Pub Date : 2024-09-16DOI: 10.1101/2024.09.14.613065
Seyede Atefe Hosseini, Viktoria Planz, Ernst HK Stelzer, Maike Windbergs, Francesco Pampaloni
We introduce a platform for the fabrication of customizable wound healing dressing. The platform integrates electrospun nanofibers, bioprinted hydrogels, and cellular spheroids into hierarchical, fiber-reinforced hybrid constructs. The construct leverages the mechanical strength of polycaprolactone (PCL) nanofibers and the ECM-like properties of GelMA/PEGDA hydrogel. These materials support the incorporation of bone marrow-derived mesenchymal stem cell (BM-hMSC) spheroids, which act as a supportive "cell niche," enhancing the viability of the hMSC during and after bioprinting, and facilitating their spreading across the construct during the maturation phase. The characterization of the hybrid constructs demonstrated strong structural integrity and enhanced mechanical properties, making them well-suited for clinical wound dressing applications. In vitro assays, including live/dead staining, MTT assays, and scratch assays, revealed increased cell attachment, proliferation, and migration. The spheroids maintained their viability over extended periods, significantly contributing to wound closure in the scratch assay. This innovative approach, which combines electrospinning and light-based bioprinting, offers a promising strategy for the development of customizable wound dressings that closely adapt to the complex architecture of human skin. The bioprinting approach allows for the creation of tailored geometries for specific clinical requirements. Future research will focus on optimizing scaffold design and conducting long-term in vivo studies to validate the platform's clinical potential.
{"title":"A hybrid bioprinting-electrospinning platform integrating nanofibers and mesenchymal cell spheroids for customizable wound healing dressings","authors":"Seyede Atefe Hosseini, Viktoria Planz, Ernst HK Stelzer, Maike Windbergs, Francesco Pampaloni","doi":"10.1101/2024.09.14.613065","DOIUrl":"https://doi.org/10.1101/2024.09.14.613065","url":null,"abstract":"We introduce a platform for the fabrication of customizable wound healing dressing. The platform integrates electrospun nanofibers, bioprinted hydrogels, and cellular spheroids into hierarchical, fiber-reinforced hybrid constructs. The construct leverages the mechanical strength of polycaprolactone (PCL) nanofibers and the ECM-like properties of GelMA/PEGDA hydrogel. These materials support the incorporation of bone marrow-derived mesenchymal stem cell (BM-hMSC) spheroids, which act as a supportive \"cell niche,\" enhancing the viability of the hMSC during and after bioprinting, and facilitating their spreading across the construct during the maturation phase. The characterization of the hybrid constructs demonstrated strong structural integrity and enhanced mechanical properties, making them well-suited for clinical wound dressing applications. In vitro assays, including live/dead staining, MTT assays, and scratch assays, revealed increased cell attachment, proliferation, and migration. The spheroids maintained their viability over extended periods, significantly contributing to wound closure in the scratch assay. This innovative approach, which combines electrospinning and light-based bioprinting, offers a promising strategy for the development of customizable wound dressings that closely adapt to the complex architecture of human skin. The bioprinting approach allows for the creation of tailored geometries for specific clinical requirements. Future research will focus on optimizing scaffold design and conducting long-term in vivo studies to validate the platform's clinical potential.","PeriodicalId":501590,"journal":{"name":"bioRxiv - Cell Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142265098","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 : 2024-09-16DOI: 10.1101/2024.09.16.613219
Krisna Prak, Christin Luft, Eliona Tsefou, Carlos Chavez-Olortegui, Janos Kriston-Vizi, Robin Ketteler, Vania Braga
Snakebite accidents are prevalent worldwide and cause a spectrum of severe clinical manifestations and reduction of patient quality of life and economic income. L-amino acid oxidase (LAAO) is a highly toxic enzyme present in various venoms that causes tissue necrosis, oedema, coagulopathies, and organ failure. Here we investigate the mechanisms of LAAO cytotoxicity preceding cell death using recombinant LAAO and a catalytic inactive LAAO mutant. Wild-type LAAO uptake by cells leads to a decrease in lysosome number and size and inhibition of autophagy flux. Mitochondria function is also impaired by severe proton leakage leading to mitochondrial fission. Despite engulfment by autophagosomes, clearance of mitochondria is prevented by the lysosomal defects. The coordinate multi-organelle dysfunction strongly perturbs energy production, cell metabolism and clearance of defective organelles by autophagy, thereby triggering an irreversible destructive path. Considering the fast organelle impairment, strategies to reduce multi-organelle injury after LAAO exposure may be effective to maintain critical cell functions and strengthen adaptive responses against cytotoxicity.
{"title":"Functional analyses and integrated mechanisms of cellular destruction by L-amino acid oxidase","authors":"Krisna Prak, Christin Luft, Eliona Tsefou, Carlos Chavez-Olortegui, Janos Kriston-Vizi, Robin Ketteler, Vania Braga","doi":"10.1101/2024.09.16.613219","DOIUrl":"https://doi.org/10.1101/2024.09.16.613219","url":null,"abstract":"Snakebite accidents are prevalent worldwide and cause a spectrum of severe clinical manifestations and reduction of patient quality of life and economic income. L-amino acid oxidase (LAAO) is a highly toxic enzyme present in various venoms that causes tissue necrosis, oedema, coagulopathies, and organ failure. Here we investigate the mechanisms of LAAO cytotoxicity preceding cell death using recombinant LAAO and a catalytic inactive LAAO mutant. Wild-type LAAO uptake by cells leads to a decrease in lysosome number and size and inhibition of autophagy flux. Mitochondria function is also impaired by severe proton leakage leading to mitochondrial fission. Despite engulfment by autophagosomes, clearance of mitochondria is prevented by the lysosomal defects. The coordinate multi-organelle dysfunction strongly perturbs energy production, cell metabolism and clearance of defective organelles by autophagy, thereby triggering an irreversible destructive path. Considering the fast organelle impairment, strategies to reduce multi-organelle injury after LAAO exposure may be effective to maintain critical cell functions and strengthen adaptive responses against cytotoxicity.","PeriodicalId":501590,"journal":{"name":"bioRxiv - Cell Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142265094","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 : 2024-09-16DOI: 10.1101/2024.09.14.613037
Wasifa Naushad, Lakmini S Premadasa, Bryson C Okeoma, Mahesh Mohan, Chioma M Okeoma
Persistence of human immunodeficiency virus (HIV) latent reservoir is the major challenge to HIV cure because the latent reservoir is not eliminated by antiretroviral therapy (ART), and they serve as sources for viral rebound upon cessation of ART. Mechanisms regulating viral persistence are not well understood. This study used model systems of post-integration latency to explore the role of basal ganglia (BG) isolated extracellular condensates (ECs) in reprogramming HIV latent cells. We found that BG ECs from uninfected macaques (VEH) and SIV infected macaques (VEH|SIV) activate latent HIV transcription in various model systems. VEH and VEH|SIV ECs significantly increased expression of viral antigen in latently infected cells. Activation of viral transcription, antigen expression, and latency reactivation was inhibited by ECs from the brain of macaques treated with Delta-9-tetrahydrocannabinol (THC) and infected with SIV (THC|SIV). Virus produced by latently infected cells treated with VEH|SIV ECs potentiated cell-cell and cell-free HIV transmission. VEH|SIV ECs also reversed dexamethasone-mediated inhibition of HIV transcription while TNFα-mediated reactivation of latency was reversed by THC|SIV ECs. Transcriptome and secretome analyses of total RNA and supernatants from latently infected cells treated with ECs revealed significant alteration in gene expression and cytokine secretion. THC|SIV ECs increased secretion of Th2 and decreased secretion of proinflammatory cytokines. Most strikingly, while VEH/SIV ECs robustly induced HIV RNA in latently HIV-infected cells, long-term low-dose THC administration enriched ECs for anti-inflammatory cargo that significantly diminished their ability to reactivate latent HIV, an indication that ECs are endogenous host factors that may regulate HIV persistence.
人类免疫缺陷病毒（HIV）潜伏库的持续存在是治愈 HIV 的主要挑战，因为抗逆转录病毒疗法（ART）无法消除潜伏库，而且在停止抗逆转录病毒疗法后，潜伏库会成为病毒反弹的源头。病毒持续存在的调节机制尚不十分清楚。本研究利用整合后潜伏的模型系统来探索基底节（BG）分离的细胞外凝集素（ECs）在重编程 HIV 潜伏细胞中的作用。我们发现，来自未感染猕猴（VEH）和SIV感染猕猴（VEH|SIV）的基底节细胞外凝集素能在各种模型系统中激活潜伏的HIV转录。VEH和VEH|SIV ECs能显著增加潜伏感染细胞中病毒抗原的表达。用δ-9-四氢大麻酚（THC）处理并感染了SIV（THC|SIV）的猕猴大脑中的ECs抑制了病毒转录的激活、抗原的表达和潜伏期的重新激活。经 VEH|SIV ECs 处理的潜伏感染细胞产生的病毒可增强细胞-细胞和无细胞 HIV 传播。VEH|SIV ECs还能逆转地塞米松介导的HIV转录抑制，而THC|SIV ECs能逆转TNFα介导的潜伏期再激活。对用ECs处理的潜伏感染细胞的总RNA和上清液进行转录组和分泌组分析，发现基因表达和细胞因子分泌发生了显著变化。THC|SIV ECs 增加了 Th2 的分泌，减少了促炎细胞因子的分泌。最引人注目的是，虽然 VEH/SIV ECs 能在潜伏的 HIV 感染细胞中强力诱导 HIV RNA，但长期低剂量 THC 给药能使 ECs 富含抗炎物质，从而显著降低其重新激活潜伏 HIV 的能力，这表明 ECs 是可能调节 HIV 持久性的内源性宿主因子。
{"title":"Extracellular condensates (ECs) are endogenous modulators of HIV transcription and latency reactivation","authors":"Wasifa Naushad, Lakmini S Premadasa, Bryson C Okeoma, Mahesh Mohan, Chioma M Okeoma","doi":"10.1101/2024.09.14.613037","DOIUrl":"https://doi.org/10.1101/2024.09.14.613037","url":null,"abstract":"Persistence of human immunodeficiency virus (HIV) latent reservoir is the major challenge to HIV cure because the latent reservoir is not eliminated by antiretroviral therapy (ART), and they serve as sources for viral rebound upon cessation of ART. Mechanisms regulating viral persistence are not well understood. This study used model systems of post-integration latency to explore the role of basal ganglia (BG) isolated extracellular condensates (ECs) in reprogramming HIV latent cells. We found that BG ECs from uninfected macaques (VEH) and SIV infected macaques (VEH|SIV) activate latent HIV transcription in various model systems. VEH and VEH|SIV ECs significantly increased expression of viral antigen in latently infected cells. Activation of viral transcription, antigen expression, and latency reactivation was inhibited by ECs from the brain of macaques treated with Delta-9-tetrahydrocannabinol (THC) and infected with SIV (THC|SIV). Virus produced by latently infected cells treated with VEH|SIV ECs potentiated cell-cell and cell-free HIV transmission. VEH|SIV ECs also reversed dexamethasone-mediated inhibition of HIV transcription while TNFα-mediated reactivation of latency was reversed by THC|SIV ECs. Transcriptome and secretome analyses of total RNA and supernatants from latently infected cells treated with ECs revealed significant alteration in gene expression and cytokine secretion. THC|SIV ECs increased secretion of Th2 and decreased secretion of proinflammatory cytokines. Most strikingly, while VEH/SIV ECs robustly induced HIV RNA in latently HIV-infected cells, long-term low-dose THC administration enriched ECs for anti-inflammatory cargo that significantly diminished their ability to reactivate latent HIV, an indication that ECs are endogenous host factors that may regulate HIV persistence.","PeriodicalId":501590,"journal":{"name":"bioRxiv - Cell Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142265137","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 : 2024-09-16DOI: 10.1101/2024.09.16.612913
Charles A. Day, Alyssa Langfald, Tana Lukes, Hanna Middlebrook, Kevin T. Vaughan, David J. Daniels, Edward H. Hinchcliffe
At anaphase, spindle microtubules (MTs) position the cleavage furrow and trigger actomyosin assembly by localizing the small GTPase RhoA and the scaffolding protein anillin to a narrow band along the equatorial cortex. Using vertebrate somatic cells we examined the temporal control of furrow assembly. Although its positioning commences at anaphase onset, furrow maturation is not complete until ~10-11 min later. The maintenance of the RhoA/anillin scaffold initially requires continuous signaling from the spindle; loss of either MTs or polo-like kinase 1 (Plk1) activity prevents proper RhoA/anillin localization to the equator, thereby disrupting furrowing. However, we find that at ~6 min post-anaphase, the cortex becomes committed to furrowing; loss of either MTs or Plk1 after this stage does not prevent eventual furrowing, even though at this point the contractile apparatus has not fully matured. Also at this stage, the RhoA/anillin scaffold at the equator becomes permanent. Surprisingly, concurrent loss of both MTs and Plk1 activity following the commitment to furrowing stage results in persistent, asymmetric half-furrows, with only one cortical hemisphere retaining RhoA/anillin, and undergoing regression. This phenotype is reminiscent of asymmetric furrows caused by a physical block between spindle and cortex, or by acentric spindle positioning. The formation of these persistent half-furrows suggests a potential feedback mechanism between the spindle and the cortex that maintains cortical competency along the presumptive equatorial region prior to the commitment to furrowing stage of cytokinesis, thereby ensuring the eventual ingression of a symmetric cleavage furrow.
{"title":"Commitment to cytokinetic furrowing requires the coordinate activity of microtubules and Plk1","authors":"Charles A. Day, Alyssa Langfald, Tana Lukes, Hanna Middlebrook, Kevin T. Vaughan, David J. Daniels, Edward H. Hinchcliffe","doi":"10.1101/2024.09.16.612913","DOIUrl":"https://doi.org/10.1101/2024.09.16.612913","url":null,"abstract":"At anaphase, spindle microtubules (MTs) position the cleavage furrow and trigger actomyosin assembly by localizing the small GTPase RhoA and the scaffolding protein anillin to a narrow band along the equatorial cortex. Using vertebrate somatic cells we examined the temporal control of furrow assembly. Although its positioning commences at anaphase onset, furrow maturation is not complete until ~10-11 min later. The maintenance of the RhoA/anillin scaffold initially requires continuous signaling from the spindle; loss of either MTs or polo-like kinase 1 (Plk1) activity prevents proper RhoA/anillin localization to the equator, thereby disrupting furrowing. However, we find that at ~6 min post-anaphase, the cortex becomes committed to furrowing; loss of either MTs or Plk1 after this stage does not prevent eventual furrowing, even though at this point the contractile apparatus has not fully matured. Also at this stage, the RhoA/anillin scaffold at the equator becomes permanent. Surprisingly, concurrent loss of both MTs and Plk1 activity following the commitment to furrowing stage results in persistent, asymmetric half-furrows, with only one cortical hemisphere retaining RhoA/anillin, and undergoing regression. This phenotype is reminiscent of asymmetric furrows caused by a physical block between spindle and cortex, or by acentric spindle positioning. The formation of these persistent half-furrows suggests a potential feedback mechanism between the spindle and the cortex that maintains cortical competency along the presumptive equatorial region prior to the commitment to furrowing stage of cytokinesis, thereby ensuring the eventual ingression of a symmetric cleavage furrow.","PeriodicalId":501590,"journal":{"name":"bioRxiv - Cell Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142265093","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 : 2024-09-16DOI: 10.1101/2024.09.16.613204
Afonso Mendes, Bruno M. Saraiva, Guillaume Jacquemet, Joao I. Mamede, Christophe Leterrier, Ricardo Henriques
From molecules to organelles, cells exhibit recurring structural motifs across multiple scales. Understanding these structures provides insights into their functional roles. While super-resolution microscopy can visualise such patterns, manual detection in large datasets is challenging and biased. We present the Structural Repetition Detector (SReD), an unsupervised computational framework that identifies repetitive biological structures by exploiting local texture repetition. SReD formulates structure detection as a similarity-matching problem between local image regions. It detects recurring patterns without prior knowledge or constraints on the imaging modality. We demonstrate SReD's capabilities on various fluorescence microscopy images. Quantitative analyses of three datasets highlight SReD's utility: estimating the periodicity of spectrin rings in neurons, detecting HIV-1 viral assembly, and evaluating microtubule dynamics modulated by EB3. Our open-source ImageJ and Fiji plugin enables unbiased analysis of repetitive structures across imaging modalities in diverse biological contexts.
{"title":"Structural Repetition Detector: multi-scale quantitative mapping of molecular complexes through microscopy","authors":"Afonso Mendes, Bruno M. Saraiva, Guillaume Jacquemet, Joao I. Mamede, Christophe Leterrier, Ricardo Henriques","doi":"10.1101/2024.09.16.613204","DOIUrl":"https://doi.org/10.1101/2024.09.16.613204","url":null,"abstract":"From molecules to organelles, cells exhibit recurring structural motifs across multiple scales. Understanding these structures provides insights into their functional roles. While super-resolution microscopy can visualise such patterns, manual detection in large datasets is challenging and biased. We present the Structural Repetition Detector (SReD), an unsupervised computational framework that identifies repetitive biological structures by exploiting local texture repetition. SReD formulates structure detection as a similarity-matching problem between local image regions. It detects recurring patterns without prior knowledge or constraints on the imaging modality. We demonstrate SReD's capabilities on various fluorescence microscopy images. Quantitative analyses of three datasets highlight SReD's utility: estimating the periodicity of spectrin rings in neurons, detecting HIV-1 viral assembly, and evaluating microtubule dynamics modulated by EB3. Our open-source ImageJ and Fiji plugin enables unbiased analysis of repetitive structures across imaging modalities in diverse biological contexts.","PeriodicalId":501590,"journal":{"name":"bioRxiv - Cell Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142265092","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 : 2024-09-16DOI: 10.1101/2024.09.15.613154
Sujan Chatterjee, Loretta Viera Preval, Zachary Sin, Nguyen Tran, Kevin Ritter, Xue Bessie Su, Jakob P. Centlivre, Saharat Jolak Ragsac, Richard Van, Seungman Park, Mira Han, Qian Liu, Brian P Hedlund, Adolfo Saiardi, Henning Jessen, Dr Prasun Guha
HDACs (histone deacetylase) play a crucial role in regulating gene expression, and the inhibition of these enzymes is gaining attention as a promising therapeutic approach for cancer treatment. Despite their significant physiological and clinical importance, the mechanisms of HDAC activation remain poorly understood. This study reveals that inositol polyphosphate multikinase (IPMK) is essential for activating HDAC1 and HDAC3 in cell lines and mice. IPMK deletion or inactivation of its kinase activity selectively impairs HDAC1/3's deacetylase activity, significantly influencing gene expression. Disruption of the IPMK-HDAC1/3 epigenetic axis results in transcriptional upregulation of matrix metalloproteinase (MMP) genes, exacerbating cell and intestinal permeability. Remarkably, treatment of IPMK KO cells with cell-permeable inositol hexaphosphate (InsP6) rescues these defects. This study elucidates the role of IPMK's kinase activity in HDAC1/3 activation and its implications for intestinal barrier function.
{"title":"Inositol Hexaphosphate (InsP6) Activates the HDAC1/3 Epigenetic Axis to Maintain Intestinal Barrier Function","authors":"Sujan Chatterjee, Loretta Viera Preval, Zachary Sin, Nguyen Tran, Kevin Ritter, Xue Bessie Su, Jakob P. Centlivre, Saharat Jolak Ragsac, Richard Van, Seungman Park, Mira Han, Qian Liu, Brian P Hedlund, Adolfo Saiardi, Henning Jessen, Dr Prasun Guha","doi":"10.1101/2024.09.15.613154","DOIUrl":"https://doi.org/10.1101/2024.09.15.613154","url":null,"abstract":"HDACs (histone deacetylase) play a crucial role in regulating gene expression, and the inhibition of these enzymes is gaining attention as a promising therapeutic approach for cancer treatment. Despite their significant physiological and clinical importance, the mechanisms of HDAC activation remain poorly understood. This study reveals that inositol polyphosphate multikinase (IPMK) is essential for activating HDAC1 and HDAC3 in cell lines and mice. IPMK deletion or inactivation of its kinase activity selectively impairs HDAC1/3's deacetylase activity, significantly influencing gene expression. Disruption of the IPMK-HDAC1/3 epigenetic axis results in transcriptional upregulation of matrix metalloproteinase (MMP) genes, exacerbating cell and intestinal permeability. Remarkably, treatment of IPMK KO cells with cell-permeable inositol hexaphosphate (InsP6) rescues these defects. This study elucidates the role of IPMK's kinase activity in HDAC1/3 activation and its implications for intestinal barrier function.","PeriodicalId":501590,"journal":{"name":"bioRxiv - Cell Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142265099","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 : 2024-09-16DOI: 10.1101/2024.09.14.613007
Julia Vorhauser, Theodoros I. Roumeliotis, Jacky K Leung, David Coupe, Lu Yu, Kristin Bohlig, Andre Nadler, Jyoti S Choudhary, Jorg Mansfeld
Cells are constantly exposed to reactive oxygen species (ROS) from both intrinsic and extrinsic sources. ROS influence proliferation and cell fate through cysteine oxidation (S-sulfenylation), but specific targets and mechanisms remain unclear. Here, we use redox proteomics to identify cell cycle-coordinated S-sulfenylation and investigate its role in cell cycle decision-making. We find that oxidation of a single cysteine (C41) on the CDK inhibitor p21 during G2 phase determines whether cells continue to proliferate. Preventing C41 oxidation redirects p21 from CDK4-Cyclin D to CDK2-Cyclin A, affecting a negative feedback loop that regulates p21 stability. When C41 cannot be oxidized in G2, daughter cells inherit more p21 from their mother, which decreases proliferation and induces senescence upon irradiation. We therefore identify a redox switch in a core cell cycle regulator that governs the decision to proliferate or exit the cell cycle and present a cell cycle-resolved S-sulfenyl proteome as a valuable resource.
{"title":"Cell cycle-dependent S-sulfenyl proteomics uncover a redox switch in p21-CDK feedback governing the proliferation-senescence decision","authors":"Julia Vorhauser, Theodoros I. Roumeliotis, Jacky K Leung, David Coupe, Lu Yu, Kristin Bohlig, Andre Nadler, Jyoti S Choudhary, Jorg Mansfeld","doi":"10.1101/2024.09.14.613007","DOIUrl":"https://doi.org/10.1101/2024.09.14.613007","url":null,"abstract":"Cells are constantly exposed to reactive oxygen species (ROS) from both intrinsic and extrinsic sources. ROS influence proliferation and cell fate through cysteine oxidation (S-sulfenylation), but specific targets and mechanisms remain unclear. Here, we use redox proteomics to identify cell cycle-coordinated S-sulfenylation and investigate its role in cell cycle decision-making. We find that oxidation of a single cysteine (C41) on the CDK inhibitor p21 during G2 phase determines whether cells continue to proliferate. Preventing C41 oxidation redirects p21 from CDK4-Cyclin D to CDK2-Cyclin A, affecting a negative feedback loop that regulates p21 stability. When C41 cannot be oxidized in G2, daughter cells inherit more p21 from their mother, which decreases proliferation and induces senescence upon irradiation. We therefore identify a redox switch in a core cell cycle regulator that governs the decision to proliferate or exit the cell cycle and present a cell cycle-resolved S-sulfenyl proteome as a valuable resource.","PeriodicalId":501590,"journal":{"name":"bioRxiv - Cell Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142265100","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 : 2024-09-16DOI: 10.1101/2024.09.16.613328
Jingchao Zhang, Greg Donahue, Michael B. Gilbert, Tomer Lapidot, Dario Nicetto, Kenneth S. Zaret
H3K9me3-heterochromatin, established by lysine methyltransferases (KMTs) and compacted by HP1 isoforms, represses alternative lineage genes and DNA repeats. Our understanding of H3K9me3-heterochromatin stability is presently limited to individual domains and DNA repeats. We engineered Suv39h2 KO mouse embryonic stem cells to degrade remaining two H3K9me3-KMTs within one hour and found that both passive dilution and active removal contribute to H3K9me3 decay within 12-24 hours. We discovered four different H3K9me3 decay rates across the genome and chromatin features and transcription factor binding patterns that predict the stability classes. A binary switch governs heterochromatin compaction, with HP1 rapidly dissociating from heterochromatin upon KMTs depletion and a particular threshold level of HP1 limiting pioneer factor binding, chromatin opening, and exit from pluripotency within 12 hr. Unexpectedly, receding H3K9me3 domains unearth residual HP1beta peaks enriched with heterochromatin-inducing proteins. Our findings reveal distinct H3K9me3-heterochromatin maintenance dynamics governing gene networks and repeats that together safeguard pluripotency.
{"title":"Distinct H3K9me3 heterochromatin maintenance dynamics govern different gene programs and repeats in pluripotent cells","authors":"Jingchao Zhang, Greg Donahue, Michael B. Gilbert, Tomer Lapidot, Dario Nicetto, Kenneth S. Zaret","doi":"10.1101/2024.09.16.613328","DOIUrl":"https://doi.org/10.1101/2024.09.16.613328","url":null,"abstract":"H3K9me3-heterochromatin, established by lysine methyltransferases (KMTs) and compacted by HP1 isoforms, represses alternative lineage genes and DNA repeats. Our understanding of H3K9me3-heterochromatin stability is presently limited to individual domains and DNA repeats. We engineered Suv39h2 KO mouse embryonic stem cells to degrade remaining two H3K9me3-KMTs within one hour and found that both passive dilution and active removal contribute to H3K9me3 decay within 12-24 hours. We discovered four different H3K9me3 decay rates across the genome and chromatin features and transcription factor binding patterns that predict the stability classes. A binary switch governs heterochromatin compaction, with HP1 rapidly dissociating from heterochromatin upon KMTs depletion and a particular threshold level of HP1 limiting pioneer factor binding, chromatin opening, and exit from pluripotency within 12 hr. Unexpectedly, receding H3K9me3 domains unearth residual HP1beta peaks enriched with heterochromatin-inducing proteins. Our findings reveal distinct H3K9me3-heterochromatin maintenance dynamics governing gene networks and repeats that together safeguard pluripotency.","PeriodicalId":501590,"journal":{"name":"bioRxiv - Cell Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142265096","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 : 2024-09-16DOI: 10.1101/2024.09.14.613056
Jiahao Zheng, Boran Li, Lanxin Jia, Jiayou Zhang, Zheng Gong, Yang Le, Xuanxuan Nian, Xuedan Li, Bo Liu, Daiguan Yu, Changgui Li, Zhegang Zhang
Madin-Darby canine kidney (MDCK) cells are the recognized cell strain for influenza vaccine production. However, the tumorigenic potential of MDCK cells raises concerns about their use in biological product manufacturing. To reduce MDCK cells’ tumorigenicity and ensure the safety of influenza vaccine production, a B-cell lymphoma extra-large (Bcl-xL) gene, which plays a pivotal role in apoptosis regulation, was knocked-out in original MDCK cells by CRISPR-Cas9 gene editing technology, so that a homozygous MDCK-Bcl-xL-/- cell strain was acquired and named as BY-02. Compared with original MDCK cells, the proliferation and migration ability of BY-02 were significantly reduced, while apoptosis level was significantly increased, the endogenous mitochondrial apoptotic pathway were also modulated after Bcl-xL knock-out in MDCK cells. For tumor formation assays in nude mouse tests, all ten mice injected with original MDCK cells presented tumors growth in the injection site, in contrast to only one mouse injected with BY-02 cells presented tumors growth. These findings suggest that Bcl-xL knock-down is an effective strategy to inhibit tumor formation in MDCK cells, making BY-02 a promising genetically engineered cell strain for influenza vaccine production.
{"title":"Tumorigencity decrease in Bcl-xL deficient MDCK cells ensuring the safety for influenza vaccine production","authors":"Jiahao Zheng, Boran Li, Lanxin Jia, Jiayou Zhang, Zheng Gong, Yang Le, Xuanxuan Nian, Xuedan Li, Bo Liu, Daiguan Yu, Changgui Li, Zhegang Zhang","doi":"10.1101/2024.09.14.613056","DOIUrl":"https://doi.org/10.1101/2024.09.14.613056","url":null,"abstract":"Madin-Darby canine kidney (MDCK) cells are the recognized cell strain for influenza vaccine production. However, the tumorigenic potential of MDCK cells raises concerns about their use in biological product manufacturing. To reduce MDCK cells’ tumorigenicity and ensure the safety of influenza vaccine production, a B-cell lymphoma extra-large (Bcl-xL) gene, which plays a pivotal role in apoptosis regulation, was knocked-out in original MDCK cells by CRISPR-Cas9 gene editing technology, so that a homozygous MDCK-Bcl-xL-/- cell strain was acquired and named as BY-02. Compared with original MDCK cells, the proliferation and migration ability of BY-02 were significantly reduced, while apoptosis level was significantly increased, the endogenous mitochondrial apoptotic pathway were also modulated after Bcl-xL knock-out in MDCK cells. For tumor formation assays in nude mouse tests, all ten mice injected with original MDCK cells presented tumors growth in the injection site, in contrast to only one mouse injected with BY-02 cells presented tumors growth. These findings suggest that Bcl-xL knock-down is an effective strategy to inhibit tumor formation in MDCK cells, making BY-02 a promising genetically engineered cell strain for influenza vaccine production.","PeriodicalId":501590,"journal":{"name":"bioRxiv - Cell Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142265101","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-15DOI: 10.1101/2024.09.14.613089
Shangming M Tang, Jennifer Koo, Mohammad Pourhosseinzadeh, Emerald Nguyen, Natalie Liu, Christopher Ma, Hanyu Lu, Monica Lee, Neil Hunter
Chromosomal linkages formed through crossover recombination are essential for accurate segregation of homologous chromosomes during meiosis1. DNA events of recombination are spatially and functionally linked to structural components of meiotic chromosomes. Imperatively, biased resolution of double-Holliday junction (dHJ) intermediates into crossovers occurs within the synaptonemal complex (SC), the meiosis-specific structure that mediates homolog synapsis during the pachytene stage. However, the SC's role in crossing over remains unclear. Here we show that SC promotes crossover-specific resolution by protecting dHJs from unscheduled and aberrant resolution. When key SC components are conditionally inactivated during pachytene, dHJs are resolved into noncrossover products by Sgs1-Top3-Rmi1 (STR), the yeast ortholog of the human BLM complex. Cohesin, the core component of SC lateral elements, plays a primary role in chromatin organization and is required to maintain both SCs and crossover recombination complexes (CRCs) during pachytene. SC central region component Zip1 is required to maintain CRCs even when dHJs are stabilized by inactivating STR. Reciprocally, SC stability requires continuous presence of CRCs, an unanticipated interdependence with important implications for SC dynamics. In conclusion, through hierarchical and interdependent functions of its key components, the SC enables crossover-specific dHJ resolution and thereby ensures the linkage and segregation of homologous chromosomes.
{"title":"Synaptonemal complex protects double-Holliday junctions during meiosis","authors":"Shangming M Tang, Jennifer Koo, Mohammad Pourhosseinzadeh, Emerald Nguyen, Natalie Liu, Christopher Ma, Hanyu Lu, Monica Lee, Neil Hunter","doi":"10.1101/2024.09.14.613089","DOIUrl":"https://doi.org/10.1101/2024.09.14.613089","url":null,"abstract":"Chromosomal linkages formed through crossover recombination are essential for accurate segregation of homologous chromosomes during meiosis1. DNA events of recombination are spatially and functionally linked to structural components of meiotic chromosomes. Imperatively, biased resolution of double-Holliday junction (dHJ) intermediates into crossovers occurs within the synaptonemal complex (SC), the meiosis-specific structure that mediates homolog synapsis during the pachytene stage. However, the SC's role in crossing over remains unclear. Here we show that SC promotes crossover-specific resolution by protecting dHJs from unscheduled and aberrant resolution. When key SC components are conditionally inactivated during pachytene, dHJs are resolved into noncrossover products by Sgs1-Top3-Rmi1 (STR), the yeast ortholog of the human BLM complex. Cohesin, the core component of SC lateral elements, plays a primary role in chromatin organization and is required to maintain both SCs and crossover recombination complexes (CRCs) during pachytene. SC central region component Zip1 is required to maintain CRCs even when dHJs are stabilized by inactivating STR. Reciprocally, SC stability requires continuous presence of CRCs, an unanticipated interdependence with important implications for SC dynamics. In conclusion, through hierarchical and interdependent functions of its key components, the SC enables crossover-specific dHJ resolution and thereby ensures the linkage and segregation of homologous chromosomes.","PeriodicalId":501590,"journal":{"name":"bioRxiv - Cell Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142265138","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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