Cytoskeletal Mechanisms of Endocytosis

描述（由申请人提供）：与环境的交流对于生物体中单个细胞的生存和正常功能至关重要。这种通讯的要素之一是细胞内和细胞外空间之间的成分交换。 它包括分泌和内吞两个主要过程，分别大致相当于人类世界中的出口和进口。该项目将重点研究主要内吞途径——网格蛋白介导的内吞作用（CME）的机制，通过该途径，细胞以高度选择性的方式吸收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）组装基于网格蛋白的外壳，将货物和内吞机器募集到内吞作用位点的多蛋白支架〜（ii）包被的质膜内陷以形成网格蛋白包被的凹坑〜（iii）坑的伸长和其颈部的收缩形成网格蛋白包被的芽〜（iv）芽颈的分裂以形成内吞囊泡〜和（v）囊泡的向内运动。所有这些过程在能量上都是不利的，需要产生力的机器才能发生。正在进行的 CME 机制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，由于缺乏有关内吞位点细胞骨架结构的高分辨率结构信息，尚未制定肌动蛋白细胞骨架在 CME 中具体作用的明确模型。这种缺陷的主要原因是与内吞位点相关的肌动蛋白斑块尺寸极小且短暂，这些斑块内单个肌动蛋白丝的密集堆积使得它们无法通过光学显微镜分辨，以及众所周知的电子显微镜保存动态肌动蛋白丝网络的困难。 利用我们在铂复制品电子显微镜方面的特殊专业知识（对于分析细胞骨架结构最有用），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 网格蛋白包被结构形成和成熟的不同阶段中细胞骨架组织的变化，并通过功能方法确定几个关键蛋白质在此过程中的作用。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凹周边成核的分支肌动蛋白网络向 所有三个表面：正在生长的芽、芽颈和芽基部的质膜，以收缩和拉长芽颈，但随后它被重新排列成彗星尾，将新形成的囊泡推入细胞质。 这些研究的结果将 显着增进了我们对 CME 期间囊泡内化的肌动蛋白依赖性机制的理解。

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.