Membrane platforms in regulated secretion from endothelial cells

血管内皮细胞通过急性暴露于血小板和白细胞黏附受体，尤其是von-Willebrand因子(VWF)和P-选择素来应答血管损伤和局部炎症。它们储存在韦贝尔-帕拉德小体(WPB)中，WPB是一种杆状分泌颗粒，在内皮激活后经历调节的胞吐作用。在这个项目中，我们将分析WPB胞吐最后几步的分子机制，重点放在WPB对接和融合位点作为细胞界面动态质膜平台的作用和组成。我们可以证明WPB融合位点的特征是磷脂酰肌醇(4，5)-二磷酸[PI(4，5)P2]的局部和瞬时富集性，我们已经鉴定了WPB相关和PI(4，5)P2结合蛋白，它们在融合位点变得特异富含。现在，我们将通过分析特定操作(脂磷酸酶或激酶的靶向表达，蛋白质耗尽或过表达)对急性WPB胞吐的影响，来探讨局部PI(4，5)P2浓缩和PI(4，5)P2结合蛋白在WPB胞吐中的作用。这将与活细胞成像方法相结合，将各自的脂肪和蛋白质动力学与实际的WPB融合部位联系起来。在第二组实验中，我们想要解决特定的Rab GTP酶在WPB胞吐的最后步骤中的作用。在一次全面的筛选中，我们确定了一些Rab蛋白，它们作为WPB胞吐的积极调节因子，以前没有与这一过程相关。我们现在计划通过确定它们的特定效应器和调节器以及它们的作用部位来表征这些新的WPB相关RAB的作用模式。总之，我们的结果将导致对作为WPB融合位点的膜平台的组成、动力学和调节的分子理解。

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.