Molecular and cellular mechanisms regulating actin dynamics

调节肌动蛋白动力学的分子和细胞机制

• 批准号: 10091492
• 负责人: Bruce L Goode
• 金额: $ 106.73万
• 依托单位: BRANDEIS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-02-01 至 2025-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10091492
• 关键词: Actins; Affect; Alzheimer' s Disease; Amyotrophic Lateral Sclerosis; Animals; Binding; Biochemical; Biological; Biological Process; Cardiovascular Diseases; Cell division; Cell physiology; Cells; Complex; Cryoelectron Microscopy; Cytoskeleton; Cytosol; Defect; Deformity; Disease; Dissociation; Endocytosis; F-Actin; Filament; Funding; Genetic; Glia Maturation Factor; Goals; Growth; Huntington Disease; In Vitro; Individual; Infertility; Intracellular Transport; Knowledge; Lead; Limb structure; Malignant Neoplasms; Mammalian Cell; Mediating; Microfilaments; Molecular; Morphogenesis; National Institute of General Medical Sciences; Nucleotides; Organism; Parkinson Disease; Process; Proteins; Regulation; Research; Role; Structure; System; Testing; Time; Tissues; Total Internal Reflection Fluorescent; Work; Yeasts; base; cell motility; cellular imaging; cofilin; coronin protein; depolymerization; developmental disease; genetic regulatory protein; hearing impairment; human disease; in vivo; inhibitor/antagonist; insight; mutant; new technology; novel strategies; protein structure; reconstitution; single molecule

Project Summary The overall goal of the NIGMS-funded research in my lab is to define the molecular and cellular mechanisms underlying dynamic rearrangements of the actin cytoskeleton, and to explore how these mechanisms are harnessed in vivo (in yeast and animal cells) to control diverse actin-based processes such as cell motility, endocytosis, intracellular transport, and cell morphogenesis. Genetic and biochemical research has been rapidly producing a ‘molecular parts list’ for the actin cytoskeleton, and many of the components have been characterized individually for their biochemical effects on actin filaments and their genetic effects on cellular actin organization and function. However, it is becoming clear that most of these proteins do not function alone, but rather in groups to perform their biological roles, and thus, new approaches are needed to define how they work in concert to perform their cellular functions. Our lab is tackling this problem using advanced single molecule TIRF microscopy to directly observe multi-component actin regulatory mechanisms in real time, and testing these mechanisms using genetic, cell biological, biochemical, and structural approaches. Through this approach, we have made fundamental new insights into actin regulation. For instance, we defined the first collaborative actin nucleation mechanisms of formins (with Bud6 & APC). We discovered that formins and Capping Protein can bind simultaneously at filament ends to accelerate each other’s dissociation. We showed that Cofilin, AIP1, and Coronin work together via an ordered mechanism to sever and disassemble F-actin. We discovered that Srv2/CAP works in conjunction with Cofilin and Twinfilin to depolymerize filament ends. In parallel, we have combined genetics, cellular imaging, and separation-of-function mutants to dissect the contributions of these mechanisms to actin-based processes in yeast and mammalian cells. Moving forward, we will ask the following questions: what are the complete regulatory cycles of the two yeast formins (Bni1 and Bnr1)? How is Arp2/3 complex-mediated actin nucleation balanced by its inhibitors (Coronin and GMF) and activators (Las17/WASP and Abp1)? How is actin nucleation at the leading edge of motile cells controlled by interactions among IQGAP1, APC and formins? How do interactions at filament ends between Capping Protein and formins (and their in vivo binding partners) control actin network growth? How do the filament severing and depolymerization mechanisms (Cofilin, AIP1, Coronin, Twinfilin, and Srv2/CAP) drive net disassembly of actin under the assembly-promoting conditions of the cytosol? Are there actin-associated proteins that accelerate the nucleotide state transition on F-actin to promote disassembly? In addition, we will introduce new technologies and directions to our research, including in vitro reconstitution of cellular actin structures, cryo-EM to study protein structure, cell-free extracts to genetically-biochemically dissect actin mechanisms, and a systems-level approach to determine how genetic disruptions in individual actin regulators affect the cellular levels, localization, and functions of the remaining actin-associated proteins.

项目摘要 我实验室NIGMS资助的研究的总体目标是确定分子和细胞机制 肌动蛋白细胞骨架的潜在动态重排，并探索这些机制是如何 在体内(在酵母和动物细胞中)控制不同的基于肌动蛋白的过程，如细胞运动， 内吞作用、细胞内运输和细胞形态发生。基因和生化研究一直是 快速生成肌动蛋白细胞骨架的分子部件清单，其中许多组件已经 它们对肌动蛋白细丝的生化效应和对细胞的遗传效应 肌动蛋白的组织和功能。然而，越来越明显的是，这些蛋白质中的大多数并不是单独发挥作用的， 而是以群体的形式发挥其生物学作用，因此，需要新的方法来定义它们是如何 协同工作以执行它们的细胞功能。我们的实验室正在使用高级单人电脑解决这个问题 分子TIRF显微镜，实时直接观察多组分肌动蛋白的调控机制，以及 使用遗传学、细胞生物学、生化和结构方法测试这些机制。通过这件事 方法，我们对肌动蛋白的调控有了全新的见解。例如，我们定义了第一个 Forins的肌动蛋白协同成核机制(与Bud6和APC)。我们发现福尔马林和 覆盖蛋白可以同时结合在丝状末端，从而加速彼此的解离。我们展示了 这种粘连蛋白、AIP1和柯罗宁通过一种有序的机制共同作用，切断和分解F-肌动蛋白。我们 发现Srv2/CAP与Cofilin和Twinfilin一起作用于解聚细丝末端。在……里面 同时，我们结合了遗传学、细胞成像和功能分离突变体来剖析 这些机制对酵母和哺乳动物细胞中基于肌动蛋白的过程的贡献。 接下来，我们将提出以下问题：这两种酵母的完整调控周期是什么 福尔马林(Bni1和Bnr1)？Arp2/3复合体介导的肌动蛋白核化是如何被其抑制物平衡的 和GMF)和激活剂(Las17/WASP和Abp1)？运动细胞前沿的肌动蛋白是如何成核的？ 受IQGAP1、APC和福尔马林相互作用的控制？细丝端部的相互作用是如何 封端蛋白和福尔马林(及其体内结合伙伴)控制肌动蛋白网络的生长？你是如何 细丝切断和解聚机制(Cofilin、AIP1、Cortin、Twinfilin和Serv2/CAP)驱动网 肌动蛋白在胞浆组装促进条件下的分解？有没有肌动蛋白相关的 加速F-肌动蛋白的核苷酸状态转换以促进拆解的蛋白质？此外，我们还将 介绍我们研究的新技术和新方向，包括细胞肌动蛋白的体外重组 结构，冷冻-EM研究蛋白质结构，无细胞提取物基因-生化剖析肌动蛋白 机制，以及系统水平的方法来确定单个肌动蛋白调节器中的遗传干扰是如何 影响剩余肌动蛋白相关蛋白的细胞水平、定位和功能。