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.

项目概要 我实验室 NIGMS 资助的研究的总体目标是定义分子和细胞机制 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发生的 利用体内（在酵母和动物细胞中）控制多种基于肌动蛋白的过程，例如细胞运动， 内吞作用、细胞内运输和细胞形态发生。遗传和生化研究已 快速生成肌动蛋白细胞骨架的“分子部件列表”，并且许多部件已被 分别针对肌动蛋白丝的生化作用及其对细胞的遗传作用进行了表征 肌动蛋白的组织和功能。然而，越来越清楚的是，大多数这些蛋白质并不单独发挥作用， 而是以群体的形式来发挥其生物学作用，因此，需要新的方法来定义它们如何 协同工作以执行其细胞功能。我们的实验室正在使用先进的单一技术来解决这个问题 分子 TIRF 显微镜直接实时观察多组分肌动蛋白调节机制，以及 使用遗传、细胞生物学、生化和结构方法测试这些机制。通过这个 通过这种方法，我们对肌动蛋白调控有了根本性的新见解。例如，我们定义了第一个 福尔明的协同肌动蛋白成核机制（与 Bud6 和 APC）。我们发现福明和 封端蛋白可以同时结合在细丝末端，加速彼此的解离。我们展示了 Cofilin、AIP1 和 Coronin 通过有序机制协同工作，切断和分解 F-肌动蛋白。我们 发现 Srv2/CAP 与 Cofilin 和 Twinfilin 协同作用，使细丝末端解聚。在 与此同时，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。 展望未来，我们将提出以下问题：两种酵母的完整调控周期是什么？ 福尔明（Bni1 和 Bnr1）？ Arp2/3 复合物介导的肌动蛋白成核如何与其抑制剂（Coronin 和 GMF）和激活剂（Las17/WASP 和 Abp1）？肌动蛋白如何在运动细胞的前缘成核 由 IQGAP1、APC 和福尔明之间的相互作用控制？之间的细丝末端如何相互作用 封盖蛋白和福明斯（及其体内结合伙伴）控制肌动蛋白网络的生长？怎样做 细丝切断和解聚机制（Cofilin、AIP1、Coronin、Twinfilin 和 Srv2/CAP）驱动网络 肌动蛋白在细胞质促进组装的条件下分解？是否存在肌动蛋白相关 加速 F-肌动蛋白上核苷酸状态转变以促进分解的蛋白质？此外，我们将 为我们的研究引入新技术和方向，包括细胞肌动蛋白的体外重建 结构、冷冻电镜用于研究蛋白质结构、无细胞提取物用于基因生化解剖肌动蛋白 机制和系统级方法来确定个体肌动蛋白调节因子的遗传破坏如何 影响其余肌动蛋白相关蛋白的细胞水平、定位和功能。