Determinants for the assembly and maturation of clathrin coated vesicles.

描述（由申请方提供）：网格蛋白包被囊泡的内吞作用是所有真核细胞用于维持其质膜、与外界交流和内化营养物质的基本机制。我们的长期目标是了解网格蛋白囊泡的形成和调节的分子基础，网格蛋白囊泡是内吞作用的重要贡献者。最近，它已经变得清楚，肌动蛋白细胞骨架与网格蛋白机制相互作用，并有助于丝，有助于形成包被囊泡。该领域的一个关键差距是缺乏一种分子机制，可以解释网格蛋白组装和肌动蛋白聚合如何在活细胞中时空协调，以有效地形成涂层囊泡。该提案的目的是定义蛋白质epsin、Hip1r和网格蛋白轻链相互作用以促进功能网格蛋白包被囊泡的形成的机制，所述囊泡偶联到活细胞中的肌动蛋白丝的聚焦阵列。以网骨藻细胞为模型系统，提出了三个具体的目标：（1）确定Hip1r如何调节肌动蛋白丝与网格蛋白的偶联我们的初步结果表明，Hip1r有助于肌动蛋白丝紧密聚集带的时间和形态。将对这些Hip1r活动的两个工作模型进行测试。信息点突变体在不同的结构域的Hip1r将进行评估，以区分的可能性，Hip1r作为一个受管制的系链之间的网格蛋白和肌动蛋白，Hip1r作为一个支架招募肌动蛋白调节蛋白网格蛋白晶格的可能性。(2)To确定磷酸化和epsin如何调节Hip1r功能最初的研究表明，epsin有助于调节Hip1r磷酸化和活性的途径。质谱法将验证候选Hip1r磷酸化位点。使用磷酸沉默和磷酸模拟形式的Hip1 r的实验将直接测试（a）磷酸化是否控制Hip1 r活性，以及（B）磷酸化如何调节Hip1 r活性。将测试epsin通过磷酸化控制Hip1r的可能性，并鉴定作为该途径中间体的epsin结合伴侣。将测试Hip1r的候选激酶（3）为了确定网格蛋白轻链对网格蛋白功能的贡献，使用快速冷冻深蚀刻显微镜，将评价网格蛋白轻链（CLC）对重链三聚化和对活细胞中组装的包被凹坑结构的贡献。总干涉显微镜将评估CLC对质膜上网格蛋白和肌动蛋白的动态组装的贡献。一个集合的五个定义截断CLC将被检查的具体方面的涂层坑形成的贡献，包括组装成一个精确的网格紧密相关的质膜，网格的耦合到一个集中的带肌动蛋白丝的网格。总的来说，这三个具体目标的结果将推进了解动态和功能网格蛋白包被的囊泡如何从质膜出现，并与肌动蛋白丝的紧密聚焦带耦合。 公共卫生相关性：总的来说，我们的研究结果将推进我们的理解动态网格蛋白包被的囊泡出现从质膜和相互作用的细胞骨架。了解网格蛋白和细胞骨架如何偶联的这种机制将有助于我们了解与这些细胞成分相关的蛋白质突变引起的疾病的基础。通过了解这些细胞过程如何发生，膜运输领域将能够理解网格蛋白囊泡如何形成，从而打开用于靶向相互作用子集的治疗策略，例如，阻断劫持网格蛋白囊泡进入的机会性病原体的进入，或者相反地，促进药物和受体的进入，所述药物和受体通过网格蛋白包被的囊泡将胆固醇或胰岛素带入细胞。

DESCRIPTION (provided by applicant): Endocytosis by clathrin coated vesicles is a fundamental mechanism used by all eukaryotic cells to maintain their plasma membrane, communicate with the outside world, and internalize nutrients. Our long-term goal is to understand the molecular basis for the formation and regulation of clathrin vesicles, important contributors to endocytosis. Recently it has become clear that the actin cytoskeleton interacts with the clathrin machinery, and contributes filaments that contribute to the formation of coated vesicles. A critical gap in the field is the lack of a molecular mechanism that could explain how clathrin assembly and actin polymerization are coordinated temporally and spatially in living cells to efficiently form a coated vesicle. The objective of this proposal is to define the mechanism by which the proteins epsin, Hip1r and the clathrin light chain interact to promote the formation of functional clathrin coated vesicles coupled to a focused array of actin filaments in living cells. Using Dictyostelium cells as a model system, three specific aims are proposed: (1) To define how Hip1r regulates coupling of actin filaments to clathrin Our initial results show that Hip1r contributes to the timing and the morphology of a tightly focused band of actin filaments with coated pits. Two working models for these Hip1r activities will be tested. Informative point mutants in distinct domains of Hip1r will be evaluated to distinguish between the possibility that Hip1r acts as a regulated tether between clathrin and actin, and the possibility that Hip1r serves as a scaffold to recruit actin-regulatory proteins to clathrin lattices. (2)To determine how phosphorylation and epsin regulate Hip1r function Initial studies suggest that epsin contributes to a pathway that regulates Hip1r phosphorylation and activity. Mass spectrometry will verify a candidate Hip1r phosphorylation site. Experiments using phosphosilent and phosphomimic versions of Hip1r will directly test (a) whether phosphorylation controls Hip1r activity, and (b) how phosphorylation may regulate Hip1r activity. The possibility that epsin controls Hip1r through phosphorylation will be tested and binding partners for epsin that serves as intermediates in this pathway will be identified. A candidate kinase for Hip1r will be tested (3) To define the contribution of the clathrin light chain contributes to clathrin function Using quick-freeze deep etch microscopy, the contribution of the clathrin light chain (CLC) to the trimerization of heavy chain and to the architecture of coated pits assembled in living cells will be evaluated. Total interference microscopy will evaluate the contribution of the CLC to the dynamic assembly of clathrin and actin on the plasma membrane. A collection of five defined truncations of CLC will be examined for contributions to specific aspects of coated pit formation, including the assembly of clathrin into a precisely shaped lattice tightly associated with the plasma membrane, and the coupling of the lattice to a focused band of actin filaments. Collectively, the outcome of the three specific aims will advance an understanding of how dynamic and functional clathrin coated vesicles emerge from the plasma membrane and couple with a tightly focused band of actin filaments. PUBLIC HEALTH RELEVANCE: Collectively, our results will advance our understanding of how dynamic clathrin-coated vesicles emerge from the plasma membrane and interact with the cytoskeleton. Understanding this mechanism of how clathrin and the cytoskeleton are coupled will contribute to our knowledge of the basis of diseases caused by mutations in proteins associated with these cellular components. With an understanding of how these cellular processes occur, the field of membrane traffic will be able to appreciate how clathrin vesicles form, opening therapeutic strategies for targeting a subset of interactions to, for example, block the entry of opportunistic pathogens that highjack clathrin vesicles for entry, or, conversely, to promote the entry of drugs and receptors that bring cholesterol or insulin into cells via clathrin coated vesicles.