Structural-Functional Basis of Actin Cytoskeleton Dynamics

肌动蛋白细胞骨架动力学的结构功能基础

基本信息

批准号：
10658930
负责人：
ROBERTO DOMINGUEZ
金额：
$ 41.17万
依托单位：
UNIVERSITY OF PENNSYLVANIA
依托单位国家：
美国
项目类别：
财政年份：
2005
资助国家：
美国
起止时间：
2005-04-01 至 2027-03-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10658930
关键词：
ATP Hydrolysis Acceleration Acidic Region Actins Address Area Binding Biochemical Biological Assay C-terminal Calorimetry Cells Coiled-Coil Domain Complex Cryoelectron Microscopy Cytoskeleton Daughter Development Dissociation EMS1 gene Environment Eukaryota F-Actin Family Filament Glia Maturation Factor Grant Human Knowledge Laboratories Link Malignant Neoplasms Measures Methods Microfilaments Microfluidics Modeling Molecular Mothers Mutagenesis N-terminal Nucleotides Organelles Play Polymers Process Protein Family Protein Isoforms Proteins Publishing Pyrenes Recombinants Reporting Research Support Resolution Role Site-Directed Mutagenesis Structure System Testing Titrations Total Internal Reflection Fluorescent Work Yeasts beta Actin cell motility coronin protein crosslink dynamic system experimental study human disease inhibitor innovation interest member monomer novel polymerization protein expression targeted treatment

项目摘要

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

概括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移动、分裂、运输细胞器并与细胞交换物质环境。许多人类疾病是由肌动蛋白细胞骨架功能障碍引起的成分。因此，人们对了解分子机制产生了浓厚的兴趣控制肌动蛋白细胞骨架过程，具有根本重要性和潜力加速开发治疗人类疾病的靶向疗法。肌动蛋白之中细胞骨架成分中，最重要的莫过于 Arp2/3 复合体（一种 7 亚基肌动蛋白）从酵母到人类的真核生物中保守的丝成核和分支系统。这赠款弥补了 Arp2/3 复合体机制的重要知识空白激活、抑制、分支稳定和分支不稳定。发表作品，应用中提出的广泛的初步研究以及实验室的新进展例如对 Arp2/3 复合物实施冷冻电子显微镜 (cryo-EM) 系统、创新的蛋白质表达方法和生化测定提供了支持研究计划的科学和技术场所。具体目标集中在三个方面主要领域：1）了解人类Arp2/3复合体分支的分子决定因素使用微流体-TIRF 显微镜测定法测定稳定性和机械感觉，2) 确定使用生化方法通过 Cortactin 稳定分支的结构-功能机制和冷冻电镜，3) 揭示 Coronin 家族的成员之一是否充当 Arp2/3 复合物抑制剂、分支稳定剂或使用生化的分支去稳定剂方法和冷冻电镜