Pub Date : 2024-09-18DOI: 10.1101/2024.09.17.613365
Nicolai D Raig, Katherine J Surridge, Marta Sanz-Murillo, Verena Dederer, Andreas Kramer, Martin P Schwalm, Lewis Elson, Deep Chatterjee, Sebastian Mathea, Thomas Hanke, Andres E Leschziner, Samara L Reck-Peterson, Stefan Knapp
Aberrant increases in kinase activity of leucine-rich repeat kinase 2 (LRRK2) are associated with Parkinson's disease (PD). Numerous LRRK2-selective type-I kinase inhibitors have been developed and some have entered clinical trials. In this study, we present the first LRRK2-selective type-II kinase inhibitors. Targeting the inactive conformation of LRRK2 is functionally distinct from targeting the active-like conformation using type-I inhibitors. We designed these inhibitors using a combinatorial chemistry approach fusing selective LRRK2 type-I and promiscuous type-II inhibitors by iterative cycles of synthesis supported by structural biology and activity testing. Our current lead structures are selective and potent LRRK2 inhibitors. Through cellular assays, cryo-electron microscopy structural analysis, and in vitro motility assays, we show that our inhibitors stabilize the open, inactive kinase conformation. These new conformation-specific compounds will be invaluable as tools to study LRRK2's function and regulation, and expand the potential therapeutic options for PD.
富亮氨酸重复激酶 2（LRRK2）激酶活性的异常增加与帕金森病（PD）有关。目前已开发出许多 LRRK2 选择性 I 型激酶抑制剂，其中一些已进入临床试验阶段。在这项研究中，我们首次提出了 LRRK2 选择性 II 型激酶抑制剂。靶向 LRRK2 的非活性构象与使用 I 型抑制剂靶向类活性构象在功能上有所不同。我们采用组合化学方法设计了这些抑制剂，通过结构生物学和活性测试支持的迭代合成循环，将选择性 LRRK2 I 型抑制剂和杂合 II 型抑制剂融合在一起。我们目前的先导结构是选择性的强效 LRRK2 抑制剂。通过细胞实验、冷冻电镜结构分析和体外运动实验，我们发现我们的抑制剂能稳定开放的、非活性激酶构象。这些新的构象特异性化合物将成为研究 LRRK2 功能和调控的宝贵工具，并扩大了对帕金森病的潜在治疗方案。
{"title":"Type-II kinase inhibitors that target Parkinson's Disease-associated LRRK2","authors":"Nicolai D Raig, Katherine J Surridge, Marta Sanz-Murillo, Verena Dederer, Andreas Kramer, Martin P Schwalm, Lewis Elson, Deep Chatterjee, Sebastian Mathea, Thomas Hanke, Andres E Leschziner, Samara L Reck-Peterson, Stefan Knapp","doi":"10.1101/2024.09.17.613365","DOIUrl":"https://doi.org/10.1101/2024.09.17.613365","url":null,"abstract":"Aberrant increases in kinase activity of leucine-rich repeat kinase 2 (LRRK2) are associated with Parkinson's disease (PD). Numerous LRRK2-selective type-I kinase inhibitors have been developed and some have entered clinical trials. In this study, we present the first LRRK2-selective type-II kinase inhibitors. Targeting the inactive conformation of LRRK2 is functionally distinct from targeting the active-like conformation using type-I inhibitors. We designed these inhibitors using a combinatorial chemistry approach fusing selective LRRK2 type-I and promiscuous type-II inhibitors by iterative cycles of synthesis supported by structural biology and activity testing. Our current lead structures are selective and potent LRRK2 inhibitors. Through cellular assays, cryo-electron microscopy structural analysis, and in vitro motility assays, we show that our inhibitors stabilize the open, inactive kinase conformation. These new conformation-specific compounds will be invaluable as tools to study LRRK2's function and regulation, and expand the potential therapeutic options for PD.","PeriodicalId":501590,"journal":{"name":"bioRxiv - Cell Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142265320","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 : 2024-09-17DOI: 10.1101/2024.09.16.613371
Darshika Bohra, Aprotim Mazumder
Double-strand breaks (DSBs) pose significant threat to genomic stability and need immediate attention from DNA Damage Response (DDR) machinery involved in Homologous Recombination (HR) or Non-homologous end joining (NHEJ). DDR in heterochromatin is challenging owing to the distinct chromatin organization. Heterochromatin Protein 1 (HP1) isoforms that contribute significantly to the organization of heterochromatin, have been shown to be involved in DDR. Mammalian HP1 has three isoforms, HP1α, HP1β, and HP1γ, which possess significant homology and yet have distinct functions. HP1α is the only isoform known to undergo liquid-liquid phase separation. We show that the minute-scale dynamics of HP1α and HP1β differ dramatically and they promote differential recruitment of HR vs. NHEJ factors at the sites of laser-induced clustered DSBs. Perturbing HP1α phase-separation abrogates both the recruitment of HR factors and readouts of HR. Our study provides a link between phase-separation and DDR-centric roles of HP1α and hints at spatial partitioning of repair pathways in response to damage in heterochromatin.
{"title":"HP1α-driven Phase Separation and Repair Pathway Choice in Response to Heterochromatin Damage","authors":"Darshika Bohra, Aprotim Mazumder","doi":"10.1101/2024.09.16.613371","DOIUrl":"https://doi.org/10.1101/2024.09.16.613371","url":null,"abstract":"Double-strand breaks (DSBs) pose significant threat to genomic stability and need immediate attention from DNA Damage Response (DDR) machinery involved in Homologous Recombination (HR) or Non-homologous end joining (NHEJ). DDR in heterochromatin is challenging owing to the distinct chromatin organization. Heterochromatin Protein 1 (HP1) isoforms that contribute significantly to the organization of heterochromatin, have been shown to be involved in DDR. Mammalian HP1 has three isoforms, HP1α, HP1β, and HP1γ, which possess significant homology and yet have distinct functions. HP1α is the only isoform known to undergo liquid-liquid phase separation. We show that the minute-scale dynamics of HP1α and HP1β differ dramatically and they promote differential recruitment of HR vs. NHEJ factors at the sites of laser-induced clustered DSBs. Perturbing HP1α phase-separation abrogates both the recruitment of HR factors and readouts of HR. Our study provides a link between phase-separation and DDR-centric roles of HP1α and hints at spatial partitioning of repair pathways in response to damage in heterochromatin.","PeriodicalId":501590,"journal":{"name":"bioRxiv - Cell Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142265089","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 : 2024-09-17DOI: 10.1101/2024.09.16.613375
Samuel McCullough, Eliene Albers, Akshata Anchan, Jane Yu, Simon Joseph O'Carroll, Bronwen Connor, Scott Graham
Background: iPSC-derived cells are increasingly used to model complex diseases in vitro because they can be patient derived and can differentiate into any cell in the adult human body. Recent studies have demonstrated the generation of brain pericytes using a neural crest-based differentiation protocol. However, the inflammatory response of these iPSC-derived brain pericytes has not been investigated. We aimed to investigate the response of iPSC-derived brain pericytes to common inflammatory stimuli, thereby assessing the suitability of these cells to study inflammatory disease. Methods: Brain pericytes were differentiated from iPSCs for 42 days. The expression of brain pericyte markers was assessed by RT-qPCR and immunofluorescent staining at days 0, 15, 21, and 42 of differentiation to validate the brain pericyte-like phenotype. Nuclear localisation of NFκB and STAT1 was assessed by immunofluorescence following IL-1β- and TNF-treatment in day 21 and day 42 iPSC-derived pericytes, and primary human pericytes. Cytometric bead array assessed the concentration of secreted inflammatory factors in the cell medium and phagocytosis was investigated using fluorescent carboxylated beads and flow cytometry. Results: At day 42 of differentiation, but not at day 21, cells expressed brain pericyte markers. Generally, iPSC-derived pericytes lacked consistent responses to inflammatory treatment compared to primary human pericytes. Day 21 and 42 iPSC-derived pericytes exhibited a NFκB response to IL-1β treatment comparable to primary human pericytes. Day 21 iPSC-derived pericytes exhibited a STAT1 response with IL-1β treatment which was absent in day 42 cells, but present in a subset of primary human pericytes. TNF treatment presented similar NFκB responses between day 21 and 42 iPSC-derived and primary human pericytes, but a STAT1 response was again present in a subset of primary human pericytes which was absent in both day 21 and day 42 iPSC-derived pericytes. Numerous differences were observed in the secretion of cytokines and chemokines following treatment of iPSC-derived and primary human pericytes with IL-1β and TNF. iPSC-derived pericytes exhibited greater rates of phagocytosis than primary human pericytes. Conclusions: With the increase in iPSC-derived cells in research, labs should undertake validation of lineage specificity when adapting an iPSC-derived differentiation protocol. In our hands, the inflammatory response of iPSC-derived pericytes was different to that of primary human pericytes, raising concern regarding the use of iPSC-derived pericytes to study neuroinflammatory disease.
{"title":"Human iPSC-derived brain pericytes exhibit differences in inflammatory activation compared to primary human brain pericytes","authors":"Samuel McCullough, Eliene Albers, Akshata Anchan, Jane Yu, Simon Joseph O'Carroll, Bronwen Connor, Scott Graham","doi":"10.1101/2024.09.16.613375","DOIUrl":"https://doi.org/10.1101/2024.09.16.613375","url":null,"abstract":"Background: iPSC-derived cells are increasingly used to model complex diseases in vitro because they can be patient derived and can differentiate into any cell in the adult human body. Recent studies have demonstrated the generation of brain pericytes using a neural crest-based differentiation protocol. However, the inflammatory response of these iPSC-derived brain pericytes has not been investigated. We aimed to investigate the response of iPSC-derived brain pericytes to common inflammatory stimuli, thereby assessing the suitability of these cells to study inflammatory disease. Methods: Brain pericytes were differentiated from iPSCs for 42 days. The expression of brain pericyte markers was assessed by RT-qPCR and immunofluorescent staining at days 0, 15, 21, and 42 of differentiation to validate the brain pericyte-like phenotype. Nuclear localisation of NFκB and STAT1 was assessed by immunofluorescence following IL-1β- and TNF-treatment in day 21 and day 42 iPSC-derived pericytes, and primary human pericytes. Cytometric bead array assessed the concentration of secreted inflammatory factors in the cell medium and phagocytosis was investigated using fluorescent carboxylated beads and flow cytometry. Results: At day 42 of differentiation, but not at day 21, cells expressed brain pericyte markers. Generally, iPSC-derived pericytes lacked consistent responses to inflammatory treatment compared to primary human pericytes. Day 21 and 42 iPSC-derived pericytes exhibited a NFκB response to IL-1β treatment comparable to primary human pericytes. Day 21 iPSC-derived pericytes exhibited a STAT1 response with IL-1β treatment which was absent in day 42 cells, but present in a subset of primary human pericytes. TNF treatment presented similar NFκB responses between day 21 and 42 iPSC-derived and primary human pericytes, but a STAT1 response was again present in a subset of primary human pericytes which was absent in both day 21 and day 42 iPSC-derived pericytes. Numerous differences were observed in the secretion of cytokines and chemokines following treatment of iPSC-derived and primary human pericytes with IL-1β and TNF. iPSC-derived pericytes exhibited greater rates of phagocytosis than primary human pericytes. Conclusions: With the increase in iPSC-derived cells in research, labs should undertake validation of lineage specificity when adapting an iPSC-derived differentiation protocol. In our hands, the inflammatory response of iPSC-derived pericytes was different to that of primary human pericytes, raising concern regarding the use of iPSC-derived pericytes to study neuroinflammatory disease.","PeriodicalId":501590,"journal":{"name":"bioRxiv - Cell Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142265328","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 : 2024-09-17DOI: 10.1101/2024.09.16.613361
Henry M Sanchez, Tomer Lapidot, Ophir Shalem
The subcellular organization of proteins carries important information on cellular state and gene function, yet currently there are no technologies that enable accurate measurement of subcellular protein localizations at scale. Here we develop an approach for pooled endogenous protein tagging using prime editing, which coupled with an optical readout and sequencing, provides a snapshot of proteome organization in a manner akin to perturbation-based CRISPR screens. We constructed a pooled library of 17,280 pegRNAs designed to exhaustively tag 60 endogenous proteins spanning diverse localization patterns and explore a large space of genomic and pegRNA design parameters. Pooled measurements of tagging efficiency uncovered both genomic and pegRNA features associated with increased efficiency, including epigenetic states and interactions with transcription. We integrate pegRNA features into a computational model with predictive value for tagging efficiency to constrain the design space of pegRNAs for large-scale peptide knock-in. Lastly, we show that combining in-situ pegRNA sequencing with high-throughput deep learning image analysis, enables exploration of subcellular protein localization patterns for many proteins in parallel following a single pooled lentiviral transduction, setting the stage for scalable studies of proteome dynamics across cell types and environmental perturbations.
{"title":"High-throughput optimized prime editing mediated endogenous protein tagging for pooled imaging of protein localization","authors":"Henry M Sanchez, Tomer Lapidot, Ophir Shalem","doi":"10.1101/2024.09.16.613361","DOIUrl":"https://doi.org/10.1101/2024.09.16.613361","url":null,"abstract":"The subcellular organization of proteins carries important information on cellular state and gene function, yet currently there are no technologies that enable accurate measurement of subcellular protein localizations at scale. Here we develop an approach for pooled endogenous protein tagging using prime editing, which coupled with an optical readout and sequencing, provides a snapshot of proteome organization in a manner akin to perturbation-based CRISPR screens. We constructed a pooled library of 17,280 pegRNAs designed to exhaustively tag 60 endogenous proteins spanning diverse localization patterns and explore a large space of genomic and pegRNA design parameters. Pooled measurements of tagging efficiency uncovered both genomic and pegRNA features associated with increased efficiency, including epigenetic states and interactions with transcription. We integrate pegRNA features into a computational model with predictive value for tagging efficiency to constrain the design space of pegRNAs for large-scale peptide knock-in. Lastly, we show that combining in-situ pegRNA sequencing with high-throughput deep learning image analysis, enables exploration of subcellular protein localization patterns for many proteins in parallel following a single pooled lentiviral transduction, setting the stage for scalable studies of proteome dynamics across cell types and environmental perturbations.","PeriodicalId":501590,"journal":{"name":"bioRxiv - Cell Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142265326","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 : 2024-09-17DOI: 10.1101/2024.09.17.613382
Klara Piletic, Amir H Kayvanjoo, Felix Clemens Richter, Mariana Borsa, Ana Victoria Lechuga-Vieco, Oliver Popp, Sacha Grenet, Jacky Ka Long Ko, Kristina Zec, Maria Kyriazi, Lada Koneva, Stephen Sansom, Philipp Mertins, Fiona Powrie, Ghada Alsaleh, Anna Katharina Simon
A hallmark of obesity is a pathological expansion of white adipose tissue (WAT), accompanied by marked tissue dysfunction and fibrosis. Autophagy promotes adipocyte differentiation and lipid homeostasis, but its role in obese adipocytes and adipose tissue dysfunction remains incompletely understood. Here, we demonstrate that autophagy is a key tissue-specific regulator of WAT remodelling in diet-induced obesity. Importantly, loss of adipocyte autophagy substantially exacerbates pericellular fibrosis in visceral WAT. Change in WAT architecture correlates with increased infiltration of macrophages with tissue-reparative, fibrotic features. We uncover that autophagy regulates purine nucleoside metabolism in obese adipocytes, preventing excessive release of the purine catabolites xanthine and hypoxanthine. Purines signal cell-extrinsically for fibrosis by driving macrophage polarisation towards a tissue reparative phenotype. Our findings reveal a novel role for adipocyte autophagy in regulating tissue purine nucleoside metabolism, thereby limiting obesity-associated fibrosis and maintaining the functional integrity of visceral WAT. Purine signals may serve as a critical balance checkpoint and therapeutic target in fibrotic diseases.
肥胖症的特征之一是白色脂肪组织（WAT）病理性扩张，并伴有明显的组织功能障碍和纤维化。自噬可促进脂肪细胞分化和脂质稳态，但其在肥胖脂肪细胞和脂肪组织功能障碍中的作用仍不完全清楚。在这里，我们证明自噬是饮食诱导肥胖中 WAT 重塑的一个关键组织特异性调节因子。重要的是，脂肪细胞自噬功能的丧失大大加剧了内脏 WAT 的细胞周围纤维化。内脏脂肪细胞结构的变化与具有组织修复和纤维化特征的巨噬细胞浸润增加有关。我们发现自噬调节肥胖脂肪细胞的嘌呤核苷代谢，防止嘌呤代谢产物黄嘌呤和次黄嘌呤的过度释放。嘌呤通过驱动巨噬细胞向组织修复表型极化，在细胞外发出纤维化信号。我们的研究结果揭示了脂肪细胞自噬在调节组织嘌呤核苷代谢中的新作用，从而限制肥胖相关的纤维化并维持内脏脂肪的功能完整性。嘌呤信号可作为纤维化疾病的关键平衡检查点和治疗靶点。
{"title":"Autophagy acts as a brake on obesity-related fibrosis by controlling purine nucleoside signalling","authors":"Klara Piletic, Amir H Kayvanjoo, Felix Clemens Richter, Mariana Borsa, Ana Victoria Lechuga-Vieco, Oliver Popp, Sacha Grenet, Jacky Ka Long Ko, Kristina Zec, Maria Kyriazi, Lada Koneva, Stephen Sansom, Philipp Mertins, Fiona Powrie, Ghada Alsaleh, Anna Katharina Simon","doi":"10.1101/2024.09.17.613382","DOIUrl":"https://doi.org/10.1101/2024.09.17.613382","url":null,"abstract":"A hallmark of obesity is a pathological expansion of white adipose tissue (WAT), accompanied by marked tissue dysfunction and fibrosis. Autophagy promotes adipocyte differentiation and lipid homeostasis, but its role in obese adipocytes and adipose tissue dysfunction remains incompletely understood. Here, we demonstrate that autophagy is a key tissue-specific regulator of WAT remodelling in diet-induced obesity. Importantly, loss of adipocyte autophagy substantially exacerbates pericellular fibrosis in visceral WAT. Change in WAT architecture correlates with increased infiltration of macrophages with tissue-reparative, fibrotic features. We uncover that autophagy regulates purine nucleoside metabolism in obese adipocytes, preventing excessive release of the purine catabolites xanthine and hypoxanthine. Purines signal cell-extrinsically for fibrosis by driving macrophage polarisation towards a tissue reparative phenotype. Our findings reveal a novel role for adipocyte autophagy in regulating tissue purine nucleoside metabolism, thereby limiting obesity-associated fibrosis and maintaining the functional integrity of visceral WAT. Purine signals may serve as a critical balance checkpoint and therapeutic target in fibrotic diseases.","PeriodicalId":501590,"journal":{"name":"bioRxiv - Cell Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142265088","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 : 2024-09-17DOI: 10.1101/2024.09.16.613379
Giulia Viola, Kyle A Jacobs, Joel Lemiere, Matthew L Kutys, Torsten Wittmann
To standardize comparison of fluorescent proteins and independently determine which monomeric StayGold variant is best for live microscopy, we analyzed fluorescent protein tagged I3-01 peptides that self-assemble into stable sixty subunit dodecahedrons inside live cells. We find mStayGold is 3-fold brighter and 3-fold more photostable compared with EGFP and superior to other monomeric variants in mammalian cytoplasm. In addition, analysis of intracellular nanocage diffusion confirms the monomeric nature of mStayGold.
{"title":"Quantitative Comparison of Monomeric StayGold Variants Using Protein Nanocages in Living Cells","authors":"Giulia Viola, Kyle A Jacobs, Joel Lemiere, Matthew L Kutys, Torsten Wittmann","doi":"10.1101/2024.09.16.613379","DOIUrl":"https://doi.org/10.1101/2024.09.16.613379","url":null,"abstract":"To standardize comparison of fluorescent proteins and independently determine which monomeric StayGold variant is best for live microscopy, we analyzed fluorescent protein tagged I3-01 peptides that self-assemble into stable sixty subunit dodecahedrons inside live cells. We find mStayGold is 3-fold brighter and 3-fold more photostable compared with EGFP and superior to other monomeric variants in mammalian cytoplasm. In addition, analysis of intracellular nanocage diffusion confirms the monomeric nature of mStayGold.","PeriodicalId":501590,"journal":{"name":"bioRxiv - Cell Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142265327","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: 10.1101/2024.09.16.613344
Moua Yang, Alexandra Boye-Doe, Salma A.S. Abosabie, Alexandra M. Barr, Lourdes M. Mendez, Anish V. Sharda
Ras-like (Ral) GTPases play essential regulatory roles in many cellular processes, including exocytosis. Cycling between GDP- and GTP-bound states, Ral GTPases function as molecular switches and regulate effectors, specifically the multi-subunit tethering complex exocyst. Here, we show that Ral isoform RalB controls regulated exocytosis of Weibel-Palade bodies (WPBs), the specialized endothelial secretory granules that store hemostatic protein von Willebrand factor. Remarkably, unlike typical small GTPase-effector interactions, RalB binds exocyst in its GDP-bound state in resting endothelium. Upon endothelial cell stimulation, exocyst is uncoupled from RalB-GTP resulting in WPB tethering and exocytosis. Furthermore, we report that PKC-dependent phosphorylation of the C-terminal hypervariable region (HVR) of RalB modulates its dynamic interaction with exocyst in endothelium. Exocyst preferentially interacts with phosphorylated RalB in resting endothelium. Dephosphorylation of RalB either by endothelial cell stimulation, or PKC inhibition, or expression of nonphosphorylatable mutant at a specific serine residue of RalB HVR, disengages exocyst and augments WPB exocytosis, resembling RalB exocyst-binding site mutant. In summary, it is the uncoupling of exocyst from RalB that mediates endothelial Weibel-Palade body exocytosis. Our data shows that Ral function may be more dynamically regulated by phosphorylation and may confer distinct functionality given high degree of homology and the shared set of effector protein between the two Ral isoforms.
{"title":"RalB uncoupled exocyst mediates endothelial Weibel-Palade body exocytosis","authors":"Moua Yang, Alexandra Boye-Doe, Salma A.S. Abosabie, Alexandra M. Barr, Lourdes M. Mendez, Anish V. Sharda","doi":"10.1101/2024.09.16.613344","DOIUrl":"https://doi.org/10.1101/2024.09.16.613344","url":null,"abstract":"Ras-like (Ral) GTPases play essential regulatory roles in many cellular processes, including exocytosis. Cycling between GDP- and GTP-bound states, Ral GTPases function as molecular switches and regulate effectors, specifically the multi-subunit tethering complex exocyst. Here, we show that Ral isoform RalB controls regulated exocytosis of Weibel-Palade bodies (WPBs), the specialized endothelial secretory granules that store hemostatic protein von Willebrand factor. Remarkably, unlike typical small GTPase-effector interactions, RalB binds exocyst in its GDP-bound state in resting endothelium. Upon endothelial cell stimulation, exocyst is uncoupled from RalB-GTP resulting in WPB tethering and exocytosis. Furthermore, we report that PKC-dependent phosphorylation of the C-terminal hypervariable region (HVR) of RalB modulates its dynamic interaction with exocyst in endothelium. Exocyst preferentially interacts with phosphorylated RalB in resting endothelium. Dephosphorylation of RalB either by endothelial cell stimulation, or PKC inhibition, or expression of nonphosphorylatable mutant at a specific serine residue of RalB HVR, disengages exocyst and augments WPB exocytosis, resembling RalB exocyst-binding site mutant. In summary, it is the uncoupling of exocyst from RalB that mediates endothelial Weibel-Palade body exocytosis. Our data shows that Ral function may be more dynamically regulated by phosphorylation and may confer distinct functionality given high degree of homology and the shared set of effector protein between the two Ral isoforms.","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":"142265090","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: 10.1101/2024.09.16.613201
Tania Sultana, Chunfeng Zheng, Garret Morton, Timothy L Megraw
Zika virus (ZIKV) is a mosquito-transmitted flavivirus that caused an epidemic in 2015-2016 in the Americas and raised serious global health concerns due to its association with congenital brain developmental defects in infected pregnancies. Upon infection, ZIKV assembles virus particles in a virus-generated toroidal compartment next to the nucleus called the replication factory, or viroplasm, which forms by remodeling the host cell endoplasmic reticulum (ER). How the viral proteins control viroplasm assembly remains unknown. Here we show that the ZIKV non-structural protein 3 (NS3) is sufficient to drive the assembly of a viroplasm-like structure (VLS) in human cells. NS3 encodes a dual-function protease and RNA helicase. The VLS is similar to the ZIKV viroplasm in its assembly near centrosomes at the nuclear periphery, its deformation of the nuclear membrane, its recruitment of ER, Golgi, and dsRNA, and its association with microtubules at its surface. While sufficient to generate a VLS, NS3 is less efficient in several aspects compared to viroplasm formation upon ZIKV infection. We further show that the helicase domain and not the protease domain is required for optimal VLS assembly and dsRNA recruitment. Overall, this work advances our understanding of the mechanism of viroplasm assembly by ZIKV and likely will extend to other flaviviruses.
{"title":"Zika virus NS3 drives the assembly of a viroplasm-like structure","authors":"Tania Sultana, Chunfeng Zheng, Garret Morton, Timothy L Megraw","doi":"10.1101/2024.09.16.613201","DOIUrl":"https://doi.org/10.1101/2024.09.16.613201","url":null,"abstract":"Zika virus (ZIKV) is a mosquito-transmitted flavivirus that caused an epidemic in 2015-2016 in the Americas and raised serious global health concerns due to its association with congenital brain developmental defects in infected pregnancies. Upon infection, ZIKV assembles virus particles in a virus-generated toroidal compartment next to the nucleus called the replication factory, or viroplasm, which forms by remodeling the host cell endoplasmic reticulum (ER). How the viral proteins control viroplasm assembly remains unknown. Here we show that the ZIKV non-structural protein 3 (NS3) is sufficient to drive the assembly of a viroplasm-like structure (VLS) in human cells. NS3 encodes a dual-function protease and RNA helicase. The VLS is similar to the ZIKV viroplasm in its assembly near centrosomes at the nuclear periphery, its deformation of the nuclear membrane, its recruitment of ER, Golgi, and dsRNA, and its association with microtubules at its surface. While sufficient to generate a VLS, NS3 is less efficient in several aspects compared to viroplasm formation upon ZIKV infection. We further show that the helicase domain and not the protease domain is required for optimal VLS assembly and dsRNA recruitment. Overall, this work advances our understanding of the mechanism of viroplasm assembly by ZIKV and likely will extend to other flaviviruses.","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":"142265097","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: 10.1101/2024.09.16.613233
Emma L Faulkner, Jeremy A Pike, Evelyn Garlick, Robert K Neely, Iain B Styles, Stephen P Watson, Natalie S Poulter, Steven G Thomas
Current microscopy approaches applied to platelet aggregates in both haemostatic and thrombotic settings indicate their structure has important implications in efficient haemostasis and in clinical treatment of thrombosis. However, current fluorescence microscopy approaches are not amenable to volumetric imaging of platelet aggregate structures. This is largely due to the small size of individual platelets and the tight packing of platelets within aggregates, resulting in optical opacity. Here we demonstrate that expansion microscopy, applied to platelet aggregates, can reveal multi-scale information about the structure of platelet aggregates. We produced volumetric images at nanoscale resolution of >700 platelet aggregates under normal and perturbed conditions, stained for cytoskeletal and membrane components. We demonstrate our custom analysis workflow provides quantitative description of platelet numbers, volumes and morphology within entire platelet aggregates. Additionally, we quantitatively describe subcellular organisation of F-actin. By comparing these measurements following treatment with the actin inhibitors, cytochalasin D and latrunculin A, we can robustly detect structural disruptions in platelet aggregates. Together these data provide a workflow to qualitatively and quantitatively describe the architecture of platelet aggregates at a range of scales (whole aggregates down to sub-cellular features within individual platelets).
目前应用于止血和血栓形成情况下血小板聚集的显微镜方法表明，其结构对有效止血和血栓形成的临床治疗具有重要意义。然而，目前的荧光显微镜方法无法对血小板聚集体结构进行体积成像。这主要是由于单个血小板的尺寸较小，而且血小板在聚集体中紧密堆积，导致光学不透明。在这里，我们证明了将膨胀显微镜应用于血小板聚集体，可以揭示血小板聚集体结构的多尺度信息。我们以纳米级分辨率制作了正常和受干扰条件下 700 个血小板聚集体的体积图像，并对细胞骨架和膜成分进行了染色。我们展示了我们的定制分析工作流程，可定量描述整个血小板聚集体内的血小板数量、体积和形态。此外，我们还定量描述了 F-肌动蛋白的亚细胞组织。通过比较使用肌动蛋白抑制剂细胞松弛素 D 和 latrunculin A 处理后的这些测量结果，我们可以稳健地检测血小板聚集体的结构破坏情况。这些数据为定性和定量描述血小板聚集体结构提供了一个工作流程（从整个聚集体到单个血小板内的亚细胞特征）。
{"title":"Expansion microscopy allows quantitative characterisation of structural organisation of platelet aggregates","authors":"Emma L Faulkner, Jeremy A Pike, Evelyn Garlick, Robert K Neely, Iain B Styles, Stephen P Watson, Natalie S Poulter, Steven G Thomas","doi":"10.1101/2024.09.16.613233","DOIUrl":"https://doi.org/10.1101/2024.09.16.613233","url":null,"abstract":"Current microscopy approaches applied to platelet aggregates in both haemostatic and thrombotic settings indicate their structure has important implications in efficient haemostasis and in clinical treatment of thrombosis. However, current fluorescence microscopy approaches are not amenable to volumetric imaging of platelet aggregate structures. This is largely due to the small size of individual platelets and the tight packing of platelets within aggregates, resulting in optical opacity.\u0000Here we demonstrate that expansion microscopy, applied to platelet aggregates, can reveal multi-scale information about the structure of platelet aggregates. We produced volumetric images at nanoscale resolution of >700 platelet aggregates under normal and perturbed conditions, stained for cytoskeletal and membrane components. We demonstrate our custom analysis workflow provides quantitative description of platelet numbers, volumes and morphology within entire platelet aggregates. Additionally, we quantitatively describe subcellular organisation of F-actin. By comparing these measurements following treatment with the actin inhibitors, cytochalasin D and latrunculin A, we can robustly detect structural disruptions in platelet aggregates. Together these data provide a workflow to qualitatively and quantitatively describe the architecture of platelet aggregates at a range of scales (whole aggregates down to sub-cellular features within individual platelets).","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":"142265095","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: 10.1101/2024.09.16.613269
Miyuki Shimoji, Hiroya Akabori
Visceral adipose tissue plays a key role in the inflammation, inducing metabolic dysfunction. The culturing system of major components, adipocytes and adipose tissue macrophages (ATM) have been improved up to date, for example, the ceiling culture system, three-dimensional collagen gels and membrane mature adipocyte aggregate cultures (MAAC). Here we applied for a novel 3D culture device, cellbed of human omental adipocytes with ATM derived from surgery and presented the first morphological report. The pilot study has the limitation of resolution due to the thickness, however, the simple method would be a convenient assay to detect their morphological alteration after surgery. In addition, the combination of cell morphological observation on the cellbed and adipocytokine secretion capability would give an insight into the mechanism of adipose tissue-derived inflammation after surgery. The cellbed composed of high-grade silica glass fiver has been already provided the morphological study with the critical signaling transduction in human oral cancer research. The novel simple 3D device will prevail to spread the practical diversity in the functional clinical research.
{"title":"Application of novel 3D culture device for human omental adipocytes and macrophages derived from surgery","authors":"Miyuki Shimoji, Hiroya Akabori","doi":"10.1101/2024.09.16.613269","DOIUrl":"https://doi.org/10.1101/2024.09.16.613269","url":null,"abstract":"Visceral adipose tissue plays a key role in the inflammation, inducing metabolic dysfunction. The culturing system of major components, adipocytes and adipose tissue macrophages (ATM) have been improved up to date, for example, the ceiling culture system, three-dimensional collagen gels and membrane mature adipocyte aggregate cultures (MAAC). Here we applied for a novel 3D culture device, cellbed of human omental adipocytes with ATM derived from surgery and presented the first morphological report. The pilot study has the limitation of resolution due to the thickness, however, the simple method would be a convenient assay to detect their morphological alteration after surgery. In addition, the combination of cell morphological observation on the cellbed and adipocytokine secretion capability would give an insight into the mechanism of adipose tissue-derived inflammation after surgery. The cellbed composed of high-grade silica glass fiver has been already provided the morphological study with the critical signaling transduction in human oral cancer research. The novel simple 3D device will prevail to spread the practical diversity in the functional clinical research.","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":"142265091","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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