Dynamics of Clathrin Coat Formation in Cells

描述（由申请方提供）：细胞需要蛋白质和脂质从一个膜结合区室有序移动到另一个膜结合区室，同时维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。网格蛋白包被的囊泡是从质膜到内体（内吞作用）的最突出的运输形式，内吞作用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核内体和高尔基体网络（trans-Golgi network，TGN）之间的交通也很重要。我们建议继续我们的研究网格蛋白介导的膜交通的分子机制，使用直接观察活细胞成像与单分子的灵敏度和高的空间和时间分辨率，回答以下问题。(1)什么是启动形成网格蛋白包被的纹孔所需的分子事件？(2)什么是被困在不同阶段的增长和关闭的外套的分子组成：早期和晚期流产的涂层坑，坑停滞在一个相对较晚的阶段，通过施加张力的膜和抑制肌动蛋白动力学？(3)Hsc 70及其辅助分子伴侣生长素介导的网格蛋白脱壳机制是什么？脱壳过程如何与组装过程协调，使这两个过程不竞争？ 公共卫生相关性：囊泡膜运输是细胞中蛋白质和脂质在膜结合区室之间移动的主要机制。网格蛋白包被的囊泡是从质膜到内体的运输（内吞作用）的最突出形式，内吞作用是配体如激素、转铁蛋白、免疫球蛋白、LDL、病毒及其受体进入细胞的途径。

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? PUBLIC HEALTH RELEVANCE: Vesicular membrane traffic is the principal mechanism for moving proteins and lipids among membrane-bound compartments in a cell. Clathrin coated vesicles are the most prominent form of traffic from the plasma membrane to endosomes (endocytosis), a pathway by which ligands such as hormones, transferrin, immunoglobulins, LDL, viruses and their receptors enter cells.