Structural-Functional Basis of Actin Cytoskeleton Dynamics

总结 肌动蛋白细胞骨架是由数百种蛋白质组成的高度动态系统。细胞利用 肌动蛋白细胞骨架移动，分裂，运输细胞器和交换材料与 环境许多人类疾病是由于肌动蛋白细胞骨架功能障碍引起的 件.因此，人们对理解分子机制有着浓厚的兴趣， 控制肌动蛋白细胞骨架过程，这既具有根本的重要性和潜力 以加速靶向疗法的发展来治疗人类疾病。在肌动蛋白 细胞骨架成分，没有一个比Arp 2/3复合物更重要，Arp 2/3复合物是一种7亚基肌动蛋白 丝成核和分支系统在从酵母到人类的真核生物中保守。这 赠款解决了Arp 2/3复合物机制的重要知识空白 活化、抑制、分支稳定化和分支不稳定化。出版的作品， 广泛的初步研究中提出的申请，并在实验室的新进展 例如对Arp 2/3复合物进行低温电子显微镜（cryo-EM） 系统和创新的蛋白质表达方法和生物化学测定提供了 支持研究计划的科学和技术前提。具体目标集中在三个方面 主要研究领域：1）了解人类Arp 2/3复合物分支的分子决定簇 使用微流控TIRF显微镜测定的稳定性和机械感觉，2）确定 生物化学方法研究玉米素稳定分支结构-功能机制 和cryo-EM，以及3）揭示冠状蛋白家族的成员之一是否充当 使用生物化学的Arp 2/3复合物抑制剂、分支稳定剂或分支去稳定剂 方法和冷冻电镜

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 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 system conserved in eukaryotes from yeast to human. This grant addresses important gaps of knowledge of the mechanisms of Arp2/3 complex activation, inhibition, branch stabilization, and branch destabilization. Published work, extensive preliminary studies presented in the application, and new advances in the laboratory such as the implementation of cryo-electron microscopy (cryo-EM) to the Arp2/3 complex system, and 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 biochemical methods and cryo-EM, 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 biochemical approaches and cryo-EM