Determinants for the assembly and maturation of clathrin coated vesicles.

网格蛋白包被的囊泡组装和成熟的决定因素。

• 批准号: 8495356
• 负责人: THERESA Joyce O'HALLORAN
• 金额: $ 27.75万
• 依托单位: UNIVERSITY OF TEXAS AT AUSTIN
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-07-01 至 2015-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8495356
• 关键词: Actins; Adipocytes; Architecture; Binding; Biochemical; Biochemistry; Biological Models; Cell membrane; Cell physiology; Cells; Cholesterol; Clathrin; Clathrin Light Chains; Clathrin-Coated Vesicles; Coated vesicle; Collection; Coupled; Coupling; Cytoskeleton; Dictyostelium; Drug Receptors; Eating; Endocytosis; Eukaryotic Cell; Event; F-Actin; Filament; Fluorescence Microscopy; Freezing; Genetic; Glucose Transporter; Goals; Health; Human; Image; Insulin; Interference Microscopy; Kinetics; Life; Link; Mass Spectrum Analysis; Membrane; Membrane Protein Traffic; Microfilaments; Microscopy; Modeling; Molecular; Morphology; Muscle Cells; Neurons; Nutrient; Outcome; Pathway interactions; Phospholipids; Phosphorylation; Phosphorylation Site; Phosphotransferases; Phylogeny; Point Mutation; Process; Property; Proteins; Recruitment Activity; Recycling; Regulation; Reporter; Research; Scaffolding Protein; Shapes; Site; Staging; Synapses; Testing; Therapeutic; Time; Vesicle; Work; base; coated pit; coronin protein; crosslink; defined contribution; disease-causing mutation; epsin; genetic regulatory protein; mutant; pathogen; polymerization; research study; scaffold; trafficking; uptake

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.

描述（由申请人提供）：网格蛋白涂层囊泡的内吞作用是所有真核细胞使用的基本机制，用于维持其质膜，与外界进行交流并内化营养素。我们的长期目标是了解网格蛋白囊泡形成和调节的分子基础，这是内吞作用的重要因素。最近，很明显，肌动蛋白细胞骨架与网格蛋白机械相互作用，并贡献有助于形成涂层囊泡的细丝。该领域的一个关键差距是缺乏分子机制，该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的时间和空间上协调以有效形成涂层囊泡。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的机制，以促进功能性网状蛋白涂层囊泡的形成，并耦合到活细胞中浓缩的肌动蛋白丝阵列。将使用Dictyostelium细胞作为模型系统，提出了三个特定的目的：（1）定义HIP1R如何调节肌动蛋白丝耦合到旋转蛋白，我们的初始结果表明，HIP1R有助于肌动蛋白丝与涂层的pits的紧密焦点和焦点带的形态。这些HIP1R活动的两个工作模型将进行测试。将评估HIP1R不同结构域中的信息点突变体，以区分HIP1R作为网格蛋白和肌动蛋白之间受调节的系绳的可能性，以及HIP1R作为募集肌动蛋白调节蛋白的支架的可能性。 （2）确定如何调节HIP1R功能的磷酸化和Epsin如何提示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HIP1R的磷和磷酸化版本的实验将直接测试（a）磷酸化是否控制HIP1R活性，以及​​（b）磷酸化如何调节HIP1R活性。将测试EPSIN通过磷酸化控制HIP1R的可能性，并将确定Epsin的Epsin伴侣的结合伴侣，该伴侣将被确定为该途径中的中间体。将测试用于HIP1R的候选激酶（3），以定义网格蛋白轻链的贡献，使用快速冻结深蚀刻显微镜对网状蛋白功能有助于克拉瑟蛋白的功能，这是克拉瑟蛋白轻链（CLC）对重链的修剪和重型链的修剪和对活细胞组合的涂层岩层组装的贡献。总干扰显微镜将评估CLC对网格蛋白和肌动蛋白动态组装对质膜的贡献。将检查五个定义的CLC截断的集合，以贡献对涂层坑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相关的精确形状的晶格中，以及将晶格的偶联与聚焦的肌动蛋白丝之间的耦合。总体而言，这三个特定目标的结果将进一步了解动态和功能性网状蛋白涂层的囊泡如何从质膜和夫妇伴以肌动蛋白丝的紧密聚焦带中出现。