Cytoskeletal Mechanisms of Endocytosis

胞吞作用的细胞骨架机制

• 批准号: 9272215
• 负责人: Tatyana Svitkina
• 金额: $ 8.26万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-07-01 至 2017-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9272215
• 关键词: Actins; Address; Architecture; Automobile Driving; Behavior; Binding Proteins; Cardiovascular Diseases; Cell membrane; Cell surface; Cells; Clathrin; Communication; Cytoplasm; Cytoskeleton; Data; Disease; Dissection; Electron Microscopy; Elements; Endocytic Vesicle; Endocytosis; Endocytosis Inhibition; Environment; Extracellular Space; Geometry; Health; Hereditary Disease; Human; Individual; Intracellular Space; Lead; Life; Malignant Neoplasms; Mediating; Membrane; Microfilaments; Modeling; Molecular; Morphogenesis; Movement; Nature; Neck; Organelles; Organism; Pathologic Processes; Pathology; Pathway interactions; Physiological Processes; Platinum; Polymers; Process; Proteins; Recruitment Activity; Research; Resolution; Rest; Role; Route; Site; Staging; Structure; Surface; System; Systems Analysis; Tail; Techniques; Testing; Vesicle; base; coated pit; constriction; electron tomography; human disease; light microscopy; nervous system disorder; novel diagnostics; protein function; reconstitution; role model; scaffold; secretion process; trafficking; treatment strategy

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.

描述（由申请人提供）：与环境的沟通对于生物体中单个细胞的生存和适当功能至关重要。这种通信的一个要素是细胞内和细胞外空间之间的组件交换。 它包括两个主要过程，分泌和内吞作用，它们在人类世界中大致相当于出口和进口。该项目将重点关注主要内吞途径，网格蛋白介导的内吞作用（CME）的机制，通过该机制，细胞以高选择性的方式摄入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）基于网状蛋白的外套的组装，一种多蛋白支架招募货物及其内吞机械的内吞作用〜（ii）涂层的质膜的内部（ii）形成了涂有粘液蛋白涂层坑的涂层（iii）的底部和clath temiv temiv temiv temiv的质子和约束〜芽颈形成囊泡的内吞囊泡〜和（v）向内运动。所有这些过程在能量上都是不利的，需要产生力的机械。关于CME机制的持续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在化的分子机械的重要组成部分。但是，由于缺乏有关内吞位点的细胞骨架结构的高分辨率结构信息，尚未制定针对CME肌动蛋白细胞骨架在CME中特定作用的明确模型。这种缺陷的主要原因是与内吞位点相关的肌动蛋白斑块的尺寸和瞬态性质，这些斑块中单个肌动蛋白丝的密集堆积，从而使它们无法通过光显微镜进行不可抵消，并为电子显微镜保存动态肌动蛋白细丝网络的众所周知的困难。 使用我们在分析细胞骨架结构最有用的铂副本电子显微镜方面的特殊专业知识，我们建议确定肌动蛋白丝阵列阵列的结构组织和分子组成与各种类型的网格蛋白涂层结构相关，以将 具有不同形成阶段的细胞骨架组织的变化和涂有网状蛋白涂层结构的成熟阶段，并通过功能方法在此过程中建立了几种关键蛋白质的作用。通过这些研究，我们将检验一个假设，即分支肌动蛋白网络围绕网状蛋白涂层的坑的周围成核，将力推到上 这三个表面：生长的芽，芽颈和芽胞膜位于芽的底部，以收缩和拉长芽颈，但随后将其重新排列成彗星尾巴，将新形成的囊泡推入细胞质。 这些研究的结果将 显着提高了我们对CME期间囊泡内在化的肌动蛋白依赖性机制的理解。