Determinants for the assembly and maturation of clathrin coated vesicles.

描述(申请人提供)：包被网状蛋白的囊泡的内吞作用是所有真核细胞维持其质膜、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状蛋白囊泡形成和调节的分子基础，网状蛋白囊泡是内吞作用的重要贡献者。最近发现，肌动蛋白细胞骨架与网状蛋白机械相互作用，并贡献有助于形成包被小泡的细丝。该领域的一个关键空白是缺乏一种分子机制来解释如何在活细胞中在时间和空间上协调网状蛋白组装和肌动蛋白聚合，从而有效地形成包被的囊泡。这项建议的目的是确定蛋白质epsin、Hip1r和clathrin轻链相互作用的机制，以促进活细胞中连接到一组聚焦的肌动蛋白细丝的功能性clathrin包被小泡的形成。以Dictyostelius细胞为模型系统，提出了三个具体目标：(1)确定Hip1r如何调节肌动蛋白细丝与clathrin的偶联。我们的初步结果表明，Hip1r有助于肌动蛋白细丝紧密聚焦的带的时间和形态。这些Hip1r活动的两个工作模型将进行测试。将对Hip1r不同结构域中的信息点突变进行评估，以区分Hip1r作为clathrin和肌动蛋白之间受调节的系链的可能性，以及Hip1r作为支架将肌动蛋白调节蛋白招募到clathrin晶格的可能性。(2)为了确定磷酸化和epsin如何调节Hip1r的功能，初步研究表明epsin参与了调节Hip1r磷酸化和活性的途径。质谱学将验证候选的Hip1r磷酸化位点。使用磷酸化沉默和磷酸化版本的Hip1r的实验将直接测试(A)磷酸化是否控制Hip1r的活性，以及(B)磷酸化如何调节Hip1r的活性。将测试Eepsin通过磷酸化控制Hip1r的可能性，并将确定作为这一途径中间体的Eepsin的结合伙伴。将测试Hip1r的候选激酶(3)利用快速冷冻深度蚀刻显微镜来确定分子筛轻链对分子筛功能的贡献，将评估分子筛轻链(CLC)对重链三聚化和活细胞中组装的涂层凹坑的结构的贡献。总干涉显微镜将评估CLC在细胞膜上动态组装网状蛋白和肌动蛋白方面的贡献。我们将研究CLC的五个已定义截断的集合，以寻找对涂层小孔形成的特定方面的贡献，包括将网状蛋白组装到与质膜紧密结合的精确形状的晶格中，以及将晶格耦合到肌动蛋白细丝的聚焦带上。总而言之，这三个特定目标的结果将促进对动态和功能笼罩的囊泡如何从质膜中出现并与紧密聚焦的肌动蛋白细丝结合的理解。

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.