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: 10549331
• 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/10549331
• 关键词: Acceleration; 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; cell motility; cellular imaging; cofilin; coronin protein; depolymerization; developmental disease; genetic regulatory protein; hearing impairment; human disease; in vivo; inhibitor; 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 在我的实验室中，由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的 利用体内（在酵母和动物细胞中）来控制基于潜水肌动蛋白的过程，例如细胞运动， 内吞作用，细胞内转运和细胞形态发生。遗传和生化研究已经 迅速为肌动蛋白细胞骨架的“分子零件清单”，许多组件已经 其对肌动蛋白丝的生化作用及其对细胞的遗传作用的单独特征 肌动蛋白组织和功能。但是，很明显，这些蛋白质中的大多数不单独起作用， 而是在小组中执行其生物学角色，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高级单曲解决这个问题 分子TIRF显微镜直接实时观察多组分肌动蛋白调节机制，并 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 方法，我们对肌动蛋白调节做出了基本的新见解。例如，我们定义了第一个 造型的协作肌动蛋白成核机制（带Bud6＆Apc）。我们发现了造型和 封盖蛋白可以在细丝末端简单地结合以加速彼此的解离。我们展示了 该Cofilin，AIP1和Coronin通过有序的机制切断和拆卸F-肌动蛋白一起起作用。我们 发现SRV2/CAP与Cofilin和Twinfilin结合使用，以解聚丝末端。在 并行，我们将遗传学，细胞成像和功能性突变体结合在一起，以剖析 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。 向前迈进，我们将提出以下问题：两个酵母的完整调节周期是什么 formins（BNI1和BNR1）？ ARP2/3复合物介导的肌动蛋白成核如何通过其抑制剂（Coronin）平衡 和GMF）和激活剂（LAS17/WASP和ABP1）？肌动蛋白成核如何在运动细胞的前缘 由IQGAP1，APC和FORMINS之间的相互作用控制？细丝的互动如何结束 限制蛋白质和formins（及其体内结合伴侣）控制肌动蛋白网络的增长？如何 细丝切断和沉积机制（Cofilin，AIP1，Coronin，Twinfilin和SRV2/CAP）驱动网 肌动蛋白在促胞胶的促进条件下拆卸？有肌动蛋白相关吗？ 加速F-肌动蛋白上核苷酸状态过渡以促进拆卸的蛋白质？此外，我们将 将新技术和方向引入我们的研究，包括细胞肌动蛋白的体外重构 结构，冷冻EM研究蛋白质结构，无细胞提取物，可用于遗传生化的肌动蛋白 机制和系统级别的方法来确定单个肌动蛋白调节剂中的遗传破坏如何 影响其余肌动蛋白相关蛋白的细胞水平，定位和功能。