-seq data to comprehensively determine cell type proportions in human atherosclerotic lesions. It facilitates future studies by providing an interactive WebUI for easy data annotation and experimental design, while supporting various downstream applications, including integration of genetic association studies and experimental planning.
{"title":"Integrated single-cell atlas of human atherosclerotic plaques","authors":"Korbinian Traeuble, Matthias Munz, Jessica Pauli, Nadja Sachs, Eshan Vafadarnejad, Tania Carrillo-Roa, Lars Maegdefessel, Peter Kastner, Matthias Heinig","doi":"10.1101/2024.09.11.612431","DOIUrl":"https://doi.org/10.1101/2024.09.11.612431","url":null,"abstract":"Atherosclerosis, a major cause of cardiovascular diseases, is characterized by the buildup of lipids and chronic inflammation in the arteries, leading to plaque formation and potential rupture. The underlying causal immune mechanisms and alterations in structural cell composition and plasticity driving plaque progression remain incompletely defined. Recent advances in single-cell transcriptomics (scRNA-seq) have provided deeper insights into the roles of immune and non-immune cells in atherosclerosis. However, existing public scRNA-seq datasets often lack comprehensive cell type coverage and consistent annotations, posing challenges for downstream analyses. In this study, we present an integrated single-cell atlas of human atherosclerotic plaques, encompassing 261,747 high-quality annotated cells from carotid, coronary, and femoral arteries. By benchmarking and applying the best-performing data integration method, scPoli, we achieved robust cell type annotations validated by expert consensus and surface protein measurements. This comprehensive atlas enables accurate automatic cell type annotation of new datasets, optimal experimental design, and deconvolution of existing as well as novel bulk RNA-seq data to comprehensively determine cell type proportions in human atherosclerotic lesions. It facilitates future studies by providing an interactive WebUI for easy data annotation and experimental design, while supporting various downstream applications, including integration of genetic association studies and experimental planning.","PeriodicalId":501590,"journal":{"name":"bioRxiv - Cell Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142189950","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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