Mechanisms governing regulated secretion in human endothelial cells

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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.