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粘附受体（最显著的是von-Willebrand因子（VWF）和P-选择素）来响应血管损伤和局部炎症。这些被储存在韦伯-帕拉德体（WPB）中，杆状分泌颗粒在内皮激活后进行调节性胞吐。在这个项目中，我们将分析WPB胞吐作用的最后步骤的分子机制，重点是WPB对接和融合位点的作用和组成，作为细胞界面的动态质膜平台。 我们可以表明，WPB融合位点的特征在于磷脂酰肌醇（4，5）-二磷酸[PI（4，5）P2]的局部和瞬时富集，并且我们已经鉴定了在融合位点特异性富集的WPB相关蛋白和PI（4，5）P2结合蛋白。我们现在将通过分析特定操作（脂质磷酸酶或激酶的靶向表达，蛋白质耗竭或过度表达）对急性WPB胞吐的影响来解决局部PI（4，5）P2富集和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，将各自的脂质和蛋白质动力学与实际的WPB融合位点相关联。在第二组实验中，我们希望解决特定的Rab GTP酶在WPB胞吐的最后步骤中的作用。在一个全面的筛选中，我们确定了一些Rab蛋白，作为WPB胞吐的正调节因子，并且以前没有与该过程相关。我们现在计划通过确定这些新的WPB相关Rab的特定效应子和调节子及其作用位点来表征其作用模式。总之，我们的研究结果将导致作为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.