Pub Date : 2024-09-10DOI: 10.1101/2024.09.09.612156
Patience O. Obi, Tamiris F. G. Souza, Ying Lao, Kirk J. McManus, Joseph W. Gordon, Richard LeDuc, Rene P. Zahedi, Ayesha Saleem
We have previously shown that skeletal muscle-derived extracellular vesicles (EVs) released post-chronic contractile activity (CCA) increased mitochondrial biogenesis in murine myoblasts, and decreased cell viability and induced apoptosis and senescence in non-small cell lung cancer cells. While the underlying mechanisms are unknown, the effects perpetuated were dependent on membrane-bound proteins. Here, we performed an extensive LC-MS/MS proteomic analysis on EVs from control and CCA myotubes. A total of 2900 proteins were identified in CON-EVs and CCA-EVs, including EV-associated proteins such as TSG101, tetraspanins (CD9, CD81, and CD63), flotillin-1, and annexins. Of these, 856 proteins are novel and not listed in EV databases (ExoCarta and Vesiclepedia), indicating that myotube-EVs harbor proteins not yet identified in EVs of different origin. Additionally, we identified 2062 unique proteins that have not yet been previously reported in myotube-EVs to date. Remarkably, of the 2900 total proteins identified, we observed 46 upregulated, and 25 downregulated differentially expressed proteins (DEPs) in CCA-EVs vs. control-EVs. Most of upregulated DEPs include EV-associated proteins. Comparing the 71 DEPs with proteins expressed in skeletal muscle indicated 61 of these as potential myokines. We identified actin cytoskeleton signaling, integrin signaling and muscle contraction as the most enriched pathways among the DEPs using different databases/software including FunRich, KEGG, STRING and Ingenuity Pathway Analysis. Using a relevance score that prioritized membrane-bound proteins with known function in mitochondrial biogenesis and inhibition of cancer growth, we identified top-scoring highly enriched DEPs of interest: IGF1R, ATP7A, PFN1, GJA1, PRKCA and ITGA6. We confirmed upregulation of these targets in EVs using immunoblotting. Among these top-scoring DEPs, PFN1, and ITGA6 are associated with EVs, with expression upregulated following acute exercise. In summary, we report the first comprehensive analysis of skeletal muscle-EV proteome following CCA, with identification of putative protein targets and signaling pathways that may execute the pro-metabolic and anti-tumorigenic effects of CCA-EVs.
{"title":"Identification of novel myokines and putative protein targets that mediate functional adaptations in response to chronic contractile activity induced skeletal muscle-extracellular vesicle treatment","authors":"Patience O. Obi, Tamiris F. G. Souza, Ying Lao, Kirk J. McManus, Joseph W. Gordon, Richard LeDuc, Rene P. Zahedi, Ayesha Saleem","doi":"10.1101/2024.09.09.612156","DOIUrl":"https://doi.org/10.1101/2024.09.09.612156","url":null,"abstract":"We have previously shown that skeletal muscle-derived extracellular vesicles (EVs) released post-chronic contractile activity (CCA) increased mitochondrial biogenesis in murine myoblasts, and decreased cell viability and induced apoptosis and senescence in non-small cell lung cancer cells. While the underlying mechanisms are unknown, the effects perpetuated were dependent on membrane-bound proteins. Here, we performed an extensive LC-MS/MS proteomic analysis on EVs from control and CCA myotubes. A total of 2900 proteins were identified in CON-EVs and CCA-EVs, including EV-associated proteins such as TSG101, tetraspanins (CD9, CD81, and CD63), flotillin-1, and annexins. Of these, 856 proteins are novel and not listed in EV databases (ExoCarta and Vesiclepedia), indicating that myotube-EVs harbor proteins not yet identified in EVs of different origin. Additionally, we identified 2062 unique proteins that have not yet been previously reported in myotube-EVs to date. Remarkably, of the 2900 total proteins identified, we observed 46 upregulated, and 25 downregulated differentially expressed proteins (DEPs) in CCA-EVs vs. control-EVs. Most of upregulated DEPs include EV-associated proteins. Comparing the 71 DEPs with proteins expressed in skeletal muscle indicated 61 of these as potential myokines. We identified actin cytoskeleton signaling, integrin signaling and muscle contraction as the most enriched pathways among the DEPs using different databases/software including FunRich, KEGG, STRING and Ingenuity Pathway Analysis. Using a relevance score that prioritized membrane-bound proteins with known function in mitochondrial biogenesis and inhibition of cancer growth, we identified top-scoring highly enriched DEPs of interest: IGF1R, ATP7A, PFN1, GJA1, PRKCA and ITGA6. We confirmed upregulation of these targets in EVs using immunoblotting. Among these top-scoring DEPs, PFN1, and ITGA6 are associated with EVs, with expression upregulated following acute exercise. In summary, we report the first comprehensive analysis of skeletal muscle-EV proteome following CCA, with identification of putative protein targets and signaling pathways that may execute the pro-metabolic and anti-tumorigenic effects of CCA-EVs.","PeriodicalId":501590,"journal":{"name":"bioRxiv - Cell Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142189984","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 : 2024-09-10DOI: 10.1101/2024.09.10.612355
Hsiao-Tang Hu, Ueh-Ting Tim Wang, Bi-Chang Chen, Yi-Ping Hsueh, Ting-Fang Wang
A hallmark of eukaryotic species is having distinct numbers of chromosomes surrounded by a nuclear envelope to separate nuclear transcription and RNA processing from cytosolic translation. In animal and plant cells undergoing open mitosis, the nuclear envelope disintegrates before the chromosomes divide into two daughter cells. To ensure faithful genome inheritance, chromosome individualization is involved in partitioning the fully condensed sister chromatids during mitosis. Ki67, a surfactant-like protein that forms a repulsive molecular brush around each pair of fully condensed sister chromatids during early mitosis, mediates chromosome individualization in vertebrates. Using a modified expansion microscopy methodology called TT-ExM for high-sensitivity and super-resolution imaging of proteins, lipids, and nuclear DNA, we found that intranuclear lipids in COS-7 cells not only are enriched at the chromosome periphery but are also excluded from the chromosome interior in a Ki67-dependent manner. Together with nuclear lipids, Ki67 forms the chromosomal envelope that mediates chromosome individualization. The transition from nuclear envelope to chromosomal envelope ensures continued separation of genetic material from cytoplasmic material during open mitosis.
{"title":"Ki67 regulates nuclear lipid accumulation at the chromosomal periphery to facilitate chromosome individualization during mitosis","authors":"Hsiao-Tang Hu, Ueh-Ting Tim Wang, Bi-Chang Chen, Yi-Ping Hsueh, Ting-Fang Wang","doi":"10.1101/2024.09.10.612355","DOIUrl":"https://doi.org/10.1101/2024.09.10.612355","url":null,"abstract":"A hallmark of eukaryotic species is having distinct numbers of chromosomes surrounded by a nuclear envelope to separate nuclear transcription and RNA processing from cytosolic translation. In animal and plant cells undergoing open mitosis, the nuclear envelope disintegrates before the chromosomes divide into two daughter cells. To ensure faithful genome inheritance, chromosome individualization is involved in partitioning the fully condensed sister chromatids during mitosis. Ki67, a surfactant-like protein that forms a repulsive molecular brush around each pair of fully condensed sister chromatids during early mitosis, mediates chromosome individualization in vertebrates. Using a modified expansion microscopy methodology called TT-ExM for high-sensitivity and super-resolution imaging of proteins, lipids, and nuclear DNA, we found that intranuclear lipids in COS-7 cells not only are enriched at the chromosome periphery but are also excluded from the chromosome interior in a Ki67-dependent manner. Together with nuclear lipids, Ki67 forms the chromosomal envelope that mediates chromosome individualization. The transition from nuclear envelope to chromosomal envelope ensures continued separation of genetic material from cytoplasmic material during open mitosis.","PeriodicalId":501590,"journal":{"name":"bioRxiv - Cell Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142189980","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 : 2024-09-09DOI: 10.1101/2024.09.09.612100
Sam Li, Jose-Jesus Fernandez, Marisa D Ruehle, Rachel A. Howard-Till, Amy S Fabritius, Chad G Pearson, David A Agard, Mark Winey
The cilium is a microtubule-based organelle critical for many cellular functions. Its assembly initiates at a basal body and continues as an axoneme that projects out of the cell to form a functional cilium. This assembly process is tightly regulated. However, our knowledge of the molecular architecture and the mechanism of assembly is limited. By applying electron cryo-tomography and subtomogram averaging, we obtained subnanometer resolution structures of the inner junction in three distinct regions of the cilium: the proximal region of the basal body, the central core of the basal body, and the flagellar axoneme. The structures allowed us to identify several basal body and axoneme components. While a few proteins are distributed throughout the entire length of the organelle, many are restricted to particular regions of the cilium, forming intricate local interaction networks and bolstering local structural stability. Finally, by knocking out a critical basal body inner junction component Poc1, we found the triplet MT was destabilized, resulting in a defective structure. Surprisingly, several axoneme-specific components were found to 'infiltrate' into the mutant basal body. Our findings provide molecular insight into cilium assembly at its inner junctions, underscoring its precise spatial regulation.
{"title":"The Structure of Cilium Inner Junctions Revealed by Electron Cryo-tomography","authors":"Sam Li, Jose-Jesus Fernandez, Marisa D Ruehle, Rachel A. Howard-Till, Amy S Fabritius, Chad G Pearson, David A Agard, Mark Winey","doi":"10.1101/2024.09.09.612100","DOIUrl":"https://doi.org/10.1101/2024.09.09.612100","url":null,"abstract":"The cilium is a microtubule-based organelle critical for many cellular functions. Its assembly initiates at a basal body and continues as an axoneme that projects out of the cell to form a functional cilium. This assembly process is tightly regulated. However, our knowledge of the molecular architecture and the mechanism of assembly is limited. By applying electron cryo-tomography and subtomogram averaging, we obtained subnanometer resolution structures of the inner junction in three distinct regions of the cilium: the proximal region of the basal body, the central core of the basal body, and the flagellar axoneme. The structures allowed us to identify several basal body and axoneme components. While a few proteins are distributed throughout the entire length of the organelle, many are restricted to particular regions of the cilium, forming intricate local interaction networks and bolstering local structural stability. Finally, by knocking out a critical basal body inner junction component Poc1, we found the triplet MT was destabilized, resulting in a defective structure. Surprisingly, several axoneme-specific components were found to 'infiltrate' into the mutant basal body. Our findings provide molecular insight into cilium assembly at its inner junctions, underscoring its precise spatial regulation.","PeriodicalId":501590,"journal":{"name":"bioRxiv - Cell Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142190067","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 : 2024-09-09DOI: 10.1101/2024.09.09.612124
Ritu Mann-Nuttel, Shivani Mandal, Marie Armbruster, Lakshmi Puttagunta, Paul Forsythe
Background: Pulmonary neuroendocrine cells (PNEC) are rare airway epithelial cells that have recently gained attention as potential amplifiers of allergic asthma. However, studying PNEC function in humans has been challenging due to a lack of cell isolation methods and little is known about human PNEC function in response to asthma relevant stimuli. Here we developed and characterized an in vitro human PNEC model and investigated the neuroendocrine response to extracts of the common aeroallergen house dust-mite. (HDM). Methods: PNEC enriched cultures were generated from human induced pluripotent stem cells (iPNEC) and primary bronchial epithelial cells (ePNEC). Characterized PNEC cultures were exposed to HDM extract, a volatile chemical odorant (Bergamot oil), or the bacterial membrane component, lipopolysaccharide (LPS) and neuroendocrine gene expression and neuropeptide release determined. Results: Both iPNEC and ePNEC models demonstrated similar baseline neuroendocrine characteristics and a stimuli specific modulation of gene expression. Most notably, exposure to HDM but not Bergamot oil or LPS, lead to dose dependent induction of the CGRP encoding gene, CALCB, and corresponding release of the neuropeptide. HDM induced CALCB expression and CGRP release could be inhibited by a protease activated receptor 1 (PAR1) antagonist or protease inhibitors and was mimicked by a PAR1 agonist. Conclusions: We have characterized a novel model of PNEC enriched human airway epithelium and utilized this model to demonstrate a previously unrecognized role for human PNEC in mediating a direct neuroendocrine response to aeroallergen exposure and highlighting CGRP production by these cells as a potential therapeutic target in allergic asthma.
{"title":"Human pulmonary neuroendocrine cells respond to House dust mite extract with PAR-1 dependent release of CGRP","authors":"Ritu Mann-Nuttel, Shivani Mandal, Marie Armbruster, Lakshmi Puttagunta, Paul Forsythe","doi":"10.1101/2024.09.09.612124","DOIUrl":"https://doi.org/10.1101/2024.09.09.612124","url":null,"abstract":"Background: Pulmonary neuroendocrine cells (PNEC) are rare airway epithelial cells that have recently gained attention as potential amplifiers of allergic asthma. However, studying PNEC function in humans has been challenging due to a lack of cell isolation methods and little is known about human PNEC function in response to asthma relevant stimuli. Here we developed and characterized an in vitro human PNEC model and investigated the neuroendocrine response to extracts of the common aeroallergen house dust-mite. (HDM).\u0000Methods: PNEC enriched cultures were generated from human induced pluripotent stem cells (iPNEC) and primary bronchial epithelial cells (ePNEC). Characterized PNEC cultures were exposed to HDM extract, a volatile chemical odorant (Bergamot oil), or the bacterial membrane component, lipopolysaccharide (LPS) and neuroendocrine gene expression and neuropeptide release determined. Results: Both iPNEC and ePNEC models demonstrated similar baseline neuroendocrine characteristics and a stimuli specific modulation of gene expression. Most notably, exposure to HDM but not Bergamot oil or LPS, lead to dose dependent induction of the CGRP encoding gene, CALCB, and corresponding release of the neuropeptide. HDM induced CALCB expression and CGRP release could be inhibited by a protease activated receptor 1 (PAR1) antagonist or protease inhibitors and was mimicked by a PAR1 agonist. Conclusions: We have characterized a novel model of PNEC enriched human airway epithelium and utilized this model to demonstrate a previously unrecognized role for human PNEC in mediating a direct neuroendocrine response to aeroallergen exposure and highlighting CGRP production by these cells as a potential therapeutic target in allergic asthma.","PeriodicalId":501590,"journal":{"name":"bioRxiv - Cell Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142189992","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}
Osteocytes, terminal-differentiated cells in bone, are now considered as more pivotal regulators of mature bone homeostasis than other bone cells, since they constitute 90-95% of the bone cell population. Given their non-migratory nature within the mineralized matrix, their unique dendrites are crucial for cell-to-cell communication in response to both intracellular and extracellular stimuli, such as bone fracture or mechanical load. Here, we showed that Osterix (Osx), usually recognized as a specific doorkeeper for osteoblast differentiation during new bone formation marked by collagen type I α 1 (Col1α1), was unexpectedly co-expressed with Col1α1 in osteocytes within the cortical bone of mice. Deleting Osx in Col1α1-positive osteocytes disrupted cortical bone structure and osteocytic dendrites in mice, thus impairing transcellular fluid flow and intercellular communication. Conversely, overexpression of Osx in osteocytes enhanced these processes. Furthermore, we identified Connexin43, a critical protein of gap junction channel, was a direct transcriptional target of Osx in regulating dendrites of osteocytes. Pharmacological restoration of Connexin43 levels rescued the dysfunction in Osx-deficient osteocytes both in vitro and in vivo. Taken together, this work demonstrated Osx's distinct role in osteocyte function through maintaining intercellular signaling, which broadened the current understanding of its role in Col1α1-positive bone cells, extending beyond osteoblasts and bone mineralization, offering new insights into bone diseases such as fracture nonunion or disuse osteoporosis.
{"title":"Osterix Facilitates Osteocytic Communication by Targeting Connexin43","authors":"zuping wu, qian chen, qian gao, muchun Liang, yumeng Zhou, Li Zhu, jiahe wang, Yang Shen, junjun Jing, Jing Xie, Xiaoheng Liu, Shujuan zou, Demao Zhang, Chenchen Zhou","doi":"10.1101/2024.09.09.611984","DOIUrl":"https://doi.org/10.1101/2024.09.09.611984","url":null,"abstract":"Osteocytes, terminal-differentiated cells in bone, are now considered as more pivotal regulators of mature bone homeostasis than other bone cells, since they constitute 90-95% of the bone cell population. Given their non-migratory nature within the mineralized matrix, their unique dendrites are crucial for cell-to-cell communication in response to both intracellular and extracellular stimuli, such as bone fracture or mechanical load. Here, we showed that Osterix (Osx), usually recognized as a specific doorkeeper for osteoblast differentiation during new bone formation marked by collagen type I α 1 (Col1α1), was unexpectedly co-expressed with Col1α1 in osteocytes within the cortical bone of mice. Deleting Osx in Col1α1-positive osteocytes disrupted cortical bone structure and osteocytic dendrites in mice, thus impairing transcellular fluid flow and intercellular communication. Conversely, overexpression of Osx in osteocytes enhanced these processes. Furthermore, we identified Connexin43, a critical protein of gap junction channel, was a direct transcriptional target of Osx in regulating dendrites of osteocytes. Pharmacological restoration of Connexin43 levels rescued the dysfunction in Osx-deficient osteocytes both in vitro and in vivo. Taken together, this work demonstrated Osx's distinct role in osteocyte function through maintaining intercellular signaling, which broadened the current understanding of its role in Col1α1-positive bone cells, extending beyond osteoblasts and bone mineralization, offering new insights into bone diseases such as fracture nonunion or disuse osteoporosis.","PeriodicalId":501590,"journal":{"name":"bioRxiv - Cell Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142190063","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}
The epithelial cell sheet maintains its integrity as a barrier while undergoing turnover of constituent cells. To sustain the barrier continuously, it's essential to preserve the 'old' tight junctions (TJs) between cells being excluded from the sheet and their neighbors while simultaneously forming de novo TJs between newly adjacent cells. However, the molecular mechanisms involved in the formation of de novo TJs remain largely unknown. This study investigates two scenarios: the formation of de novo TJs during the removal of apoptotic cells from monolayer epithelial sheets and during the differentiation of the granular layer in stratified epidermis. We revealed that rapid claudin assembly is achieved by actively regulating the dissociation of the EpCAM/TROP2-claudin complex in both situations. Furthermore, we found that the Rho-ROCK pathway initiates the activation of matriptase, which cleaves EpCAM/TROP2, resulting in the supply of polymerizable claudin from the stockpiled EpCAM/TROP2-claudin complex at the plasma membrane to induce rapid de novo TJ formation.
{"title":"Rho-ROCK liberates sequestered claudin for rapid de novo tight junction formation","authors":"Yuma Cho, Akari Taniguchi, Akiharu Kubo, Junichi Ikenouchi","doi":"10.1101/2024.09.09.612007","DOIUrl":"https://doi.org/10.1101/2024.09.09.612007","url":null,"abstract":"The epithelial cell sheet maintains its integrity as a barrier while undergoing turnover of constituent cells. To sustain the barrier continuously, it's essential to preserve the 'old' tight junctions (TJs) between cells being excluded from the sheet and their neighbors while simultaneously forming <em>de novo</em> TJs between newly adjacent cells. However, the molecular mechanisms involved in the formation of <em>de novo</em> TJs remain largely unknown. This study investigates two scenarios: the formation of <em>de novo</em> TJs during the removal of apoptotic cells from monolayer epithelial sheets and during the differentiation of the granular layer in stratified epidermis. We revealed that rapid claudin assembly is achieved by actively regulating the dissociation of the EpCAM/TROP2-claudin complex in both situations. Furthermore, we found that the Rho-ROCK pathway initiates the activation of matriptase, which cleaves EpCAM/TROP2, resulting in the supply of polymerizable claudin from the stockpiled EpCAM/TROP2-claudin complex at the plasma membrane to induce rapid <em>de novo</em> TJ formation.","PeriodicalId":501590,"journal":{"name":"bioRxiv - Cell Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142189991","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 : 2024-09-09DOI: 10.1101/2024.09.09.611943
Wai Kit Lam, Runa S. J. Lindblom, Bridget Milky, Paris Mazzachi, Marjan Hadian-Jazi, Catharina Kung, Grace Khuu, Louise Uoselis, Thanh Ngoc Nguyen, Marvin Skulsuppaisarn, Tahnee L Saunders, Marlene F Schmidt, Grant Dewson, Adam I Fogel, Cedric Bardy, Michael Lazarou
Defects in neuronal mitophagy have been linked to neurodegenerative diseases including Parkinson's disease. However, despite the importance of mitophagy in neuronal homeostasis, the mechanistic basis for neurodegeneration when mitophagy is defective is unclear. Here, using human neurons, we discover that presynapses are mitophagy pit stops for damaged axonal mitochondria. We show that while mitochondrial damage and PINK1/Parkin activation events are distributed throughout axons, mitophagy initiation and autophagosome formation are restricted to presynapses, which we show contain the machineries required for mitophagy. Being the primary sites of axonal mitophagy, presynapses were vulnerable when PINK1/Parkin mitophagy was defective. We observed local cytochrome c release within presynapses from an accumulation of damaged mitochondria. This resulted in downstream degradative caspase activation, defining a mechanism for neurodegeneration. Pharmacological rescue of axon degeneration was achieved through synthetic upregulation of receptor mediated mitophagy with the clinically approved compound Roxadustat, revealing a potential therapeutic avenue for disease.
{"title":"Presynapses are mitophagy pit stops that prevent axon degeneration","authors":"Wai Kit Lam, Runa S. J. Lindblom, Bridget Milky, Paris Mazzachi, Marjan Hadian-Jazi, Catharina Kung, Grace Khuu, Louise Uoselis, Thanh Ngoc Nguyen, Marvin Skulsuppaisarn, Tahnee L Saunders, Marlene F Schmidt, Grant Dewson, Adam I Fogel, Cedric Bardy, Michael Lazarou","doi":"10.1101/2024.09.09.611943","DOIUrl":"https://doi.org/10.1101/2024.09.09.611943","url":null,"abstract":"Defects in neuronal mitophagy have been linked to neurodegenerative diseases including Parkinson's disease. However, despite the importance of mitophagy in neuronal homeostasis, the mechanistic basis for neurodegeneration when mitophagy is defective is unclear. Here, using human neurons, we discover that presynapses are mitophagy pit stops for damaged axonal mitochondria. We show that while mitochondrial damage and PINK1/Parkin activation events are distributed throughout axons, mitophagy initiation and autophagosome formation are restricted to presynapses, which we show contain the machineries required for mitophagy. Being the primary sites of axonal mitophagy, presynapses were vulnerable when PINK1/Parkin mitophagy was defective. We observed local cytochrome c release within presynapses from an accumulation of damaged mitochondria. This resulted in downstream degradative caspase activation, defining a mechanism for neurodegeneration. Pharmacological rescue of axon degeneration was achieved through synthetic upregulation of receptor mediated mitophagy with the clinically approved compound Roxadustat, revealing a potential therapeutic avenue for disease.","PeriodicalId":501590,"journal":{"name":"bioRxiv - Cell Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142189989","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 : 2024-09-09DOI: 10.1101/2024.09.09.612135
Shannon Sim, Sean Moore, Khalid Al-Naemi, Ziad El-Hajj, Jackie Vogel
The formation of a stable mitotic spindle is critical to the accurate partitioning of the chromosomes during mitosis. Interpolar microtubules of the spindle midzone consist of antiparallel microtubules crosslinked by kinesin-5 and stabilize the spindle by opposing forces produced when sister chromatids are attached to microtubules and under tension. Despite the importance of the interpolar microtubules, how and when they form and what determines their number remain unknown. In this study, we report that a γ-tubulin mutation (γtub-Y445D) disrupts the localization of kinesin-5 and the formation of the interpolar microtubules, resulting in spindle instability. We find that kinesin-5 crosslinking is intact in this mutant, but that it is incapable of the subsequent kinesin-5 microtubule sliding needed to stabilize the nascent spindle. Early activation of the PRC1 homolog Ase1 restores nascent spindle stability to the γtub-Y445D mutant but cannot stabilize spindles during centromere attachment. Our work shows that midzone assembly begins with the formation of interpolar microtubule precursors in monopolar spindles that persist until early metaphase and limit the formation of kinetochore attachments in new spindles.
{"title":"Early events in midzone formation stabilize nascent bipolar spindles","authors":"Shannon Sim, Sean Moore, Khalid Al-Naemi, Ziad El-Hajj, Jackie Vogel","doi":"10.1101/2024.09.09.612135","DOIUrl":"https://doi.org/10.1101/2024.09.09.612135","url":null,"abstract":"The formation of a stable mitotic spindle is critical to the accurate partitioning of the chromosomes during mitosis. Interpolar microtubules of the spindle midzone consist of antiparallel microtubules crosslinked by kinesin-5 and stabilize the spindle by opposing forces produced when sister chromatids are attached to microtubules and under tension. Despite the importance of the interpolar microtubules, how and when they form and what determines their number remain unknown. In this study, we report that a γ-tubulin mutation (γtub-Y445D) disrupts the localization of kinesin-5 and the formation of the interpolar microtubules, resulting in spindle instability. We find that kinesin-5 crosslinking is intact in this mutant, but that it is incapable of the subsequent kinesin-5 microtubule sliding needed to stabilize the nascent spindle. Early activation of the PRC1 homolog Ase1 restores nascent spindle stability to the γtub-Y445D mutant but cannot stabilize spindles during centromere attachment. Our work shows that midzone assembly begins with the formation of interpolar microtubule precursors in monopolar spindles that persist until early metaphase and limit the formation of kinetochore attachments in new spindles.","PeriodicalId":501590,"journal":{"name":"bioRxiv - Cell Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142190062","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 : 2024-09-09DOI: 10.1101/2024.09.09.612098
Christophe Guérin, Anne-Betty N'Diaye, Laurène Gressin, Alex Mogilner, Manuel Théry, Laurent Blanchoin, Alexandra Colin
In cells, multiple actin networks coexist in a dynamic manner. These networks compete for a common pool of actin monomers and actin-binding proteins. Interestingly, this competition does not result in the mere survival of the more consuming networks. Moreover, the co-existence of networks with various strengths is key to cell adaption to external changes. However, a comprehensive view of how these networks coexist in this competitive environment, where resources are limited, is still lacking. To address this question, we used a reconstituted system, in closed microwells, consisting of beads propelled by actin polymerization or micropatterns functionalized with lipids capable of initiating polymerization close to a membrane. This system enabled us to build dynamic actin architectures, competing for a limited pool of proteins, over a period of hours. We demonstrated the importance of protein turnover for the coexistence of actin networks, showing it ensures resource distribution between weak and strong networks. However, when competition becomes too intense, turnover alone is insufficient, leading to a selection process that favors the strongest networks. Consequently, we emphasize the importance of competition strength, which is defined by the turnover rate, the amount of available protein, and the number of competing structures. More generally, this work illustrates how turnover allows biological populations with various competition strengths to coexist despite resource constraints.
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Pub Date : 2024-09-09DOI: 10.1101/2024.09.09.612097
Jayasri Nanduri, Ning Wang, Matthew Hildreth, Nanduri R Prabhakar
Histones play a crucial role in regulating gene expression through post translational modifications (PTMS) which include acetylation, methylation and phosphorylation. We have previously identified histone 3 acetylation (H3Kac) and methylation (H3Kme) as an early epigenetic mechanism associated with intermittent hypoxia (IH), a hallmark feature of sleep apnea. The goal of the present study was to determine the molecular mechanisms underlying IH increased H3 acetylation. IH-induced H3 acetylation was blocked by an antioxidant. Conversely, reactive oxygen species (ROS) mimetics, increased H3 acetylated protein expression similar to IH, suggesting a role for ROS. Trichostatin A (TSA), an HDAC (histone deacetylase) inhibitor mimicked IH-induced H3 acetylation under normoxic conditions, while pharmacological blockade of p300/CBP (HAT, histone acetylase) with CTK7A abolished IH-induced H3 acetylation. These results suggest that interplay between HATs and HDACs regulate ROS-dependent H3 acetylation by IH. Lysine 27 (H3K27) on H3 was one of the lysines specifically acetylated by IH and this acetylation was associated with dephsophorylation of H3 at serine 28 (H3S28). Inhibition of S28 dephosphorylation by protein phosphatase inhibitors (PIC or Calyculin A), prevented H3K27 acetylation by IH. Conversely, inhibiting K27 acetylation with CTK7A, increased S28 phosphorylation in IH-exposed cells. These findings highlight the intricate balance between H3 acetylation and phosphorylation in response to IH, shedding light on epigenetic mechanism regulating gene expression. (Supported by NIH-PO1-HL90554).
组蛋白通过翻译后修饰（PTMS）（包括乙酰化、甲基化和磷酸化）在调节基因表达方面发挥着至关重要的作用。我们以前曾发现组蛋白 3 乙酰化（H3Kac）和甲基化（H3Kme）是与间歇性缺氧（IH）相关的早期表观遗传机制，而间歇性缺氧是睡眠呼吸暂停的一个标志性特征。本研究的目的是确定 IH 增加 H3 乙酰化的分子机制。抗氧化剂阻断了 IH 诱导的 H3 乙酰化。相反，活性氧（ROS）模拟物增加了 H3 乙酰化蛋白的表达，与 IH 相似，这表明 ROS 起了作用。在常氧条件下，HDAC（组蛋白去乙酰化酶）抑制剂 Trichostatin A（TSA）模拟了 IH 诱导的 H3 乙酰化，而 CTK7A 对 p300/CBP（HAT，组蛋白乙酰化酶）的药理阻断则消除了 IH 诱导的 H3 乙酰化。这些结果表明，HATs 和 HDACs 之间的相互作用调节着 IH 依赖于 ROS 的 H3 乙酰化。H3上的赖氨酸27（H3K27）是被IH特异性乙酰化的赖氨酸之一，这种乙酰化与H3上丝氨酸28（H3S28）的去磷酸化有关。蛋白磷酸酶抑制剂（PIC 或 Calyculin A）可抑制 S28 的去磷酸化，从而阻止 IH 对 H3K27 的乙酰化。相反，用 CTK7A 抑制 K27 乙酰化会增加 IH 暴露细胞中的 S28 磷酸化。这些发现突显了 H3 乙酰化和磷酸化在 IH 反应中的复杂平衡，揭示了调控基因表达的表观遗传学机制。(美国国立卫生研究院-PO1-HL90554资助）。
{"title":"Reactive Oxygen species dependent increase in H3K27 acetylation by intermittent hypoxia is regulated by H3S28 phosphorylation","authors":"Jayasri Nanduri, Ning Wang, Matthew Hildreth, Nanduri R Prabhakar","doi":"10.1101/2024.09.09.612097","DOIUrl":"https://doi.org/10.1101/2024.09.09.612097","url":null,"abstract":"Histones play a crucial role in regulating gene expression through post translational modifications (PTMS) which include acetylation, methylation and phosphorylation. We have previously identified histone 3 acetylation (H3Kac) and methylation (H3Kme) as an early epigenetic mechanism associated with intermittent hypoxia (IH), a hallmark feature of sleep apnea. The goal of the present study was to determine the molecular mechanisms underlying IH increased H3 acetylation. IH-induced H3 acetylation was blocked by an antioxidant. Conversely, reactive oxygen species (ROS) mimetics, increased H3 acetylated protein expression similar to IH, suggesting a role for ROS. Trichostatin A (TSA), an HDAC (histone deacetylase) inhibitor mimicked IH-induced H3 acetylation under normoxic conditions, while pharmacological blockade of p300/CBP (HAT, histone acetylase) with CTK7A abolished IH-induced H3 acetylation. These results suggest that interplay between HATs and HDACs regulate ROS-dependent H3 acetylation by IH. Lysine 27 (H3K27) on H3 was one of the lysines specifically acetylated by IH and this acetylation was associated with dephsophorylation of H3 at serine 28 (H3S28). Inhibition of S28 dephosphorylation by protein phosphatase inhibitors (PIC or Calyculin A), prevented H3K27 acetylation by IH. Conversely, inhibiting K27 acetylation with CTK7A, increased S28 phosphorylation in IH-exposed cells. These findings highlight the intricate balance between H3 acetylation and phosphorylation in response to IH, shedding light on epigenetic mechanism regulating gene expression. (Supported by NIH-PO1-HL90554).","PeriodicalId":501590,"journal":{"name":"bioRxiv - Cell Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142190065","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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