Dynamics of Clathrin Coat Formation in Cells

描述(由申请人提供)：细胞需要蛋白质和脂类有序地从一个膜结合的隔室到另一个膜结合的隔室，同时保持供体和受体膜的组织、功能和异质性；囊泡膜运输执行这些功能。笼蛋白包裹的囊泡是从质膜到内吞体(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一条途径。包被网状蛋白的囊泡对于内体和反式高尔基体网络(TGN)之间的交通也很重要。我们建议继续我们对分子筛蛋白介导的膜运输的分子机制的研究，利用具有单分子灵敏度和高空间和时间分辨率的活细胞成像直接观察，以回答以下问题。(1)通过施加膜的张力和抑制肌动蛋白动力学，在生长和闭合的不同阶段捕获的涂层的分子组成是什么？(1)需要什么分子事件才能启动笼状蛋白涂层的形成？(3)通过施加膜的张力和抑制肌动蛋白动力学，涂层涂层早期和后期停滞的涂层涂层的分子组成是什么？(3)Hsc70及其辅助伴侣生长素介导的网格蛋白脱涂层的机制是什么，脱涂层与组装是如何协调的，从而使这两个过程不相互竞争？

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?