Actin Dynamics, Interactions and Function

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络的快速重塑是通过它们与肌动蛋白结合蛋白的相互作用进行的。本提案的总体目标是提供对这些相互作用的结构和功能理解。细胞中肌动蛋白丝动力学的关键调节剂是肌动蛋白解聚因子（ADF）/cofilin蛋白家族的成员。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，并在某些条件下使其稳定。我们过去的工作有助于目前的描述cofilin的肌动蛋白丝的行动，并导致其切断变构，合作模式。目标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光谱，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。我们的目标也将是在结构水平上澄清不同的肌动蛋白丝切割活性的cofilin亚型。切丝蛋白的切断活性在细胞中受到高度调节，并且越来越多的证据表明冠蛋白在该调节中起重要作用。目标2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。目的是使用多方法方法阐明冠蛋白调节cofilin功能的ATP和ADP依赖性差异，类似于目标1中列出的方法。对细胞中肌动蛋白丝切断的一般理解的兴趣使我们的焦点-在目标3中-元黏着斑蛋白在肌动蛋白细胞骨架动力学中的作用。肌动蛋白和后黏着斑蛋白在肌肉细胞中将肌动蛋白细胞骨架连接到细胞膜，但最近的研究结果表明肌动蛋白丝被后黏着斑蛋白的尾部结构域切断。这项工作将通过电子显微镜确定肌动蛋白-后黏着斑蛋白复合物的高分辨率结构。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的变化的详细描述引起的metavinculin，特别是在interprotomer相互作用的网站，确定丝的稳定性。在目标4中提出的关于神经元肌动蛋白结合蛋白碎片的工作，是由树突棘在树突棘的形成和功能中的关键作用激发的，树突棘与轴突的突触进行通信。拟议的工作将澄清与肌动蛋白丝和dreplant的肌动蛋白丝成核，分支，切断，动力学和结构的影响dreplant的相互作用的结构和功能方面-在协同作用或竞争与其他神经元肌动蛋白结合蛋白。重点将是在肌动蛋白丝的interprotomer接触区域的变化和映射的相互作用界面上的两种蛋白质的交联和质谱方法。 公共卫生相关性：我们对cofilin和coronin作用于肌动蛋白丝的机制的研究将加深对肌动蛋白动力学和重塑的理解，从而导致转移过程中潜在的治疗干预。阐明metavinculin-actin相互作用及其复合物的结构的目标是有助于更好地理解扩张型心肌病和肌细胞中的其他疾病过程。树突状细胞是神经细胞中肌动蛋白细胞骨架的重要组成部分，我们对它与肌动蛋白相互作用的研究可以揭示干预这些细胞疾病状态的潜在途径。

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.