Actin Dynamics, Interactions and Function

肌动蛋白动力学、相互作用和功能

• 批准号: 8187753
• 负责人: EMIL REISLER
• 金额: $ 51.83万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 1978
• 资助国家: 美国
• 起止时间: 1978-07-01 至 2015-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8187753
• 关键词: Actin-Binding Protein; Actins; Address; Axon; Binding Proteins; Cell membrane; Cells; Chemicals; Complex; Cryoelectron Microscopy; Crystallography; Cytoskeleton; Dendritic Spines; Dilated Cardiomyopathy; Disease; Electron Microscopy; Electron Spin Resonance Spectroscopy; Elements; F-Actin; Filament; Fluorescence; Gelsolin; Goals; Grant; Intervention; Investigation; Lead; Length; Life; Link; Maps; Mass Spectrum Analysis; Measurement; Methods; Microfilaments; Modeling; Muscle Cells; Mutation; Nature; Neurons; Nucleotides; Process; Protein Family; Protein Isoforms; Proteins; Regulation; Resolution; Role; Route; Site; Solutions; Structure; Structure-Activity Relationship; Synapses; Tail; Testing; Therapeutic Intervention; Vinculin; Work; Yeasts; actin depolymerizing factor; base; cell motility; cofilin; coronin protein; crosslink; depolymerization; drebrins; experience; fluorescence imaging; human disease; interest; member; metastatic process; metavinculin; migration; mutant; pathogen; polymerization; therapeutic target

DESCRIPTION (provided by applicant): The dynamic remodeling of the actin cytoskeleton is a critical component of nearly all aspects of cell motility, including migration of healthy and metastatic cells and host-pathogen interactions. The rapid remodeling of actin filament networks in cells proceeds through their interactions with actin binding proteins. The overall goal of this proposal is to provide structural and functional understanding of these interactions. Key modulators of actin filaments dynamics in cells are members of the actin-depolymerizing factor (ADF)/cofilin family of proteins. They increase the turnover of actin filaments by severing and depolymerizing them, and also stabilizing them under some conditions. Our past work contributed to the current description of cofilin's action on actin filaments and led to the allosteric, cooperative model of their severing. The work proposed in Aim 1 will bring together multiple approaches, including cryo-electron microscopy, crystallography, fluorescence and electron paramagnetic resonance spectroscopies, fluorescence imaging, mutational work, chemical cross-linking and mass spectrometry methods, to provide structural understanding of the mechanism of cofilin's interactions with actin. The goal will also be to clarify at a structural level the different actin filament severing activities of cofilin isoforms. The severing activity of cofilin is highly regulated in cells, and there is mounting evidence for an important role of coronin in that regulation. The work proposed in Aim 2 will determine the structures of coronin-actin complexes by high resolution cryo-electron microscopy. The goal is to clarify the ATP and ADP dependent differences in the regulation of cofilin function by coronin using multi-method approaches, similar to those listed in Aim 1. The interest in the general understanding of actin filaments severing in cells brings to our focus - in Aim 3 - the role of metavinculin in actin cytoskeleton dynamics. Vinculin and metavinculin connect the actin cytoskeleton to cell membranes in muscle cells, but recent findings show actins filaments severing by the tail domain of metavinculin. The proposed work will determine the high resolution structure of actin- metavinculin complexes by electron microscopy. The parallel goal is to obtain from solution studies a detailed description of the changes in actin filament structure and dynamics caused by metavinculin, and in particular at the interprotomer interaction sites that determine filament stability. The work proposed in Aim 4, on the neuronal actin binding protein debris, is motivated by drebrin's critical role in the formation and function of dendritic spines, which communicate with the synapses of axons. The proposed work will clarify structural and functional aspects of drebrin interactions with actin filaments and drebrin's effect on actin filaments nucleation, branching, severing, dynamics, and structure - in synergy or competition with other neuronal actin binding proteins. The emphasis will be on the changes in the interprotomer contact regions in actin filaments and on mapping the interaction interface on both proteins by cross-linking and mass spectrometry methods. PUBLIC HEALTH RELEVANCE: Our investigation of the mechanism of cofilin and coronin action on actin filaments will deepen the understanding of actin dynamics and remodeling, leading to potential therapeutic intervention in metastatic processes. The goal of clarifying metavinculin-actin interactions and the structure of their complexes is to contribute to better understanding of dilated cardiomyopathy and other disease processes in muscle cells. Drebrin is a critical component of actins cytoskeleton in neuronal cells and our work on its interactions with actin can reveal potential routes for intervention in the diseased state of these cells.

描述（由申请人提供）：肌动蛋白细胞骨架的动态重塑是细胞运动几乎所有方面的关键组成部分，包括健康和转移细胞的迁移以及宿主-病原体相互作用。细胞内肌动蛋白丝网的快速重塑是通过与肌动蛋白结合蛋白的相互作用进行的。本建议的总体目标是提供对这些交互的结构和功能理解。细胞中肌动蛋白丝动力学的关键调节因子是肌动蛋白解聚因子(ADF)/cofilin家族蛋白的成员。它们通过切断和解聚肌动蛋白丝来增加肌动蛋白丝的周转，并在某些条件下稳定它们。我们过去的工作有助于目前对cofilin对肌动蛋白丝的作用的描述，并导致了它们断裂的变构，合作模型。在Aim 1中提出的工作将汇集多种方法，包括冷冻电子显微镜，晶体学，荧光和电子顺磁共振波谱，荧光成像，突变工作，化学交联和质谱方法，以提供对cofilin与肌动蛋白相互作用机制的结构理解。目标还将是澄清在结构水平上不同的肌动蛋白丝切断活动的cofilin亚型。在细胞中，cofilin的切断活性受到高度调控，越来越多的证据表明，cofilin在这种调控中起着重要作用。在第二部分中提出的工作将通过高分辨率冷冻电子显微镜确定冠状蛋白-肌动蛋白复合物的结构。目的是利用多种方法澄清冠状蛋白对cofilin功能调节中ATP和ADP依赖性的差异，类似于Aim 1中列出的方法。对细胞中肌动蛋白丝断裂的一般理解的兴趣使我们的重点-在Aim 3中-元静脉素在肌动蛋白细胞骨架动力学中的作用。在肌细胞中，肌动蛋白骨架与肌动蛋白的细胞膜连接在一起，但最近的研究发现肌动蛋白丝被肌动蛋白的尾部区域切断。提出的工作将确定高分辨率结构的肌动蛋白-元静脉球蛋白配合物的电子显微镜。平行的目标是从溶液研究中获得肌动蛋白丝结构和动力学变化的详细描述，特别是在决定丝稳定性的原蛋白体相互作用位点。在Aim 4中提出的关于神经元肌动蛋白结合蛋白碎片的工作，是由drebrin在树突棘的形成和功能中的关键作用所激发的，树突棘与轴突突触进行通信。这项工作将阐明drebrin与肌动蛋白丝相互作用的结构和功能方面，以及drebrin在与其他神经元肌动蛋白结合蛋白协同或竞争时对肌动蛋白丝成核、分支、切断、动力学和结构的影响。重点将放在肌动蛋白丝中蛋白质间接触区域的变化，以及通过交联和质谱方法绘制两种蛋白质的相互作用界面。