The Clathrin Coated Vesicle Cycle

描述（由申请人提供）：网格蛋白介导的内吞作用（CME）是细胞选择性摄取表面受体及其结合配体的主要途径。因此，CME控制着细胞稳态的许多方面，包括营养摄取、离子、糖和其他代谢物转运体的表面表达、与免疫系统的相互作用、信号受体的调节、细胞粘附受体的调节等。在突触，CME是突触囊泡循环的主要途径，从而维持神经传递。大多数（如果不是全部的话）网格蛋白介导的内吞机制的组成部分已被确定，但其中的确切功能尚不清楚。研究得最好的成分是外壳蛋白、网格蛋白和接头蛋白以及GTPase动力蛋白。许多其他部分是根据它们与这些主要成分的相互作用确定的。我们已经开发了内吞网格蛋白包被囊泡（CCV）形成的定量和互补的体内和体外分析，并将利用这些来探索CME的基本机制。体内实验利用全内反射荧光显微镜，结合新颖的跟踪软件和统计分析，使我们能够全面定量地分析网格蛋白包覆坑（CCP）动力学和活细胞中CCV形成的动力学。体外分析利用支持脂质双层和多余的膜储层或“SUPER”模板，为囊泡形成提供了一个强大而便捷的分析系统，适用于生化分析和实时成像。通过这些实验，我们将追求三个主要目标：1)定量定义体内调节网格蛋白包覆坑的形成和成熟的因素，包括cargo、PI4、52、v-SNAREs和AP2相互作用；2)明确含SH3结构域的动力蛋白-1效应器在体内和体外CCV形成过程中作为动力蛋白功能调节剂的作用；3)利用纯化的蛋白组分从SUPER模板中重建CCV形成。通过定义CCV形成所需的最小机制，并建立一个强大的、定量的和生理相关的测定方法来测量这一过程，我们将建立充分理解网格蛋白介导的内吞作用的机制基础的手段。

DESCRIPTION (provided by applicant): Clathrin-mediated endocytosis (CME) is the major pathway for selective uptake of surface receptors and their bound ligands into cells. As such, CME controls many aspects of cellular homeostasis, including nutrient uptake, the surface expression of ion, sugar and other metabolite transporters, interactions with the immune system, regulation of signaling receptors, regulation of cell adhesion receptors, etc. At the synapse, CME is the major pathway for synaptic vesicle recycling and thus for maintaining neurotransmission. Most, if not all, of the component parts of the clathrin- mediated endocytotic machinery have been identified but the exact of function of few of these are known. The best studied components are the coat proteins, clathrin and adaptor proteins, and the GTPase dynamin. Many of the other parts were identified based on their interactions with these major constituents. We have developed quantitative and complementary in vivo and in vitro assays for endocytic clathrin coated vesicle (CCV) formation and will use these to probe the fundamental mechanisms underlying CME. The in vivo assays utilize total internal reflection fluorescence microscopy coupled with novel tracking software and statistical analyses that allow us to comprehensively and quantitatively analyze clathrin coated pit (CCP) dynamics and the kinetics of CCV formation in living cells. The in vitro assay utilizes supported lipid bilayers with excess membrane reservoir or 'SUPER' templates, which provide a robust and facile assay system for vesicle formation that is amenable to both biochemical analysis and real-time imaging. Using these assays, we will pursue three major aims: 1) To quantitatively define factors that regulate clathrin coated pit initiation and maturation in vivo, including cargo, PI4,52, v-SNAREs and AP2 interactions; 2) To define the role of SH3 domain-containing dynamin-1 effectors as regulators of dynamin function in CCV formation in vivo and in vitro; and 3) To reconstitute CCV formation from SUPER templates using purified protein components. By defining the minimum machinery required for CCV formation and establishing a robust, quantitative and physiologically relevant assay for measuring this process we will have established the means to fully understand the mechanistic underpinnings of clathrin-mediated endocytosis. )