Mechanisms of nucleation and stabilization in clathrin-mediated endocytosis

描述（由申请人提供）：网格蛋白介导的内吞作用的成核和稳定机制网格蛋白介导的内吞作用（CME）是细胞内化细胞外分子的主要机制。因此，它参与调节细胞内稳态的所有功能。CME可以组成性发生，也可以由配体结合受体刺激。CME开始于膜上网格蛋白包覆的凹坑（CCPs）的组装，这些凹坑稳定、成熟、内陷并夹住，形成密封的网格蛋白包覆的囊泡（CCVs）。在成熟过程中，核穴积累货物、接头、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，内吞坑在分子组成、寿命、大小甚至是否形成ccv方面都有变化。我们还不知道CME中这种异质性的来源或功能意义。虽然观测到的异质性可能来自许多不同的来源，但这种变异性很可能反映了CME调节的内在异质性。来自Danuser和Schmid实验室的数据表明，CME的大部分调节发生在难以研究的内吞坑生命的早期阶段。特别是，观察到的大部分非均质性很可能是在成核过程中传递的。本研究的目的是建立调控ccp成核的机制，并确定这些起始事件中异质性的来源和影响。我建议从研究成核的空间调节作为CME异质性的可能来源开始。稳定成核机制的概念，即在一个被称为热点的地方产生多个成核，以前已经提出过，但在该领域是一个争论的来源。我建议解决ccp的异质性是否可以部分解释为它们在质膜上的空间组织。我建议找出在空间上调控成核的蛋白质。我建议观察各种成核资源在重复成核位置的停留时间，以确定是否存在稳定的成核结构。CME成核和稳定阶段的非均质性和规律性的另一个可能来源是这些早期阶段的坑的组成。我建议测量形成成核坑的蛋白质的化学计量。

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.