Membrane platforms in regulated secretion from endothelial cells

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Vascular endothelial cells respond to blood vessel injury and local inflammation by the acute exposure of platelet and leukocyte adhesion receptors, most notably von-Willebrand factor (VWF) and P-selectin. These are stored in Weibel-Palade bodies (WPB), rod-shaped secretory granules that undergo regulated exocytosis following endothelial activation. In this project we will analyze the molecular mechanisms that underlie the final steps of WPB exocytosis focussing on the role and composition of WPB docking and fusion sites as dynamic plasma membrane platforms at the cellular interface. We could show that WPB fusion sites are characterized by a local and transient enrichment of phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P2] and we have identified WPB-associated and PI(4,5)P2-binding proteins that become specifically enriched at the fusion sites. We will now address the role of local PI(4,5)P2 enrichment and of PI(4,5)P2 binding proteins in WPB exocytosis by analyzing the effects of specific manipulations (targeted expression of lipid phosphatases or kinases, protein depletion or overexpression) on acute WPB exocytosis. This will be combined with live cell imaging approaches relating the respective lipid and protein dynamics to the actual WPB fusion sites. In a second set of experiments we want to address the role of specific Rab GTPases in the final steps of WPB exocytosis. In a comprehensive screen we identified a number of Rab proteins that function as positive regulators of WPB exocytosis and have not been linked to this process before. We now plan to characterize the mode of action of these novel, WPB associated Rabs by identifying their specific effectors and regulators and their site of action. Together, our results will lead to a molecular understanding of the composition, dynamics and regulation of membrane platforms that serve as WPB fusion sites.