Actin Dynamics, Interactions and Function

描述(申请人提供)：肌动蛋白细胞骨架的动态重构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与病原体的相互作用。细胞内肌动蛋白细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。这项提案的总体目标是提供对这些相互作用的结构和功能的理解。细胞内肌动蛋白细丝动力学的关键调节者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细丝，并在一定条件下稳定肌动蛋白细丝来增加肌动蛋白细丝的周转率。我们过去的工作有助于目前对cofilin对肌动蛋白细丝作用的描述，并导致了它们被切断的变构、合作模型。目标1中提出的工作将结合多种方法，包括冷冻电子显微镜、结晶学、荧光和电子顺磁共振光谱、荧光成像、突变工作、化学交联和质谱学方法，以提供对cofilin与肌动蛋白相互作用机制的结构性理解。目标还将是在结构水平上阐明不同的肌动蛋白细丝切断Cofilin异构体的活动。粘附素的切断活性在细胞中受到高度调控，越来越多的证据表明，冠状病毒在这一调控中发挥着重要作用。目标2中提出的工作将通过高分辨率冷冻电子显微镜来确定柯拉宁-肌动蛋白复合体的结构。我们的目标是用多种方法阐明ATP和ADP依赖的差异，这些差异与目标1中列出的方法类似。对细胞内肌动蛋白细丝断裂的一般理解的兴趣使我们的重点-在目标3-Metavinculin在肌动蛋白细胞骨架动力学中的作用。纽蛋白和Metavinculin将肌动蛋白细胞骨架连接到细胞膜上，但最近的发现表明，肌动蛋白细丝被Metavinculin的尾部区域切断。这项拟议的工作将通过电子显微镜确定肌动蛋白-Metavinculin复合体的高分辨结构。平行的目标是从溶液研究中获得对Metavinculin引起的肌动蛋白微丝结构和动力学变化的详细描述，特别是在决定微丝稳定性的原核间相互作用部位。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，是由于Drebrin在树突棘的形成和功能中扮演的关键角色，树突棘与轴突的突触相连。这项拟议的工作将阐明Drebrin与肌动蛋白细丝相互作用的结构和功能方面，以及Drebrin对肌动蛋白细丝的成核、分支、切断、动力学和结构的影响--与其他神经元肌动蛋白结合蛋白协同或竞争。重点将放在肌动蛋白细丝中原体间接触区的变化，以及通过交联法和质谱学方法绘制两种蛋白质之间的相互作用界面。 公共卫生相关性：我们对cofilin和cortin对肌动蛋白细丝作用机制的研究将加深对肌动蛋白动力学和重塑的理解，导致潜在的转移过程中的治疗干预。阐明Metavinculin-肌动蛋白相互作用及其复合体结构的目的是为了更好地了解扩张型心肌病和肌肉细胞中的其他疾病过程。Drebrin是神经细胞中肌动蛋白细胞骨架的关键组成部分，我们对其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.