Pub Date : 2024-09-11DOI: 10.1101/2024.09.11.612427
Julian M Carosi, Alexis Martin, Leanne K Hein, Sofia Hassiotis, Kathryn J Hattersley, Celia Fourrier, Julien Bensalem, Timothy J Sargeant
Autophagy is a waste-disposal pathway that protects against age-related pathology. It is widely accepted that autophagy declines with age, yet role that sex and diet-related obesity play during aging remain unknown. Here, we present the most comprehensive in vivo study of autophagic flux to date. We employed transgenic mice overexpressing tandem-florescent LC3B (RFP-GFP-LC3B) to measure autophagic flux in the blood (PBMCs), heart, and motor cortex of aging mice that were fed regular chow or a high-fat diet for 6-, 12- or 18-months. In male mice, aging reduced autophagic flux in the heart and brain, but increased it in the blood. Age-dependent changes in female autophagic flux was less pronounced. Autophagic flux was modified by a high-fat diet in the blood and heart of male but not female mice. Overall, we uncovered sexual dimorphisms that underpin how autophagy changes with age across different tissues and in response to a high-fat diet.
{"title":"Autophagy across tissues of aging mice","authors":"Julian M Carosi, Alexis Martin, Leanne K Hein, Sofia Hassiotis, Kathryn J Hattersley, Celia Fourrier, Julien Bensalem, Timothy J Sargeant","doi":"10.1101/2024.09.11.612427","DOIUrl":"https://doi.org/10.1101/2024.09.11.612427","url":null,"abstract":"Autophagy is a waste-disposal pathway that protects against age-related pathology. It is widely accepted that autophagy declines with age, yet role that sex and diet-related obesity play during aging remain unknown. Here, we present the most comprehensive in vivo study of autophagic flux to date. We employed transgenic mice overexpressing tandem-florescent LC3B (RFP-GFP-LC3B) to measure autophagic flux in the blood (PBMCs), heart, and motor cortex of aging mice that were fed regular chow or a high-fat diet for 6-, 12- or 18-months. In male mice, aging reduced autophagic flux in the heart and brain, but increased it in the blood. Age-dependent changes in female autophagic flux was less pronounced. Autophagic flux was modified by a high-fat diet in the blood and heart of male but not female mice. Overall, we uncovered sexual dimorphisms that underpin how autophagy changes with age across different tissues and in response to a high-fat diet.","PeriodicalId":501590,"journal":{"name":"bioRxiv - Cell Biology","volume":"2011 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142189958","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 : 2024-09-11DOI: 10.1101/2024.09.11.612005
Gabriel Guilloux, Maiko Kitaoka, Karel Mocaer, Claire Heichette, Laurence Duchesne, Rebecca Heald, Thierry Pécot, Romain Gibeaux
The spindle is a key structure in cell division as it orchestrates the accurate segregation of genetic material. While its assembly and function are well-studied, the mechanisms regulating spindle architecture remain elusive. In this study, we focus on the differences in spindle organization between Xenopus laevis and Xenopus tropicalis, leveraging expansion microscopy (ExM) to overcome the limitations of conventional imaging techniques. We optimized an ExM protocol tailored for Xenopus egg extract spindles, improving upon fixation, denaturation and gelation methods to achieve higher resolution imaging of spindles. Our protocol preserves spindle integrity and allows effective pre-expansion immunofluorescence. This method enabled detailed analysis of the differences in microtubule organization between the two species. X. laevis spindles overall exhibit a broader range of bundle sizes, while X. tropicalis spindles are more limited to smaller bundles. Moreover, while both species favor larger bundle sizes near and at the spindle center, X. tropicalis spindles otherwise prefer very small bundles, and X. laevis spindles medium-sized bundles. By enhancing resolution and minimizing distortions and fixation artifacts, our optimized ExM approach offers new insights into spindle morphology and provides a robust tool for studying the structural intricacies of these large cellular assemblies. This work advances our understanding of spindle architecture and opens up new avenues for exploring spindle-related questions.
{"title":"Optimized expansion microscopy reveals species-specific spindle microtubule organization in Xenopus egg extracts","authors":"Gabriel Guilloux, Maiko Kitaoka, Karel Mocaer, Claire Heichette, Laurence Duchesne, Rebecca Heald, Thierry Pécot, Romain Gibeaux","doi":"10.1101/2024.09.11.612005","DOIUrl":"https://doi.org/10.1101/2024.09.11.612005","url":null,"abstract":"The spindle is a key structure in cell division as it orchestrates the accurate segregation of genetic material. While its assembly and function are well-studied, the mechanisms regulating spindle architecture remain elusive. In this study, we focus on the differences in spindle organization between <em>Xenopus laevis</em> and <em>Xenopus tropicalis</em>, leveraging expansion microscopy (ExM) to overcome the limitations of conventional imaging techniques. We optimized an ExM protocol tailored for <em>Xenopus</em> egg extract spindles, improving upon fixation, denaturation and gelation methods to achieve higher resolution imaging of spindles. Our protocol preserves spindle integrity and allows effective pre-expansion immunofluorescence. This method enabled detailed analysis of the differences in microtubule organization between the two species. <em>X. laevis</em> spindles overall exhibit a broader range of bundle sizes, while <em>X. tropicalis</em> spindles are more limited to smaller bundles. Moreover, while both species favor larger bundle sizes near and at the spindle center, <em>X. tropicalis</em> spindles otherwise prefer very small bundles, and <em>X. laevis</em> spindles medium-sized bundles. By enhancing resolution and minimizing distortions and fixation artifacts, our optimized ExM approach offers new insights into spindle morphology and provides a robust tool for studying the structural intricacies of these large cellular assemblies. This work advances our understanding of spindle architecture and opens up new avenues for exploring spindle-related questions.","PeriodicalId":501590,"journal":{"name":"bioRxiv - Cell Biology","volume":"68 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142189981","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 : 2024-09-11DOI: 10.1101/2024.09.11.612445
Sara Ambjoern, Bob Meeusen, Johanna Kliche, Juanjuan Wang, Dimitriya Garvanska, Thomas Kruse, Blanca Lopez-Mendez, Matthias Mann, Niels Mailand, Emil Hertz, Norman E Davey, Jakob Nilsson
Short linear motifs (SLiMs) are the most ubiquitous protein interaction modules in the unstructured regions of the human proteome. Despite their central role in protein function, our understanding of the contribution of SLiMs to cellular homeostasis remains limited. To address this, we designed base editor libraries to precisely mutate all curated SLiMs and a set of computationally predicted instances defined by SLiM-like evolutionary patterns. By targeting 7,293 SLiM containing regions with 80,473 mutations, we define a SLiM dependency map identifying 450 known and 264 predicted SLiMs required for normal cell proliferation. Notably, the vast majority of essential predicted SLiMs belong to novel classes of SLiMs. We also uncover the binding partners of several predicted SLiMs and provide mechanistic insight into disease causing mutations. Our study provides a proteome-wide resource on SLiM essentiality and highlights the presence of numerous uncharacterised essential SLiMs in the human proteome.
{"title":"A proteome-wide dependency map of protein interaction motifs","authors":"Sara Ambjoern, Bob Meeusen, Johanna Kliche, Juanjuan Wang, Dimitriya Garvanska, Thomas Kruse, Blanca Lopez-Mendez, Matthias Mann, Niels Mailand, Emil Hertz, Norman E Davey, Jakob Nilsson","doi":"10.1101/2024.09.11.612445","DOIUrl":"https://doi.org/10.1101/2024.09.11.612445","url":null,"abstract":"Short linear motifs (SLiMs) are the most ubiquitous protein interaction modules in the unstructured regions of the human proteome. Despite their central role in protein function, our understanding of the contribution of SLiMs to cellular homeostasis remains limited. To address this, we designed base editor libraries to precisely mutate all curated SLiMs and a set of computationally predicted instances defined by SLiM-like evolutionary patterns. By targeting 7,293 SLiM containing regions with 80,473 mutations, we define a SLiM dependency map identifying 450 known and 264 predicted SLiMs required for normal cell proliferation. Notably, the vast majority of essential predicted SLiMs belong to novel classes of SLiMs. We also uncover the binding partners of several predicted SLiMs and provide mechanistic insight into disease causing mutations. Our study provides a proteome-wide resource on SLiM essentiality and highlights the presence of numerous uncharacterised essential SLiMs in the human proteome.","PeriodicalId":501590,"journal":{"name":"bioRxiv - Cell Biology","volume":"12 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142189961","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}
Abstract: Like a photoreceptor cilium, the sensory cilia have a complex bipartite architecture containing 9+0 connecting cilium at the base and a singlet microtubule-supported, highly membranous outer segment, essential for the receptor display. How such diverse cilia morphology and underlying microtubule cytoskeleton develops remains unclear. Here we show that individual olfactory cilium, inside the large basiconic sensilla in developing Drosophila antenna, grows in episodic steps following several pulsatile influxes of tubulin. Each tubulin influx event is preceded by transient elevations of a microtubule-stabilising protein, the End-binding protein 1 (EB1). Additionally, EB1 is found to specifically interact with the tail domain of Drosophila KLP68D, an orthologue of the kinesin-2beta motor subunit, in vitro. Finally, the loss of EB1 in olfactory neurons preceding the growth surges reduces the tubulin influx as well as arrests the olfactory cilia assembly and stability. These findings suggest a novel mechanism of bipartite cilia assembly.
{"title":"Active EB1 surges promote tubulin influx into the growing outer segments of the bipartite olfactory cilia in Drosophila","authors":"Riddhi Girdhar Agarwal, Saishree Iyer, Ayan Barbora, Yogesh Gadgil, Swadhin Jana, Krishanu Ray","doi":"10.1101/2024.09.10.612170","DOIUrl":"https://doi.org/10.1101/2024.09.10.612170","url":null,"abstract":"Abstract:\u0000Like a photoreceptor cilium, the sensory cilia have a complex bipartite architecture containing 9+0 connecting cilium at the base and a singlet microtubule-supported, highly membranous outer segment, essential for the receptor display. How such diverse cilia morphology and underlying microtubule cytoskeleton develops remains unclear. Here we show that individual olfactory cilium, inside the large basiconic sensilla in developing Drosophila antenna, grows in episodic steps following several pulsatile influxes of tubulin. Each tubulin influx event is preceded by transient elevations of a microtubule-stabilising protein, the End-binding protein 1 (EB1). Additionally, EB1 is found to specifically interact with the tail domain of Drosophila KLP68D, an orthologue of the kinesin-2beta motor subunit, in vitro. Finally, the loss of EB1 in olfactory neurons preceding the growth surges reduces the tubulin influx as well as arrests the olfactory cilia assembly and stability. These findings suggest a novel mechanism of bipartite cilia assembly.","PeriodicalId":501590,"journal":{"name":"bioRxiv - Cell Biology","volume":"73 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142189983","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: 10.1101/2024.09.10.612178
Chang Wang, Ioan Iacovache, Benoit Zuber
Cryo-electron tomography (cryoET) is an important imaging technique that can provide 3D datasets of organelles and proteins at nanometer and sub-nanometer resolution. Recently, combining cryoET with subtomogram averaging has pushed the resolution to 3-4 A. However, one main challenge for cryoET is locating target proteins in live cells. Conventional methods such as fluorescent protein tagging and immunogold labeling are not entirely suitable to label small structures in live cells with molecular resolution in vitrified samples. If large proteins, which can be visually identified in cryoET, are directly linked to the target protein, the large tag may alter the target protein structure, localization and function. To address this challenge, we used the rapamycin-induced oligomer formation system, which involves two tags (FKBP and FRB) that can bind together within rapamycin. In our system, the FKBP tag is linked to target protein and the FRB tag is linked to a large protein to create a marker. We chose ferritin as the marker protein because it is a large complex (10-12 nm) and can bind iron to create strong contrast in cryoET. After adding rapamycin to the cell medium, the iron-loaded ferritin accurately indicates the location of the target protein. Recently, in-situ cryoET with subtomogram averaging has been rapidly developing. However, it is still challenging to locate target proteins in live cells, and this method provides a much-needed solution.
{"title":"Genetically Encoded FerriTag as a Specific Label for Cryo-Electron Tomography","authors":"Chang Wang, Ioan Iacovache, Benoit Zuber","doi":"10.1101/2024.09.10.612178","DOIUrl":"https://doi.org/10.1101/2024.09.10.612178","url":null,"abstract":"Cryo-electron tomography (cryoET) is an important imaging technique that can provide 3D datasets of organelles and proteins at nanometer and sub-nanometer resolution. Recently, combining cryoET with subtomogram averaging has pushed the resolution to 3-4 A. However, one main challenge for cryoET is locating target proteins in live cells. Conventional methods such as fluorescent protein tagging and immunogold labeling are not entirely suitable to label small structures in live cells with molecular resolution in vitrified samples. If large proteins, which can be visually identified in cryoET, are directly linked to the target protein, the large tag may alter the target protein structure, localization and function. To address this challenge, we used the rapamycin-induced oligomer formation system, which involves two tags (FKBP and FRB) that can bind together within rapamycin. In our system, the FKBP tag is linked to target protein and the FRB tag is linked to a large protein to create a marker. We chose ferritin as the marker protein because it is a large complex (10-12 nm) and can bind iron to create strong contrast in cryoET. After adding rapamycin to the cell medium, the iron-loaded ferritin accurately indicates the location of the target protein. Recently, in-situ cryoET with subtomogram averaging has been rapidly developing. However, it is still challenging to locate target proteins in live cells, and this method provides a much-needed solution.","PeriodicalId":501590,"journal":{"name":"bioRxiv - Cell Biology","volume":"23 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142189985","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: 10.1101/2024.09.10.612293
Quanyi Zhao, Albert Pedroza, Disha Sharma, Wenduo Gu, Alex Dalal, Chad Weldy, William Jackson, Daniel Yuhang Li, Yana Ryan, Trieu Nguyen, Rohan Shad, Brian T. Palmisano, Joao P. Monteiro, Matthew Worssam, Alexa Berezwitz, Meghana Iyer, Huitong Shi, Ramendra Kundu, Lasemahang Limbu, Juyong Brian Kim, Anshul Kundaje, Michael Fischbein, Robert Wirka, Thomas Quertermous, Paul Cheng
Vascular beds show different propensities for different vascular pathologies, yet mechanisms explaining these fundamental differences remain unknown. We sought to build a transcriptomic, cellular, and spatial atlas of human arterial cells across multiple different arterial segments to understand this phenomenon. We found significant cell type-specific segmental heterogeneity. Determinants of arterial identity are predominantly encoded in fibroblasts and smooth muscle cells, and their differentially expressed genes are particularly enriched for vascular disease-associated loci and genes. Adventitial fibroblast-specific heterogeneity in gene expression coincides with numerous vascular disease risk genes, suggesting a previously unrecognized role for this cell type in disease risk. Adult arterial cells from different segments cluster not by anatomical proximity but by embryonic origin, with differentially regulated genes heavily influenced by developmental master regulators. Non-coding transcriptomes across arterial cells contain extensive variation in lnc-RNAs expressed in cell type- and segment-specific patterns, rivaling heterogeneity in protein coding transcriptomes, and show enrichment for non-coding genetic signals for vascular diseases.
{"title":"A cell and transcriptome atlas of the human arterial vasculature","authors":"Quanyi Zhao, Albert Pedroza, Disha Sharma, Wenduo Gu, Alex Dalal, Chad Weldy, William Jackson, Daniel Yuhang Li, Yana Ryan, Trieu Nguyen, Rohan Shad, Brian T. Palmisano, Joao P. Monteiro, Matthew Worssam, Alexa Berezwitz, Meghana Iyer, Huitong Shi, Ramendra Kundu, Lasemahang Limbu, Juyong Brian Kim, Anshul Kundaje, Michael Fischbein, Robert Wirka, Thomas Quertermous, Paul Cheng","doi":"10.1101/2024.09.10.612293","DOIUrl":"https://doi.org/10.1101/2024.09.10.612293","url":null,"abstract":"Vascular beds show different propensities for different vascular pathologies, yet mechanisms explaining these fundamental differences remain unknown. We sought to build a transcriptomic, cellular, and spatial atlas of human arterial cells across multiple different arterial segments to understand this phenomenon. We found significant cell type-specific segmental heterogeneity. Determinants of arterial identity are predominantly encoded in fibroblasts and smooth muscle cells, and their differentially expressed genes are particularly enriched for vascular disease-associated loci and genes. Adventitial fibroblast-specific heterogeneity in gene expression coincides with numerous vascular disease risk genes, suggesting a previously unrecognized role for this cell type in disease risk. Adult arterial cells from different segments cluster not by anatomical proximity but by embryonic origin, with differentially regulated genes heavily influenced by developmental master regulators. Non-coding transcriptomes across arterial cells contain extensive variation in lnc-RNAs expressed in cell type- and segment-specific patterns, rivaling heterogeneity in protein coding transcriptomes, and show enrichment for non-coding genetic signals for vascular diseases.","PeriodicalId":501590,"journal":{"name":"bioRxiv - Cell Biology","volume":"1 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142189962","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}
Cellular senescence is a complex stress response that results in the permanent arrest of cell proliferation. The accumulation of senescent cells occurs during aging in living organisms, and contributes to tissue dysfunction. Although there are growing lines of evidence that various metabolic changes occur in senescent cells, the link between cellular metabolism and senescence is not yet fully understood. In this study, we demonstrate that alterations in the metabolism of branched-chain amino acids (BCAAs) play a crucial role in establishing cellular senescence. Furthermore, we identified mitochondrial BCAA transamination as a crucial step in this process. Our findings show that various types of cellular stress lead to a reduction in the expression of BCAA aminotransferase 2 (BCAT2), one of the BCAA catabolic enzymes, resulting in decreased catabolism of BCAAs and reduced synthesis of glutamate. The reduction of BCAA catabolites, together with the consequent limitation in glutathione production from glutamate, triggers cellular senescence. Furthermore, we demonstrate that a reduction in BCAT2 levels alone is sufficient to induce cellular senescence, both in cultured cells and in mice. Additionally, our results demonstrate that aging alters BCAA metabolism in both mice and humans. Our findings provide new insights into the metabolic mechanisms underlying cellular senescence, with a particular focus on the role of BCAAs.
{"title":"Branched-chain amino acid metabolism is a crucial modulator of cellular senescence","authors":"Yuma Aramaki, Kazuki Irie, Hideru Obinata, Shinya Honda, Takuro Horii, Satoko Arakawa, Aiko Tsuchida, Junki Hoshino, Ryosuke Kobayashi, Takashi Izumi, Izuho Hatada, Shigeomi Shimizu, Yoji A. Minamishima, Akimitsu Konishi","doi":"10.1101/2024.09.10.612139","DOIUrl":"https://doi.org/10.1101/2024.09.10.612139","url":null,"abstract":"Cellular senescence is a complex stress response that results in the permanent arrest of cell proliferation. The accumulation of senescent cells occurs during aging in living organisms, and contributes to tissue dysfunction. Although there are growing lines of evidence that various metabolic changes occur in senescent cells, the link between cellular metabolism and senescence is not yet fully understood. In this study, we demonstrate that alterations in the metabolism of branched-chain amino acids (BCAAs) play a crucial role in establishing cellular senescence. Furthermore, we identified mitochondrial BCAA transamination as a crucial step in this process. Our findings show that various types of cellular stress lead to a reduction in the expression of BCAA aminotransferase 2 (BCAT2), one of the BCAA catabolic enzymes, resulting in decreased catabolism of BCAAs and reduced synthesis of glutamate. The reduction of BCAA catabolites, together with the consequent limitation in glutathione production from glutamate, triggers cellular senescence. Furthermore, we demonstrate that a reduction in BCAT2 levels alone is sufficient to induce cellular senescence, both in cultured cells and in mice. Additionally, our results demonstrate that aging alters BCAA metabolism in both mice and humans. Our findings provide new insights into the metabolic mechanisms underlying cellular senescence, with a particular focus on the role of BCAAs.","PeriodicalId":501590,"journal":{"name":"bioRxiv - Cell Biology","volume":"1 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142189986","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: 10.1101/2024.09.09.611999
Hongwei Zhang, Qixia Xu, Zhirui Jiang, Rong Sun, Sanhong Liu, James L. Kirkland, Weidong Zhang, Yu Sun
Cellular senescence is a cell fate triggered by inherent or environmental stress and characterized by stable cell cycle arrest accompanied by a hypersecretory feature, termed as the senescence-associated secretory phenotype (SASP). Senescent cell burden increases with natural aging, functionally contributing to age-related organ dysfunction and multiple disorders. In this study, we performed a large scale screening of a natural product library for senotherapeutic candidates by assessing their effects on human senescent cells. Apigenin, a dietary flavonoid previously reported with antioxidant and anti-inflammatory activities, exhibited a prominent capacity in targeting senescent cells as a senomorphic agent. In senescent cells, apigenin blocks the interactions between ATM/p38 and HSPA8, thus preventing transition of the acute stress-associated phenotype (ASAP) towards the SASP. Mechanistically, apigenin targets peroxiredoxin 6 (PRDX6), an intracellular redox-active molecule, suppressing the iPLA2 activity of PRDX6 and disrupting downstream reactions underlying the SASP development. Without reversing cellular senescence, apigenin deprives cancer cells of malignancy acquired from senescent stromal cells in culture, while reducing chemoresistance upon combination with chemotherapy in anticancer regimens. In preclinical trials, apigenin administration improves physical function of animals prematurely aged after whole body irradiation, alleviating physical frailty and cognitive impairment. Overall, our study demonstrates the potential of exploiting a naturally derived compound with senomorphic capacity to achieve geroprotective effects by modulating the SASP, thus providing a research platform for future exploration of novel natural agents against age-related conditions.
{"title":"Repurposing the plant-derived compound apigenin for senomorphic effect in antiaging pipelines","authors":"Hongwei Zhang, Qixia Xu, Zhirui Jiang, Rong Sun, Sanhong Liu, James L. Kirkland, Weidong Zhang, Yu Sun","doi":"10.1101/2024.09.09.611999","DOIUrl":"https://doi.org/10.1101/2024.09.09.611999","url":null,"abstract":"Cellular senescence is a cell fate triggered by inherent or environmental stress and characterized by stable cell cycle arrest accompanied by a hypersecretory feature, termed as the senescence-associated secretory phenotype (SASP). Senescent cell burden increases with natural aging, functionally contributing to age-related organ dysfunction and multiple disorders. In this study, we performed a large scale screening of a natural product library for senotherapeutic candidates by assessing their effects on human senescent cells. Apigenin, a dietary flavonoid previously reported with antioxidant and anti-inflammatory activities, exhibited a prominent capacity in targeting senescent cells as a senomorphic agent. In senescent cells, apigenin blocks the interactions between ATM/p38 and HSPA8, thus preventing transition of the acute stress-associated phenotype (ASAP) towards the SASP. Mechanistically, apigenin targets peroxiredoxin 6 (PRDX6), an intracellular redox-active molecule, suppressing the iPLA2 activity of PRDX6 and disrupting downstream reactions underlying the SASP development. Without reversing cellular senescence, apigenin deprives cancer cells of malignancy acquired from senescent stromal cells in culture, while reducing chemoresistance upon combination with chemotherapy in anticancer regimens. In preclinical trials, apigenin administration improves physical function of animals prematurely aged after whole body irradiation, alleviating physical frailty and cognitive impairment. Overall, our study demonstrates the potential of exploiting a naturally derived compound with senomorphic capacity to achieve geroprotective effects by modulating the SASP, thus providing a research platform for future exploration of novel natural agents against age-related conditions.","PeriodicalId":501590,"journal":{"name":"bioRxiv - Cell Biology","volume":"9 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142189982","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: 10.1101/2024.09.09.612041
Judith A Sharp, Emily Sparago, Rachael Thomas, Kaitlyn Alimenti, Wei Wang, Michael D Blower
SAF-A is conserved throughout vertebrates and has emerged as an important factor regulating a multitude of nuclear functions, including lncRNA localization, gene expression, and splicing. SAF-A has several functional domains, including an N-terminal SAP domain that binds directly to DNA. Phosphorylation of SAP domain serines S14 and S26 are important for SAF-A localization and function during mitosis, however whether these serines are involved in interphase functions of SAF-A is not known. In this study we tested for the role of the SAP domain, and SAP domain serines S14 and S26 in X chromosome inactivation, protein dynamics, gene expression, splicing, and cell proliferation. Here we show that the SAP domain serines S14 and S26 are required to maintain XIST RNA localization and polycomb-dependent histone modifications on the inactive X chromosome in female cells. In addition, we present evidence that an Xi localization signal resides in the SAP domain. We found that that the SAP domain is not required to maintain gene expression and plays only a minor role in mRNA splicing. In contrast, the SAF-A SAP domain, in particular serines S14 and S26, are required for normal protein dynamics, and to maintain normal cell proliferation. We propose a model whereby dynamic phosphorylation of SAF-A serines S14 and S26 mediates rapid turnover of SAF-A interactions with DNA during interphase.
SAF-A在整个脊椎动物中都是保守的，它已成为调节多种核功能的重要因子，包括lncRNA定位、基因表达和剪接。SAF-A 有几个功能域，包括一个直接与 DNA 结合的 N 端 SAP 结构域。SAP结构域丝氨酸S14和S26的磷酸化对有丝分裂期间SAF-A的定位和功能非常重要，但这些丝氨酸是否参与了SAF-A的间期功能尚不清楚。在本研究中，我们检测了 SAP 结构域、SAP 结构域丝氨酸 S14 和 S26 在 X 染色体失活、蛋白质动力学、基因表达、剪接和细胞增殖中的作用。在这里，我们发现 SAP 结构域丝氨酸 S14 和 S26 是维持雌性细胞中无活性 X 染色体上 XIST RNA 定位和多聚酶依赖性组蛋白修饰所必需的。此外，我们还提出了Xi定位信号存在于SAP结构域的证据。我们发现，SAP结构域不是维持基因表达所必需的，而且在mRNA剪接过程中只起次要作用。与此相反，SAF-A 的 SAP 结构域，尤其是丝氨酸 S14 和 S26，是正常蛋白质动态和维持正常细胞增殖所必需的。我们提出了一个模型，根据该模型，SAF-A 丝氨酸 S14 和 S26 的动态磷酸化介导了 SAF-A 在间期与 DNA 相互作用的快速转换。
{"title":"Role of the SAF-A SAP domain in X inactivation, transcription, splicing, and cell proliferation","authors":"Judith A Sharp, Emily Sparago, Rachael Thomas, Kaitlyn Alimenti, Wei Wang, Michael D Blower","doi":"10.1101/2024.09.09.612041","DOIUrl":"https://doi.org/10.1101/2024.09.09.612041","url":null,"abstract":"SAF-A is conserved throughout vertebrates and has emerged as an important factor regulating a multitude of nuclear functions, including lncRNA localization, gene expression, and splicing. SAF-A has several functional domains, including an N-terminal SAP domain that binds directly to DNA. Phosphorylation of SAP domain serines S14 and S26 are important for SAF-A localization and function during mitosis, however whether these serines are involved in interphase functions of SAF-A is not known. In this study we tested for the role of the SAP domain, and SAP domain serines S14 and S26 in X chromosome inactivation, protein dynamics, gene expression, splicing, and cell proliferation. Here we show that the SAP domain serines S14 and S26 are required to maintain XIST RNA localization and polycomb-dependent histone modifications on the inactive X chromosome in female cells. In addition, we present evidence that an Xi localization signal resides in the SAP domain. We found that that the SAP domain is not required to maintain gene expression and plays only a minor role in mRNA splicing. In contrast, the SAF-A SAP domain, in particular serines S14 and S26, are required for normal protein dynamics, and to maintain normal cell proliferation. We propose a model whereby dynamic phosphorylation of SAF-A serines S14 and S26 mediates rapid turnover of SAF-A interactions with DNA during interphase.","PeriodicalId":501590,"journal":{"name":"bioRxiv - Cell Biology","volume":"30 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142189988","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: 10.1101/2024.09.09.612156
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":"11 1","pages":""},"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}
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