Mechanisms governing regulated secretion in human endothelial cells

内皮细胞通过向血液供应调节血栓形成、纤溶和局部炎症事件的因子来控制血管动态平衡。这些因子包括P-选择素和von-Willebrand因子，它们分别作为白细胞和血小板的受体，控制局部炎症激活和血管损伤的反应。VWF和P-选择素储存在内皮细胞的大分泌颗粒--韦氏小体(WPB)中，受复杂的生物发生途径的影响。未成熟的WPB起源于跨高尔基体网络，然后被运输到细胞外围，在那里它们与肌动蛋白皮质相连，并经历短距离的肌动蛋白依赖的运动。WPB的成熟发生在运输和外周锚定过程中，涉及内体成分的转移，特别是P-选择素辅助因子CD63。在内皮细胞激活后，皮质锚定和成熟的WPB的胞吐作用通过分泌剂升高胞内钙离子或cAMP水平而被触发。在之前的资金支持期间，参与调节WPB胞吐的后期步骤的几个成分，包括GTPase Rab35和拴系/启动因子Munc13-4已被鉴定和鉴定。此外，我们可以记录融合后WPB的动态肌动蛋白重组，并通过进行邻近蛋白质组筛选确定新的WPB相关因子。基于这些发现，下一个资助阶段的重点将放在这些新因素的表征和WPB胞吐过程中皮质肌动蛋白重排的调节上。此外，我们计划分析WPB成熟背后的大部分未知机制。具体地说，我们将集中于以下目标：1.描述与早期WPB的生成和成熟有关的过程。这里的焦点将集中在Rab43及其效应器在TGN形成早期WPB中的作用，以及内体蛋白，特别是CD63，转移到成熟的WPB中。分析皮质肌动蛋白重排在调节WPB运动和货物释放中的作用。这一部分将集中于肌动蛋白在WPB运动中的作用，以及融合后WPB中肌动蛋白重组的调控和功能。因此，本项目旨在阐明内皮细胞WPB成熟、运输和分泌诱导的胞吐作用的关键机制。这种详细的理解最终可能导致开发工具来专门改变病理情况下内皮细胞的血栓和炎症反应。

Endothelial cells control vascular homeostasis by supplying the blood with factors that regulate thrombosis, fibrinolysis and local inflammatory events. These factors include P-selectin and von-Willebrand factor (VWF), which serve as receptors for leukocytes and platelets, respectively, and control responses to local inflammatory activation and blood vessel injury. VWF and P-selectin are stored in large secretory granules of endothelial cells, the Weibel Palade bodies (WPB), that are subject to a complex biogenesis pathway. Immature WPB originate at the trans-Golgi network and are then transported to the cell periphery where they are linked to the actin cortex and undergo short range, actin-dependent movements. Maturation of WPB occurs during transport and peripheral anchorage and involves transfer of endosomal components, in particular CD63, a P-selectin cofactor, to WPB. Exocytosis of cortically anchored and matured WPB is then triggered following endothelial activation by secretagogues elevating cytosolic Ca2+ or cAMP levels.Several components participating in late steps of regulated WPB exocytosis, including the GTPase Rab35 and the tethering/priming factor Munc13-4, have been identified and characterized in the previous funding period. Moreover, we could document dynamic actin reorganizations at post-fusion WPB and we identified novel WPB-associated factors by performing proximity proteome screens. Based on these findings, the focus in a next funding period will be on the characterization of these novel factors and the regulation of cortical actin rearrangements during WPB exocytosis. Moreover, we plan to analyze the largely unkown mechanisms underlying WPB maturation. Specifically, we will focus on the following objectives:1. Characterization of processes that are involved in the generation and maturation of early WPB. Here the focus will be on the role of Rab43 and its effectors in the formation of early WPB at the TGN, and the transfer of endosomal proteins, in particular CD63, to maturing WPB.2. Analysis of the role of cortical actin rearrangements in regulating WPB motility and cargo release. This part will focus on the actin nucleator Spire1 in the actin-based movement of WPB and the regulation and function of actin reorganizations at post-fusion WPB.Thus, the project aims at elucidating key mechanisms underlying the maturation, transport and secretagogue-evoked exocytosis of endothelial WPB. This detailed understanding could eventually lead to the development of tools to specifically alter thrombotic and inflammatory responses of endothelial cells in pathological scenarios.