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: 8186514
• 负责人: THERESA Joyce O'HALLORAN
• 金额: $ 28.76万
• 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/8186514
• 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. 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.

描述（由申请人提供）：网格蛋白包被的囊泡的内吞作用是所有真核细胞用来维持其质膜、与外界沟通以及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。最近已经清楚，肌动蛋白细胞骨架与网格蛋白机制相互作用，并贡献有助于形成包被囊泡的细丝。该领域的一个关键差距是缺乏可以解释网格蛋白组装和肌动蛋白聚合如何在活细胞中在时间和空间上协调以有效形成包被囊泡的分子机制。该提案的目的是确定蛋白质 epsin、Hip1r 和网格蛋白轻链相互作用的机制，以促进功能性网格蛋白包被囊泡的形成，该囊泡与活细胞中肌动蛋白丝的聚焦阵列偶联。使用盘基网柄菌细胞作为模型系统，提出了三个具体目标：（1）定义 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焦带的时间和形态涂层坑。将测试这些 Hip1r 活动的两个工作模型。将评估 Hip1r 不同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的调节系链的可能性，以及 Hip1r 作为将肌动蛋白调节蛋白招募到网格蛋白晶格的支架的可能性。 (2)确定磷酸化和epsin如何调节Hip1r功能初步研究表明epsin有助于调节Hip1r磷酸化和活性的途径。质谱分析将验证候选 Hip1r 磷酸化位点。使用 Hip1r 的磷酸沉默和拟磷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。我们将测试epsin 通过磷酸化控制Hip1r 的可能性，并确定作为该途径中间体的epsin 的结合伴侣。将测试 Hip1r 的候选激酶 (3) 确定网格蛋白轻链对网格蛋白功能的贡献 使用速冻深蚀刻显微镜，确定网格蛋白轻链 (CLC) 对重链三聚化和对网格蛋白功能的贡献将评估活细胞中组装的涂层凹坑的结构。全干涉显微镜将评估 CLC 对质膜上网格蛋白和肌动蛋白动态组装的贡献。将检查五个定义的 CLC 截断的集合，以了解其对包被凹坑形成的特定方面的贡献，包括将网格蛋白组装成与质膜紧密相关的精确形状的晶格，以及晶格与肌动蛋白聚焦带的耦合细丝。总的来说，这三个具体目标的结果将促进对动态和功能性网格蛋白包被的囊泡如何从质膜中出现并与紧密聚焦的肌动蛋白丝带结合的理解。 公共健康相关性：总的来说，我们的结果将增进我们对动态网格蛋白包被的囊泡如何从质膜中出现并与细胞骨架相互作用的理解。了解网格蛋白和细胞骨架如何耦合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，膜交通领域将能够了解网格蛋白囊泡是如何形成的，从而开辟针对相互作用子集的治疗策略，例如，阻止劫持网格蛋白囊泡的机会性病原体的进入进入，或者相反，促进药物和受体进入，通过网格蛋白包被的囊泡将胆固醇或胰岛素带入细胞。