Mechanisms of nucleation and stabilization in clathrin-mediated endocytosis

网格蛋白介导的内吞作用中的成核和稳定机制

• 批准号: 8212917
• 负责人: Daniel Nunez
• 金额: $ 2.75万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-01-14 至 2013-01-13
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8212917
• 关键词: Actins; Adaptor Signaling Protein; Binding; Biological Assay; Cell membrane; Cells; Clathrin; Clathrin-Coated Vesicles; Data; Disease; Dissociation; Endocytosis; Event; Extracellular Space; Goals; Heterogeneity; Homeostasis; Hot Spot; Individual; Laboratories; Ligand Binding; Measures; Mediating; Membrane; Methods; Molecular; Phase; Proteins; Regulation; Research; Resources; Site; Source; Spottings; Staging; Structure; Testing; Time; coated pit; public health relevance; receptor; residence; seal; stoichiometry

DESCRIPTION (provided by applicant): Mechanisms of nucleation and stabilization in clathrin-mediated endocytosis Clathrin-mediated endocytosis (CME) is the main mechanism by which cells internalize molecules from the extracellular space. As such, it is involved in all functions regulating cellular homeostasis. CME can occur constitutively or can be stimulated by ligand-bound receptors. CME begins with the assembly of clathrin-coated pits (CCPs) at the membrane which stabilize, mature, invaginate, and pinch-off as sealed clathrin-coated vesicles (CCVs). During maturation the pits accumulate cargo, adaptors, accessory proteins, and clathrin. An emerging theme in the field of mammalian endocytosis is the notion that endocytic pits vary in molecular composition, lifetime, size, and even on whether they form CCVs or not. We do not yet understand what is the source or the functional significance of this heterogeneity in CME. While it is likely that the observed heterogeneity could result from many different sources, it is probable that the variability reflects the intrinsic heterogeneity in the regulation of CME. Data from the Danuser and Schmid laboratories suggest that most of the regulation of CME occurs during the difficult to study early stages of an endocytic pit's lifetime. In particular, it is probable that a large part of the observed heterogeneity is imparted during nucleation. The goal of this research is to establish the mechanisms which regulate the nucleation of CCPs, and to determine the sources and effects of heterogeneity during these initiation events. I propose to begin by studying the spatial regulation of nucleation as a possible source of heterogeneity in CME. The concept of stable nucleation machinery that creates multiple nucleations in one spot, called the hot-spot, has been previously suggested, but is a source of debate in the field. I propose to resolve whether the heterogeneity of CCPs can be explained in part by their spatial organization at the plasma membrane. I propose to identify the proteins that spatially regulate nucleation. I propose to look at the residence time of various nucleation resources at sites of repeated nucleation to determine if a stable nucleation structure exists. Another possible source of heterogeneity and regulation during the nucleation and stabilization phases of CME is the composition of pits at these early stages. I propose to measure the stoichiometry of the proteins that form the nucleating pit. PUBLIC HEALTH RELEVANCE: Clathrin-mediated endocytosis (CME) is the main mechanism by which cells internalize molecules from the extracellular space. As such, it is involved in all functions regulating cellular homeostasis. Deregulation of CME is therefore relevant in many diseases.

描述（由申请人提供）：网格蛋白介导的内吞作用中的成核和稳定机制网格蛋白介导的内吞作用（CME）是细胞从细胞外空间内化分子的主要机制。因此，它参与调节细胞稳态的所有功能。CME可以组成性地发生或可以由配体结合受体刺激。CME开始于网格蛋白包被的小凹（CCP）在膜上的组装，其稳定、成熟、内陷和夹断为密封的网格蛋白包被的囊泡（CCV）。在成熟过程中，凹坑积累货物，衔接子，辅助蛋白和网格蛋白。哺乳动物内吞作用领域的一个新兴主题是内吞凹在分子组成、寿命、大小甚至它们是否形成CCV上变化的概念。我们还不知道CME中这种异质性的来源或功能意义是什么。虽然观察到的异质性可能来自许多不同的来源，但这种变异性可能反映了CME调控的内在异质性。来自Danuser和Schmid实验室的数据表明，大多数CME的调节发生在难以研究的内吞陷窝生命早期阶段。特别是，很可能观察到的异质性的大部分是在成核过程中赋予的。本研究的目的是建立调节CCP成核的机制，并确定在这些引发事件的异质性的来源和影响。我建议开始通过研究的空间调控的成核作为一个可能的来源的异质性在CME。稳定的成核机制，在一个点，称为热点，创造多个成核的概念，以前已经提出，但在该领域的辩论的来源。我建议解决CCP的异质性是否可以部分解释其空间组织在质膜。我建议确定空间调节成核的蛋白质。我建议看看在重复成核的网站，以确定是否存在一个稳定的成核结构的各种成核资源的停留时间。在日冕物质抛射的成核和稳定阶段，另一个可能的不均匀性和调节来源是这些早期阶段的凹坑成分。我建议测量形成核孔的蛋白质的化学计量。 公共卫生相关性：网格蛋白介导的内吞作用（CME）是细胞从细胞外空间内化分子的主要机制。因此，它参与调节细胞稳态的所有功能。因此，CME的放松管制与许多疾病有关。