Cytoskeletal Mechanisms of Endocytosis

描述（由申请人提供）：与环境的沟通对于生物体内单个细胞的生存和正常功能至关重要。这种通讯的一个要素是细胞内和细胞外空间之间的组分交换。 它包括两个主要的过程，分泌和内吞，在人类世界中分别大致相当于输出和输入。本项目将着重于主要的内吞途径，网格蛋白介导的内吞作用（CME）的机制，通过该机制，细胞以高度选择性的方式摄取外源分子和细胞表面成分。CME的关键步骤是内吞囊泡的初始形成。 该过程包括：（i）基于网格蛋白的涂层的组装，一种多蛋白支架，将货物和内吞机器募集到内吞位点~（ii）被膜的内陷形成被网格蛋白包被的小窝~（iii）小窝的伸长和颈部的收缩不形成被网格蛋白包被的芽~（iv）芽颈断裂形成内吞小泡;（v）小泡向内移动。所有这些过程在能量上都是不利的，需要力产生机制才能发生。目前对CME机制的研究越来越多地指出，肌动蛋白细胞骨架是驱动内吞囊泡内化的分子机制的重要组成部分。然而，一个明确的模型，肌动蛋白细胞骨架在CME的具体作用还没有制定，因为缺乏高分辨率的结构信息的细胞骨架结构的内吞网站。这种缺陷的主要原因是与内吞位点相关的肌动蛋白斑块的极小尺寸和瞬时性质，这些斑块内的单个肌动蛋白丝的致密包装使得它们通过光学显微镜无法分辨，以及众所周知的保存动态肌动蛋白丝网络用于电子显微镜的困难。 利用我们在铂复型电子显微镜方面的特殊专长，这对分析细胞骨架结构是最有用的，我们建议确定与各种类型的网格蛋白涂层结构相关的肌动蛋白丝阵列的结构组织和分子组成，以将细胞骨架结构与细胞骨架结构相关联。 在网格蛋白包被结构的形成和成熟的不同阶段的细胞骨架组织的变化，并建立几个关键蛋白在这一过程中的作用，通过功能的方法。通过这些研究，我们将检验一个假设，即围绕网格蛋白包被的小坑周边成核的分支肌动蛋白网络对细胞施加推力。 三个表面：生长中的芽、芽颈和芽基部的质膜，以便收缩和拉长芽颈，但随后它被重新排列成彗星尾，将新形成的小泡推进细胞质。 这些研究的结果将 显著推进我们对CME期间囊泡内化的肌动蛋白依赖机制的理解。

DESCRIPTION (provided by applicant): Communication with the environment is essential for survival and proper functionality of individual cells within an organism. One element of such communication is exchange of components between the intracellular and extracellular space. It includes two major processes, secretion and endocytosis, which are roughly equivalent to export and import, respectively, in the human world. This project will focus on the mechanisms of the major endocytic pathway, clathrin-mediated endocytosis (CME), by which cells take up exogenous molecules and cell surface components in a highly selective way. The key step of CME is the initial formation of an endocytic vesicle. This process consists of: (i) Assembly of the clathrin-based coat, a multiprotein scaffold recruiting the cargo and the endocytic machinery to the sites of endocytosis~ (ii) invagination of the coated plasma membrane to form a clathrin-coated pit~ (iii) elongation of the pit and constrictions of its neck t form a clathrin-coated bud~ (iv) scission of the bud neck to form an endocytic vesicle~ and (v) inward movement of the vesicle. All these processes are energetically unfavorable and require force-generating machinery to occur. The ongoing research on the mechanisms of CME increasingly points to the actin cytoskeleton as an important component of the molecular machinery driving endocytic vesicle internalization. However, an explicit model for the specific roles of actin cytoskeleton in CME has not been formulated because of a lack of high resolution structural information about the cytoskeletal architecture at endocytic sites. The major reasons for this deficiency are an extremely small size and transient nature of actin patches associated with the endocytic sites, dense packing of individual actin filaments within these patche making them irresolvable by light microscopy, and a well-known difficulty of preserving dynamic actin filament networks for electron microscopy. Using our special expertise in platinum replica electron microscopy that is most useful for the analysis of the cytoskeletal architecture, we propose to determine the structural organization and molecular composition of actin filament arrays associated with various types of clathrin-coated structures, to correlate the changes in the cytoskeleton organization with different stages of formation and maturation of clathrin-coated structures, and establish roles of several key proteins in this process by functional approaches. By these studies, we will test a hypothesis that a branched actin network nucleated around the perimeter of a clathrin-coated pit exerts pushing force onto all three surfaces: the growing bud, the bud neck, and the plasma membrane at the base of a bud, in order to constrict and elongate the bud neck, but then it is rearranged into a comet tail that propels the newly formed vesicle into the cytoplasm. The results of these studies will significantly advance our understanding of the actin-dependent mechanisms of vesicle internalization during CME.