Dynamics of Clathrin Coat Formation in Cells

描述（由申请人提供）：细胞需要蛋白质和脂质从一个膜结合室有序地移动到另一个膜结合室，同时保持供体和受体膜的组织、功能和异质性；囊泡膜交通执行这些功能。网格蛋白包被的囊泡是从质膜到核内体（内吞作用）的最主要的运输形式，是各种配体及其受体进入细胞的途径。网格蛋白包被的囊泡对于内体和反式高尔基网络（TGN）之间的交通也很重要。我们建议继续研究网格蛋白介导的膜交通的分子机制，使用单分子灵敏度和高时空分辨率的活细胞成像直接观察，以回答以下问题。(1)形成网格蛋白包覆坑需要哪些分子事件？(2)通过施加膜张力和抑制肌动蛋白动力学，在生长和闭合的不同阶段被捕获的被膜分子成分是什么：早期和晚期流产的包膜坑，以及在相对较晚的阶段停滞的坑？(3) Hsc70及其协同伴侣auxilin介导的网格蛋白脱膜的机制是什么，脱膜如何与组装协调，使两者不竞争？

DESCRIPTION (provided by applicant): Cells require ordered movement of proteins and lipids from one membrane-bound compartment to another, while maintaining the organization, function and heterogeneity of the donor and acceptor membranes; vesicular membrane traffic carries out these functions. Clathrin coated vesicles are the most prominent form of traffic from the plasma membrane to endosomes (endocytosis), a pathway by which various ligands and their receptors enter cells. Clathrin coated vesicles are also important for traffic between endosomes and the trans-Golgi network (TGN). We propose to continue our studies of molecular mechanisms in clathrin-mediated membrane traffic, using direct observation by live-cell imaging with single-molecular sensitivity and high spatial and temporal resolution, to answer the following questions. (1) What are the molecular events required to initiate formation of a clathrin coated pit? (2) What are the molecular components of coats trapped at various stages of growth and closure: early and late abortive coated pits, and pits stalled at a relatively late stage, by imposing tension of the membrane and by inhibiting actin dynamics? (3) What is the mechanism of clathrin uncoating mediated by Hsc70 and its co-chaperone auxilin and how is the uncoating coordinated with assembly so that the two processes do not compete?