The Clathrin Coated Vesicle Cycle

描述(申请人提供)：网状蛋白介导的内吞作用(CME)是选择性地将表面受体及其结合配体吸收到细胞中的主要途径。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、细胞黏附受体的调节等。在突触，CME是突触小泡循环的主要途径，从而维持神经传递。大多数，如果不是全部的话，由分子筛介导的内吞机制的组成部分已经被确定，但其中少数的确切功能是已知的。研究得最好的成分是外壳蛋白、网状蛋白和接头蛋白，以及GTP酶动力蛋白。许多其他部分是根据它们与这些主要成分的相互作用而确定的。我们在体内和体外开发了定量的和互补的内细胞膜包裹小泡(CCV)形成的检测方法，并将用这些方法来探索CME的基本机制。体内检测采用全内反射荧光显微镜，结合新的跟踪软件和统计分析，使我们能够全面和定量地分析笼状蛋白涂层凹坑(CCP)的动力学和活细胞中CCV的形成动力学。体外检测利用具有超量膜储存库的支持脂质双分子层或“超级”模板，这为囊泡的形成提供了一种健壮而简便的检测系统，既可用于生化分析，也可用于实时成像。利用这些分析方法，我们将追求三个主要目标：1)定量定义调节笼状蛋白包裹的凹坑启动和成熟的因素，包括Cargo、PI4、52、v-SNARES和AP2相互作用；2)确定含有SH3结构域的Dynamin-1效应器在体内和体外作为Dynamin功能调节因子在CCV形成中的作用；以及3)使用纯化的蛋白质组分从超级模板重建CCV形成。通过定义CCV形成所需的最小机制，并建立一种稳健的、定量的和生理上相关的检测方法来测量这一过程，我们将建立充分理解网状蛋白介导的内吞作用的机制基础的方法。) 与公共卫生相关：网状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小泡循环的主要途径。CME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、眼部和神经退行性变以及肾脏疾病。了解这一基本的细胞过程将有助于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. )