Mechanisms governing regulated secretion in human endothelial cells

内皮细胞通过向血液提供调节血栓形成、纤维蛋白溶解和局部炎症事件的因子来控制血管稳态。这些因子包括p -选择素和范氏血友病因子（VWF），它们分别作为白细胞和血小板的受体，并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分泌大颗粒中，即韦贝尔帕拉德体（WPB），这是一个复杂的生物发生途径。未成熟的WPB起源于反式高尔基网络，然后被运送到细胞外周，在那里它们与肌动蛋白皮层相连，并经历短距离的、依赖于肌动蛋白的运动。WPB的成熟发生在运输和外周锚定过程中，涉及到内体成分的转移，特别是CD63（一种p选择素辅助因子）向WPB的转移。皮层锚定和成熟的WPB在内皮细胞激活后，通过分泌剂提高细胞质Ca2+或cAMP水平触发胞外分泌。参与WPB胞吐调节后期步骤的几个成分，包括GTPase Rab35和tethering/priming factor Munc13-4，已经在之前的资助期被确定和表征。此外，我们可以记录融合后WPB的动态肌动蛋白重组，我们通过进行近距离蛋白质组筛选确定了新的WPB相关因子。基于这些发现，下一个资助期的重点将放在这些新因素的特征和WPB胞吐过程中皮质肌动蛋白重排的调节上。此外，我们计划分析大部分未知的WPB成熟机制。具体而言，我们将重点实现以下目标：早期WPB发生和成熟过程的表征。这里的重点是Rab43及其效应物在TGN早期WPB形成中的作用，以及内体蛋白（特别是CD63）向成熟WPB的转移。皮质肌动蛋白重排在调节WPB运动和货物释放中的作用分析。本部分将重点介绍肌动蛋白成核子Spire1在WPB中基于肌动蛋白的运动，以及融合后WPB中肌动蛋白重组的调控和功能。因此，该项目旨在阐明内皮细胞的成熟、运输和分泌诱导的胞吐的关键机制。这种详细的了解可能最终导致工具的发展，以特异性地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.