Structural-Functional Basis of Actin Cytoskeleton Dynamics

摘要 肌动蛋白细胞骨架是一个由数百种蛋白质组成的高度动态的系统。细胞使用 肌动蛋白细胞骨架与细胞移动、分离、运输细胞器和交换物质 环境。许多人类疾病是由肌动蛋白细胞骨架的故障引起的。 组件。因此，人们对了解分子机制产生了浓厚的兴趣 它控制着肌动蛋白的细胞骨架过程，这既具有根本的重要性，也具有 有可能加快开发治疗人类疾病的靶向疗法。其中 肌动蛋白的细胞骨架成分，没有比Arp2/3复合体更重要的了，Arp2/3复合体是一种7亚单位肌动蛋白 从酵母到人类，真核生物中保守的细丝成核和分支机制。 这项资助解决了Arp2/3复合体机制中的重要知识空白 激活、抑制、分支稳定和分支失稳。出版的作品， 广泛的初步研究，低温电子显微镜的实施进展 (Cryo-EM)研究Arp2/3复杂调控系统，创新蛋白质表达 方法和生化分析提供了科学和技术前提支持 研究计划。具体目标集中在三个主要方面：1)了解分子 人Arp2/3复合体分支稳定性和机械感觉的决定因素 微流控-TIRF显微分析，2)确定其结构-功能机制 使用冷冻-EM和生化方法使用Cortactin稳定分支，以及3)发现 冠状病毒家族中的一个成员是否作为Arp2/3复合体抑制物，一个分支 稳定剂，或使用低温EM和生化方法的分支失稳剂。

Summary The actin cytoskeleton is a highly dynamic system consisting of hundreds of proteins. Cells use the actin cytoskeleton to move, divide, transport organelles and exchange materials with the environment. Many human diseases result from malfunctioning of actin cytoskeletal components. There is therefore intense interest in understanding the molecular mechanisms that control actin cytoskeletal processes, which has both fundamental importance and the potential to accelerate the development of targeted therapies to treat human diseases. Among actin cytoskeletal components, none is more important than Arp2/3 complex, a 7-subunit actin filament nucleation and branching machinery conserved in eukaryotes from yeast to human. This grant addresses important gaps of knowledge in the mechanisms of Arp2/3 complex activation, inhibition, branch stabilization, and branch destabilization. Published work, extensive preliminary studies, advances in the implementation of cryo-electron microscopy (cryo-EM) to the study of the Arp2/3 complex regulatory system, innovative protein expression methods, and biochemical assays provide the scientific and technical premises supporting the research plans. The specific aims focus on three major areas: 1) understand the molecular determinants of human Arp2/3 complex branch stability and mechanosensation using a microfluidics-TIRF microscopy assay, 2) determine the structural-functional mechanism of branch stabilization by Cortactin using cryo-EM and biochemical methods, and 3) uncover whether one of the members of the coronin family acts as an Arp2/3 complex inhibitor, a branch stabilizer, or a branch destabilizer using cryo-EM and biochemical approaches.