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控制细胞内稳态的许多方面，包括营养摄取，离子，糖和其他代谢物转运蛋白的表面表达，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是突触囊泡回收的主要途径，从而维持神经传递。网格蛋白介导的内吞机制的大多数（如果不是全部）组成部分已经被鉴定，但其中很少有确切的功能是已知的。研究得最好的成分是外壳蛋白、网格蛋白和衔接蛋白以及GTdR发动蛋白。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。我们已经开发了定量和互补的内吞网格蛋白包被囊泡（CCV）的形成在体内和体外测定，并将使用这些探测CME的基本机制。体内试验利用全内反射荧光显微镜加上新的跟踪软件和统计分析，使我们能够全面和定量分析网格蛋白包被的坑（CCP）的动力学和CCV形成的动力学活细胞。该体外测定利用具有过量膜储库或“SUPER”模板的支持的脂质双层，其提供了用于囊泡形成的稳健且容易的测定系统，该系统适合于生化分析和实时成像。利用这些分析，我们将追求三个主要目标：1）定量确定在体内调控网格蛋白包被的小窝起始和成熟的因素，包括货物，PI 4，52，v-SNARE和AP 2的相互作用; 2）确定在体内和体外含有SH 3结构域的dynamin-1效应物作为dynamin功能的调节剂在CCV形成中的作用;和3）使用纯化的蛋白质组分从SUPER模板重建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个过程，我们将建立的手段，以充分了解网格蛋白介导的内吞作用的机制基础。) 公共卫生相关性：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，也是维持神经传递所需的突触囊泡再循环的主要途径。CME的缺陷与许多人类疾病有关，包括高胆固醇血症、白血病和乳腺癌、肌肉肌病、眼部和神经变性以及肾脏疾病。了解这一基本的细胞过程将提供深入了解这些疾病的发病机制，并为寻求新的治疗方法提供必要的基础。)

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. ) PUBLIC HEALTH RELEVANCE: Clathrin-mediated endocytosis (CME) is the major pathway for selective uptake of surface receptors and their bound ligands into cells and the major pathway for synaptic vesicle recycling required to maintain neurotransmission. Defects in CME have been linked to many human diseases, including hypercholesterolemia, leukemia and breast cancers, muscle myopathies, ocular and neurodegeneration, and kidney disease. Understanding this fundamental cellular process will provide insight into the pathogenesis of these diseases and provide the necessary foundation for seeking new therapeutic approaches towards their treatment. )