Pub Date : 2024-09-09DOI: 10.1101/2024.09.09.611003
Genta Ishikawa, Xueyan Peng, John McGovern, Alexander Ghincea, Samuel Woo, Daisuke Okuno, Sheeline Yu, Chris J. Lee, Angela Liu, Tina Saber, Buqu Hu, Ying Sun, Huanxing Sun, Karam Al Jumaily, Carol Feghali-Bostwick, Tomokazu S. Sumida, Maor Sauler, Changwan Ryu, Erica L. Herzog
Tissue fibrosis contributes to pathology in vital organs including the lung. Curative therapies are scant. Myofibroblasts, pivotal effector cells in tissue fibrosis, accumulate via incompletely understood interactions with their microenvironment. In an investigative platform grounded in experimental lung biology, we find that sympathetic innervation stimulates fibrotic remodeling via noradrenergic α1-adrenergic receptor engagement in myofibroblasts. We demonstrate the anti-fibrotic potential of targeted sympathetic denervation and pharmacological disruption of noradrenergic neurotransmitter functions mediated by α1-adrenoreceptors (α1-ARs). Using the α1-adrenoreceptor subtype D as a representative α1-AR, we discover direct noradrenergic input from sympathetic nerves to lung myofibroblasts utilizing established mouse models, genetic denervation, pharmacologic interventions, a newly invented transgenic mouse line, advanced tissue mimetics, and samples from patients with diverse forms of pulmonary fibrosis. The discovery of this previously unappreciated nerve-fibroblast axis in the lung demonstrates the crucial contribution of nerves to tissue repair and heralds a novel paradigm in fibrosis research.
组织纤维化会导致包括肺部在内的重要器官发生病变。治疗方法很少。肌成纤维细胞是组织纤维化的关键效应细胞，它们通过与其微环境的相互作用而积聚，但人们对其作用机制还不完全了解。在一个以实验肺生物学为基础的研究平台上，我们发现交感神经支配通过去甲肾上腺素能α1-肾上腺素能受体参与肌成纤维细胞，刺激纤维化重塑。我们证明了有针对性的交感神经去神经支配和药理学破坏由α1-肾上腺素受体（α1-ARs）介导的去肾上腺素能神经递质功能的抗纤维化潜力。以α1-肾上腺素受体亚型 D 为代表的α1-AR，我们利用已建立的小鼠模型、基因去势、药物干预、新发明的转基因小鼠品系、先进的组织模拟物以及不同形式肺纤维化患者的样本，发现了交感神经对肺肌成纤维细胞的去甲肾上腺素能直接输入。肺部神经-成纤维细胞轴的发现证明了神经对组织修复的重要贡献，并预示着纤维化研究的新范例。
{"title":"A Nerve-Fibroblast Axis in Mammalian Lung Fibrosis","authors":"Genta Ishikawa, Xueyan Peng, John McGovern, Alexander Ghincea, Samuel Woo, Daisuke Okuno, Sheeline Yu, Chris J. Lee, Angela Liu, Tina Saber, Buqu Hu, Ying Sun, Huanxing Sun, Karam Al Jumaily, Carol Feghali-Bostwick, Tomokazu S. Sumida, Maor Sauler, Changwan Ryu, Erica L. Herzog","doi":"10.1101/2024.09.09.611003","DOIUrl":"https://doi.org/10.1101/2024.09.09.611003","url":null,"abstract":"Tissue fibrosis contributes to pathology in vital organs including the lung. Curative therapies are scant. Myofibroblasts, pivotal effector cells in tissue fibrosis, accumulate via incompletely understood interactions with their microenvironment. In an investigative platform grounded in experimental lung biology, we find that sympathetic innervation stimulates fibrotic remodeling via noradrenergic α1-adrenergic receptor engagement in myofibroblasts. We demonstrate the anti-fibrotic potential of targeted sympathetic denervation and pharmacological disruption of noradrenergic neurotransmitter functions mediated by α1-adrenoreceptors (α1-ARs). Using the α1-adrenoreceptor subtype D as a representative α1-AR, we discover direct noradrenergic input from sympathetic nerves to lung myofibroblasts utilizing established mouse models, genetic denervation, pharmacologic interventions, a newly invented transgenic mouse line, advanced tissue mimetics, and samples from patients with diverse forms of pulmonary fibrosis. The discovery of this previously unappreciated nerve-fibroblast axis in the lung demonstrates the crucial contribution of nerves to tissue repair and heralds a novel paradigm in fibrosis research.","PeriodicalId":501590,"journal":{"name":"bioRxiv - Cell Biology","volume":"23 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142190066","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: 10.1101/2024.09.09.612036
Michelle M McWilliams, Faezeh Koohestani, Wendy N Jefferson, Sumedha Gunewardena, Kavya Shivashankar, Riley A Wertenberger, Carmen J Williams, T. Rajendra Kumar, Vargheese Mani Chennathukuzhi
Uterine fibroids (leiomyomas), benign tumors of the myometrial smooth muscle layer, are present in over 75% of women, often causing severe pain, menorrhagia and reproductive dysfunction. The molecular pathogenesis of fibroids is poorly understood. We previously showed that the loss of REST (RE-1 Silencing Transcription factor), a tumor suppressor, in fibroids leads to activation of PI3K/AKT-mTOR pathway. We report here a critical link between estrogen receptor alpha (ERα) and the loss of REST, via PRICKLE1. PRICKLE1 expression is markedly lower in leiomyomas, and the suppression of PRICKLE1 significantly down regulates REST protein levels. Conversely, overexpression of PRICKLE1 resulted in the restoration of REST in cultured primary leiomyoma smooth muscle cells (LSMCs). Crucially, mice exposed neonatally to environmental estrogens, proven risk factors for fibroids, expressed lower levels of PRICKLE1 and REST in the myometrium. Using mice that lack either endogenous estrogen (Lhb-/- mice) or ERα (Esr1-/- mice), we demonstrate that Prickle1 expression in the myometrium is suppressed by estrogen through ERα. Enhancer of zeste homolog 2 (EZH2) is known to participate in the repression of specific ERα target genes. Uterine leiomyomas express increased levels of EZH2 that inversely correlate with the expression of PRICKLE1. Using chromatin immunoprecipitation, we provide evidence for association of EZH2 with the PRICKLE1 promoter and for hypermethylation of H3K27 within the regulatory region of PRICKLE1 in leiomyomas. Additionally, siRNA mediated knockdown of EZH2 leads to restoration of PRICKLE1 in LSMCs. Collectively, our results identify a novel link between estrogen exposure and PRICKLE1/REST-regulated tumorigenic pathways in leiomyomas.
{"title":"Estrogen receptor alpha mediated repression of PRICKLE1 destabilizes REST and promotes uterine fibroid pathogenesis","authors":"Michelle M McWilliams, Faezeh Koohestani, Wendy N Jefferson, Sumedha Gunewardena, Kavya Shivashankar, Riley A Wertenberger, Carmen J Williams, T. Rajendra Kumar, Vargheese Mani Chennathukuzhi","doi":"10.1101/2024.09.09.612036","DOIUrl":"https://doi.org/10.1101/2024.09.09.612036","url":null,"abstract":"Uterine fibroids (leiomyomas), benign tumors of the myometrial smooth muscle layer, are present in over 75% of women, often causing severe pain, menorrhagia and reproductive dysfunction. The molecular pathogenesis of fibroids is poorly understood. We previously showed that the loss of REST (RE-1 Silencing Transcription factor), a tumor suppressor, in fibroids leads to activation of PI3K/AKT-mTOR pathway. We report here a critical link between estrogen receptor alpha (ERα) and the loss of REST, via PRICKLE1. PRICKLE1 expression is markedly lower in leiomyomas, and the suppression of PRICKLE1 significantly down regulates REST protein levels. Conversely, overexpression of PRICKLE1 resulted in the restoration of REST in cultured primary leiomyoma smooth muscle cells (LSMCs). Crucially, mice exposed neonatally to environmental estrogens, proven risk factors for fibroids, expressed lower levels of PRICKLE1 and REST in the myometrium. Using mice that lack either endogenous estrogen (Lhb-/- mice) or ERα (Esr1-/- mice), we demonstrate that Prickle1 expression in the myometrium is suppressed by estrogen through ERα. Enhancer of zeste homolog 2 (EZH2) is known to participate in the repression of specific ERα target genes. Uterine leiomyomas express increased levels of EZH2 that inversely correlate with the expression of PRICKLE1. Using chromatin immunoprecipitation, we provide evidence for association of EZH2 with the PRICKLE1 promoter and for hypermethylation of H3K27 within the regulatory region of PRICKLE1 in leiomyomas. Additionally, siRNA mediated knockdown of EZH2 leads to restoration of PRICKLE1 in LSMCs. Collectively, our results identify a novel link between estrogen exposure and PRICKLE1/REST-regulated tumorigenic pathways in leiomyomas.","PeriodicalId":501590,"journal":{"name":"bioRxiv - Cell Biology","volume":"14 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142189987","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: 10.1101/2024.09.09.611904
Sayantika Ghosh, John James, Badeer Ummat, Darius Vasco Köster
Adherens junctions (AJ) are E-cadherin-based adhesions at cell-cell contacts that connect the actin cytoskeleton of epithelial cells. The formation and maturation of these junctions is important in development, e.g. for the generation of epithelial tissues, and loss of adherens junctions is linked to metastasis in cancer. It is well established that AJ is a mechano-sensitive process involving the clustering of E-cadherins within the plasma membrane of cells and across adjacent cells, and the mechanical activation of α-catenins that connect E-cadherins with the actin cytoskeleton. However, how membrane mobility of E-cadherins and their organisation in time and space influence this process is less well understood, partly due to limitations to control the physical properties of cell membranes and perform high resolution in model organisms or cell monolayers. Here we place MCF7 cells labelled with fluorescent actin, e-cadherin, and α-catenin, on fluid-supported lipid bilayers containing the extracellular domain of cadherin as a biomimetic system to enable super resolution TIRF-SIM imaging of AJ. We found that MCF7 cells were able to attach and spread on these substrates, recruiting E-cadherin and α-catenin to form AJs that can mature and are mobile. Interestingly, we found that, depending on the mobility of E-cadherin within the SLB, distinct types of actin architecture emerge over time. Low mobility substrates support formin-based linear polymerisation while high mobility substrates support Arp2/3 -based branched actin polymerisation. These regions are spatially delimited within the cell and can change over time, giving rise to a mature state containing regions of both branched and linear actin.
{"title":"Membrane-tethered cadherin substrates reveal actin architecture at adherens junctions","authors":"Sayantika Ghosh, John James, Badeer Ummat, Darius Vasco Köster","doi":"10.1101/2024.09.09.611904","DOIUrl":"https://doi.org/10.1101/2024.09.09.611904","url":null,"abstract":"Adherens junctions (AJ) are E-cadherin-based adhesions at cell-cell contacts that connect the actin cytoskeleton of epithelial cells. The formation and maturation of these junctions is important in development, e.g. for the generation of epithelial tissues, and loss of adherens junctions is linked to metastasis in cancer. It is well established that AJ is a mechano-sensitive process involving the clustering of E-cadherins within the plasma membrane of cells and across adjacent cells, and the mechanical activation of α-catenins that connect E-cadherins with the actin cytoskeleton. However, how membrane mobility of E-cadherins and their organisation in time and space influence this process is less well understood, partly due to limitations to control the physical properties of cell membranes and perform high resolution in model organisms or cell monolayers. Here we place MCF7 cells labelled with fluorescent actin, e-cadherin, and α-catenin, on fluid-supported lipid bilayers containing the extracellular domain of cadherin as a biomimetic system to enable super resolution TIRF-SIM imaging of AJ. We found that MCF7 cells were able to attach and spread on these substrates, recruiting E-cadherin and α-catenin to form AJs that can mature and are mobile. Interestingly, we found that, depending on the mobility of E-cadherin within the SLB, distinct types of actin architecture emerge over time. Low mobility substrates support formin-based linear polymerisation while high mobility substrates support Arp2/3 -based branched actin polymerisation. These regions are spatially delimited within the cell and can change over time, giving rise to a mature state containing regions of both branched and linear actin.","PeriodicalId":501590,"journal":{"name":"bioRxiv - Cell Biology","volume":"54 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142189990","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: 10.1101/2024.09.09.612024
Luiz da Silva, Lia Paim, Ana Paula Menezes, Julia PC da Cunha, Susanne Bechstedt, Nathalie Cella
Maspin/SerpinB5 is an abundant and pleiotropic protein mostly expressed by epithelia. Initially described as a tumor suppressor, it has been reported as a regulator of cell adhesion, migration, and invasion. How intracellular Maspin orchestrates these processes is poorly understood. In this study, we utilized Affinity purification-Mass spectrometry (AP/MS) alongside in vitro reconstitution assays to establish that Maspin directly interacts with microtubules and microfilaments. Additionally, CRISPR/Cas9-mediated GFP tagging of endogenous Maspin, combined with immunostaining, revealed its localization at the cortical cytoskeleton and the mitotic spindle. Depletion of Maspin by RNAi and CRISPR/Cas9 in three distinct epithelial cell lines disrupts cell-cell adhesion, reorganizes the cytoskeleton and results in upregulation of mesenchymal markers during interphase. In mitotic cells, loss of Maspin induces abnormal cell rounding and rearrangement of cortical F-actin. Moreover, Maspin suppresses microtubule growth in vitro and in cells. Collectively, these results demonstrate that Maspin acts at the interface between the cytoskeleton and adhesion sites, directly modulating cell shape and preventing epithelial-mesenchymal transition.
{"title":"Maspin/SerpinB5 is a cytoskeleton-binding protein that regulates epithelial cell shape","authors":"Luiz da Silva, Lia Paim, Ana Paula Menezes, Julia PC da Cunha, Susanne Bechstedt, Nathalie Cella","doi":"10.1101/2024.09.09.612024","DOIUrl":"https://doi.org/10.1101/2024.09.09.612024","url":null,"abstract":"Maspin/SerpinB5 is an abundant and pleiotropic protein mostly expressed by epithelia. Initially described as a tumor suppressor, it has been reported as a regulator of cell adhesion, migration, and invasion. How intracellular Maspin orchestrates these processes is poorly understood. In this study, we utilized Affinity purification-Mass spectrometry (AP/MS) alongside in vitro reconstitution assays to establish that Maspin directly interacts with microtubules and microfilaments. Additionally, CRISPR/Cas9-mediated GFP tagging of endogenous Maspin, combined with immunostaining, revealed its localization at the cortical cytoskeleton and the mitotic spindle. Depletion of Maspin by RNAi and CRISPR/Cas9 in three distinct epithelial cell lines disrupts cell-cell adhesion, reorganizes the cytoskeleton and results in upregulation of mesenchymal markers during interphase. In mitotic cells, loss of Maspin induces abnormal cell rounding and rearrangement of cortical F-actin. Moreover, Maspin suppresses microtubule growth in vitro and in cells. Collectively, these results demonstrate that Maspin acts at the interface between the cytoskeleton and adhesion sites, directly modulating cell shape and preventing epithelial-mesenchymal transition.","PeriodicalId":501590,"journal":{"name":"bioRxiv - Cell Biology","volume":"14 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142189828","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 : 2024-09-08DOI: 10.1101/2024.09.08.611854
Aaran Vijayakumaran, Christopher Godbehere, Analle Abuammar, Sophia Y Breusegem, Leah R. Hurst, Nobuhiro Morone, Jaime Llodra, Melis T. Dalbay, Niaj M. Tanvir, Kirsty MacLellan-Gibson, Chris O'Callaghan, Esben Lorentzen, CellMap Project Team, FIB-SEM Technology, Andrew Murray, Kedar Narayan, Vito Mennella
A major frontier in single cell biology is decoding how transcriptional states result in cellular-level architectural changes, ultimately driving function. A remarkable example of this cellular remodelling program is the differentiation of airway stem cells into the human respiratory multiciliated epithelium, a tissue barrier protecting against bacteria, viruses and particulate matter. Here, we present the first isotropic three-dimensional map of the airway epithelium at the nanometre scale unveiling the coordinated changes in cellular organisation, organelle topology and contacts, occurring during multiciliogenesis. This analysis led us to discover a cellular mechanism of communication whereby motile cilia relay mechanical information to mitochondria through striated cytoskeletal fibres, the rootlets, to promote effective ciliary motility and ATP generation. Altogether, this study integrates nanometre-scale structural, functional and dynamic insights to elucidate fundamental mechanisms responsible for airway defence.
单细胞生物学的一个重要前沿领域是解码转录状态如何导致细胞级结构变化，并最终驱动功能。气道干细胞分化成人类呼吸道多纤毛上皮细胞就是这种细胞重塑程序的一个杰出范例，多纤毛上皮细胞是抵御细菌、病毒和微粒物质的组织屏障。在这里，我们首次展示了气道上皮纳米尺度的各向同性三维图，揭示了多纤毛发生过程中细胞组织、细胞器拓扑和接触的协调变化。这项分析使我们发现了一种细胞通信机制，即运动纤毛通过条状细胞骨架纤维（小根）向线粒体传递机械信息，以促进有效的纤毛运动和 ATP 生成。总之，这项研究综合了纳米尺度的结构、功能和动态见解，阐明了气道防御的基本机制。
{"title":"3D nanoscale architecture of the respiratory epithelium reveals motile cilia-rootlets-mitochondria axis of communication","authors":"Aaran Vijayakumaran, Christopher Godbehere, Analle Abuammar, Sophia Y Breusegem, Leah R. Hurst, Nobuhiro Morone, Jaime Llodra, Melis T. Dalbay, Niaj M. Tanvir, Kirsty MacLellan-Gibson, Chris O'Callaghan, Esben Lorentzen, CellMap Project Team, FIB-SEM Technology, Andrew Murray, Kedar Narayan, Vito Mennella","doi":"10.1101/2024.09.08.611854","DOIUrl":"https://doi.org/10.1101/2024.09.08.611854","url":null,"abstract":"A major frontier in single cell biology is decoding how transcriptional states result in cellular-level architectural changes, ultimately driving function. A remarkable example of this cellular remodelling program is the differentiation of airway stem cells into the human respiratory multiciliated epithelium, a tissue barrier protecting against bacteria, viruses and particulate matter. Here, we present the first isotropic three-dimensional map of the airway epithelium at the nanometre scale unveiling the coordinated changes in cellular organisation, organelle topology and contacts, occurring during multiciliogenesis. This analysis led us to discover a cellular mechanism of communication whereby motile cilia relay mechanical information to mitochondria through striated cytoskeletal fibres, the rootlets, to promote effective ciliary motility and ATP generation. Altogether, this study integrates nanometre-scale structural, functional and dynamic insights to elucidate fundamental mechanisms responsible for airway defence.","PeriodicalId":501590,"journal":{"name":"bioRxiv - Cell Biology","volume":"68 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142190069","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 : 2024-09-08DOI: 10.1101/2024.09.07.611681
Evgeny I. Bakhmet, Anna S. Zinovyeva, Andrey A. Kuzmin, Daria V. Smirnova, Mikhail N. Gordeev, Ekaterina E. Petrenko, Nikolay D. Aksenov, Alexey N. Tomilin
Oct4 is a commonly known marker of pluripotent stem cells as well as one of the key factors required for pluripotency induction. Its gene (Pou5f1) is subject to complicated regulation through distal and proximal enhancers. Noteworthy, this protein also plays an important role in primitive endoderm (PrE) specification, though the mechanisms driving its expression during this process are still unknown. Here we show that KH-domain protein Pcbp1 occupies poly(C)-sites of the Pou5f1 enhancers, but Pcbp1 knockout does not affect the Oct4 expression level in ESCs. On the contrary, Pcbp1 is essential for timely Oct4 downregulation upon differentiation signals. Residual Oct4 expression in turn leads to PrE specification, and this phenotype is reminiscent of that in ESCs constitutively expressing Oct4. Overall, our results point to Pcbp1 is a transcriptional regulator of Pou5f1, purported to synchronize Oct4 expression decline with the pluripotency network shutdown during differentiation. Oct4 being outside of this network loss its functions as factor of pluripotency and acts as PrE specifier.
{"title":"Pcbp1 constrains Oct4 expression in the context of pluripotency","authors":"Evgeny I. Bakhmet, Anna S. Zinovyeva, Andrey A. Kuzmin, Daria V. Smirnova, Mikhail N. Gordeev, Ekaterina E. Petrenko, Nikolay D. Aksenov, Alexey N. Tomilin","doi":"10.1101/2024.09.07.611681","DOIUrl":"https://doi.org/10.1101/2024.09.07.611681","url":null,"abstract":"Oct4 is a commonly known marker of pluripotent stem cells as well as one of the key factors required for pluripotency induction. Its gene (Pou5f1) is subject to complicated regulation through distal and proximal enhancers. Noteworthy, this protein also plays an important role in primitive endoderm (PrE) specification, though the mechanisms driving its expression during this process are still unknown. Here we show that KH-domain protein Pcbp1 occupies poly(C)-sites of the Pou5f1 enhancers, but Pcbp1 knockout does not affect the Oct4 expression level in ESCs. On the contrary, Pcbp1 is essential for timely Oct4 downregulation upon differentiation signals. Residual Oct4 expression in turn leads to PrE specification, and this phenotype is reminiscent of that in ESCs constitutively expressing Oct4. Overall, our results point to Pcbp1 is a transcriptional regulator of Pou5f1, purported to synchronize Oct4 expression decline with the pluripotency network shutdown during differentiation. Oct4 being outside of this network loss its functions as factor of pluripotency and acts as PrE specifier.","PeriodicalId":501590,"journal":{"name":"bioRxiv - Cell Biology","volume":"30 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142190070","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 : 2024-09-08DOI: 10.1101/2024.09.08.611912
Peng Sheng, zhe Li Bai, Hong Cao, Dan Li
The organelles of yeast demonstrate diverse morphological traits in response to different stress stimuli. However, there is a lack of systematic reports on the structural changes induced by stress stimuli in all membrane-based organelles. Here, we utilized a set of fluorescent protein-based organelle markers to highlight the distinct characteristics of yeast under various stress triggers, including high temperature, hydrogen peroxide, acetic acid, and ethyl alcohol. We found that all of these organelles undergo alterations in structure or function in response to the four stress triggers we tested. Specifically, filamentous mitochondria rupture into smaller segments when exposed to the above four stress conditions. The structure of the endoplasmic reticulum (ER) remains relatively unchanged, but its function is affected. Additionally, high temperature and hydrogen peroxide can induce the Ire1p-mediated ER unfolded protein response (UPR). The translocation of most nuclear-localized proteins to the cytosol is dependent on the specific stress conditions employed. Under the above stress conditions, the vacuole undergoes fusion, resulting in the formation of a larger vacuole from multiple smaller ones. Meanwhile, acetic acid-induced stress leads to the translocation of vacuole-localized proteins Prc1p and Pep4p to unknown puncta, while Ybh3p relocates from the inner vacuole to the vacuole membrane. Proteins localized in the early Golgi, late Golgi, and late endosomes exhibit distinct traits, such as fading away or mis-localization. The structure and function of peroxisomes, lipid droplets, and autophagosomes also undergo modifications. Furthermore, upon exposure to high temperature and ethanol, apoptosis-related proteins Yca1, Aif1, and Mmi1 aggregate instead of remaining dispersed.
{"title":"Stress response of membrane-based cell organelles in budding yeast","authors":"Peng Sheng, zhe Li Bai, Hong Cao, Dan Li","doi":"10.1101/2024.09.08.611912","DOIUrl":"https://doi.org/10.1101/2024.09.08.611912","url":null,"abstract":"The organelles of yeast demonstrate diverse morphological traits in response to different stress stimuli. However, there is a lack of systematic reports on the structural changes induced by stress stimuli in all membrane-based organelles. Here, we utilized a set of fluorescent protein-based organelle markers to highlight the distinct characteristics of yeast under various stress triggers, including high temperature, hydrogen peroxide, acetic acid, and ethyl alcohol. We found that all of these organelles undergo alterations in structure or function in response to the four stress triggers we tested. Specifically, filamentous mitochondria rupture into smaller segments when exposed to the above four stress conditions. The structure of the endoplasmic reticulum (ER) remains relatively unchanged, but its function is affected. Additionally, high temperature and hydrogen peroxide can induce the Ire1p-mediated ER unfolded protein response (UPR). The translocation of most nuclear-localized proteins to the cytosol is dependent on the specific stress conditions employed. Under the above stress conditions, the vacuole undergoes fusion, resulting in the formation of a larger vacuole from multiple smaller ones. Meanwhile, acetic acid-induced stress leads to the translocation of vacuole-localized proteins Prc1p and Pep4p to unknown puncta, while Ybh3p relocates from the inner vacuole to the vacuole membrane. Proteins localized in the early Golgi, late Golgi, and late endosomes exhibit distinct traits, such as fading away or mis-localization. The structure and function of peroxisomes, lipid droplets, and autophagosomes also undergo modifications. Furthermore, upon exposure to high temperature and ethanol, apoptosis-related proteins Yca1, Aif1, and Mmi1 aggregate instead of remaining dispersed.","PeriodicalId":501590,"journal":{"name":"bioRxiv - Cell Biology","volume":"30 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142190068","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 : 2024-08-12DOI: 10.1101/2024.08.12.607533
Yuge Ji, Alejandro Tejada-Lapuerta, Niklas A Schmacke, Zihe Zheng, Xinyue Zhang, Simrah Khan, Ina Rothenaigner, Juliane Tschuck, Kamyar Hadian, Fabian J Theis
Biological systems can be understood by perturbing individual components and studying the system's response. Cell biology experiments are defined by the applied treatment, cellular state, and the assayed phenotype. Given the vast number of possible combinations, testing every scenario is impractical. We present Prophet, a transformer-based computational model for cellular phenotype prediction. Prophet learns a representation of the cell biology experiment space, enabling it to predict the outcomes of untested small molecule or genetic perturbations in new cellular contexts across diverse phenotypes including gene expression, cell viability, and cell morphology. Its scalable architecture facilitates training across independent assays, using transfer learning to enhance performance across phenotypes. In vitro validation shows Prophet's potential to guide experimental design, making it a valuable tool for accelerating biological discovery.
{"title":"Scalable and universal prediction of cellular phenotypes","authors":"Yuge Ji, Alejandro Tejada-Lapuerta, Niklas A Schmacke, Zihe Zheng, Xinyue Zhang, Simrah Khan, Ina Rothenaigner, Juliane Tschuck, Kamyar Hadian, Fabian J Theis","doi":"10.1101/2024.08.12.607533","DOIUrl":"https://doi.org/10.1101/2024.08.12.607533","url":null,"abstract":"Biological systems can be understood by perturbing individual components and studying the system's response. Cell biology experiments are defined by the applied treatment, cellular state, and the assayed phenotype. Given the vast number of possible combinations, testing every scenario is impractical. We present Prophet, a transformer-based computational model for cellular phenotype prediction. Prophet learns a representation of the cell biology experiment space, enabling it to predict the outcomes of untested small molecule or genetic perturbations in new cellular contexts across diverse phenotypes including gene expression, cell viability, and cell morphology. Its scalable architecture facilitates training across independent assays, using transfer learning to enhance performance across phenotypes. In vitro validation shows Prophet's potential to guide experimental design, making it a valuable tool for accelerating biological discovery.","PeriodicalId":501590,"journal":{"name":"bioRxiv - Cell Biology","volume":"369 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-08-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141930639","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 : 2024-08-11DOI: 10.1101/2024.08.11.607506
Benjamin Arnold, Laurell Kessler, Ellen Kazumi Okuda, Ricarda R Rieger, Maria Clara Hernandez Canas, Ewelina Zebrowska, Cem Bakisoglu, Helder Y Nagasse, David Stanek, Dorothee Dormann, Kathi Zarnack, Mike Heilemann, Michaela Mueller-McNicoll
Nuclear speckles (NS) and paraspeckles (PS) are adjacent condensates with distinct protein composition, with serine-arginine-rich splicing factors (SRSFs) concentrated in NS. Surprisingly, we find that SRSF5 is present in both. Combining super-resolution imaging, proximity proteomics and iCLIP, we show that SRSF5 binds with PS core proteins to the PS-scaffold RNA NEAT1 and locates between PS spheres. Acute SRSF5 depletion results in reduced PS with differently packaged NEAT1. Under stress, SRSF5's association with PS increases, and without SRSF5, PS cluster assembly is impaired. Interfering with binding to purine-rich RNAs even causes PS-NS fusion. In an intriguing over-compensation, longer SRSF5 depletion reduces TDP-43 levels via premature polyadenylation, leading to NEAT1 isoform switching and more PS. We propose that SRSF5 forms a stress-specific PS shell and acts as a glue for PS clusters. Additionally, we uncover SRSF5 as a novel regulator of TDP-43 and demonstrate how acute depletion distinguishes direct from compensatory effects.
{"title":"Rapid depletion and super-resolution microscopy reveal an unexpected role of the nuclear-speckle protein SRSF5 in paraspeckle assembly and dynamics during cellular stress","authors":"Benjamin Arnold, Laurell Kessler, Ellen Kazumi Okuda, Ricarda R Rieger, Maria Clara Hernandez Canas, Ewelina Zebrowska, Cem Bakisoglu, Helder Y Nagasse, David Stanek, Dorothee Dormann, Kathi Zarnack, Mike Heilemann, Michaela Mueller-McNicoll","doi":"10.1101/2024.08.11.607506","DOIUrl":"https://doi.org/10.1101/2024.08.11.607506","url":null,"abstract":"Nuclear speckles (NS) and paraspeckles (PS) are adjacent condensates with distinct protein composition, with serine-arginine-rich splicing factors (SRSFs) concentrated in NS. Surprisingly, we find that SRSF5 is present in both. Combining super-resolution imaging, proximity proteomics and iCLIP, we show that SRSF5 binds with PS core proteins to the PS-scaffold RNA NEAT1 and locates between PS spheres. Acute SRSF5 depletion results in reduced PS with differently packaged NEAT1. Under stress, SRSF5's association with PS increases, and without SRSF5, PS cluster assembly is impaired. Interfering with binding to purine-rich RNAs even causes PS-NS fusion. In an intriguing over-compensation, longer SRSF5 depletion reduces TDP-43 levels via premature polyadenylation, leading to NEAT1 isoform switching and more PS. We propose that SRSF5 forms a stress-specific PS shell and acts as a glue for PS clusters. Additionally, we uncover SRSF5 as a novel regulator of TDP-43 and demonstrate how acute depletion distinguishes direct from compensatory effects.","PeriodicalId":501590,"journal":{"name":"bioRxiv - Cell Biology","volume":"12 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-08-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141949474","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 : 2024-08-11DOI: 10.1101/2024.08.10.607465
Anand Desai, Zinger Yang Loureiro, Tiffany DeSouza, Qin Yang, Javier Solivan-Rivera, Silvia Corvera
Objective: The uncoupling protein 1 (UCP1) is induced in brown or beige adipocytes through catecholamine-induced cAMP signaling, which activates diverse transcription factors. UCP1 expression can also be enhanced by PPARγ agonists such as rosiglitazone (Rsg). However, it is unclear whether this upregulation results from de-novo differentiation of beige adipocytes from progenitor cells, or from the induction of UCP1 in pre-existing adipocytes. To explore this, we employed human adipocytes differentiated from progenitor cells and examined their acute response to Rsg, to the adenylate-cyclase activator forskolin (Fsk), or to both simultaneously. Methods: Adipocytes generated from primary human progenitor cells were differentiated without exposure to PPARγ agonists, and treated for 3, 6 or 78 hours to Fsk, to Rsg, or to both simultaneously. Bulk RNASeq, RNAScope, RT-PCR, CRISPR-Cas9 mediated knockout, oxygen consumption and western blotting were used to assess cellular responses. Results: UCP1 mRNA expression was induced within 3 hours of exposure to either Rsg or Fsk, indicating that the Rsg effect is independent on additional adipocyte differentiation. Although Rsg and Fsk induced distinct overall transcriptional responses, both induced genes associated with calcium metabolism, lipid droplet assembly, and mitochondrial remodeling, denoting core features of human adipocyte beiging. Unexpectedly, we found that Fsk-induced UCP1 expression was reduced by approximately 80% following CRISPR-Cas9-mediated knockout of PNPLA2, the gene encoding the triglyceride lipase ATGL. As anticipated, ATGL knockout suppressed lipolysis; however, the associated suppression of UCP1 induction indicates that maximal cAMP-mediated UCP1 induction requires products of ATGL-catalyzed lipolysis. Supporting this, we observed that the reduction in Fsk-stimulated UCP1 induction caused by ATGL knockout was reversed by Rsg, implying that the role of lipolysis in this process is to generate natural PPARγ agonists. Conclusion: UCP1 transcription is known to be stimulated by transcription factors activated downstream of cAMP-dependent protein kinases. Here we demonstrate that UCP1 transcription can also be acutely induced through PPARγ-activation. Moreover, both pathways are activated in human adipocytes in response to cAMP, synergistically inducing UCP1 expression. The stimulation of PPARγ in response to cAMP occurs as a result of the production of natural PPARγ-activating ligands through ATGL-mediated lipolysis.
{"title":"PPARγ activation by lipolysis-generated ligands is required for cAMP dependent UCP1 induction in human thermogenic adipocytes","authors":"Anand Desai, Zinger Yang Loureiro, Tiffany DeSouza, Qin Yang, Javier Solivan-Rivera, Silvia Corvera","doi":"10.1101/2024.08.10.607465","DOIUrl":"https://doi.org/10.1101/2024.08.10.607465","url":null,"abstract":"Objective: The uncoupling protein 1 (UCP1) is induced in brown or beige adipocytes through catecholamine-induced cAMP signaling, which activates diverse transcription factors. UCP1 expression can also be enhanced by PPARγ agonists such as rosiglitazone (Rsg). However, it is unclear whether this upregulation results from de-novo differentiation of beige adipocytes from progenitor cells, or from the induction of UCP1 in pre-existing adipocytes. To explore this, we employed human adipocytes differentiated from progenitor cells and examined their acute response to Rsg, to the adenylate-cyclase activator forskolin (Fsk), or to both simultaneously. Methods: Adipocytes generated from primary human progenitor cells were differentiated without exposure to PPARγ agonists, and treated for 3, 6 or 78 hours to Fsk, to Rsg, or to both simultaneously. Bulk RNASeq, RNAScope, RT-PCR, CRISPR-Cas9 mediated knockout, oxygen consumption and western blotting were used to assess cellular responses. Results: UCP1 mRNA expression was induced within 3 hours of exposure to either Rsg or Fsk, indicating that the Rsg effect is independent on additional adipocyte differentiation. Although Rsg and Fsk induced distinct overall transcriptional responses, both induced genes associated with calcium metabolism, lipid droplet assembly, and mitochondrial remodeling, denoting core features of human adipocyte beiging. Unexpectedly, we found that Fsk-induced UCP1 expression was reduced by approximately 80% following CRISPR-Cas9-mediated knockout of PNPLA2, the gene encoding the triglyceride lipase ATGL. As anticipated, ATGL knockout suppressed lipolysis; however, the associated suppression of UCP1 induction indicates that maximal cAMP-mediated UCP1 induction requires products of ATGL-catalyzed lipolysis. Supporting this, we observed that the reduction in Fsk-stimulated UCP1 induction caused by ATGL knockout was reversed by Rsg, implying that the role of lipolysis in this process is to generate natural PPARγ agonists. Conclusion: UCP1 transcription is known to be stimulated by transcription factors activated downstream of cAMP-dependent protein kinases. Here we demonstrate that UCP1 transcription can also be acutely induced through PPARγ-activation. Moreover, both pathways are activated in human adipocytes in response to cAMP, synergistically inducing UCP1 expression. The stimulation of PPARγ in response to cAMP occurs as a result of the production of natural PPARγ-activating ligands through ATGL-mediated lipolysis.","PeriodicalId":501590,"journal":{"name":"bioRxiv - Cell Biology","volume":"15 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-08-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141930640","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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