Coordination of multiple ADF-H family proteins in controlling actin cytoskeleton dynamics

多个 ADF-H 家族蛋白在控制肌动蛋白细胞骨架动力学中的协调

• 批准号: 10212353
• 负责人: Reynaldo Aguilar Lopez
• 金额: $ 3.21万
• 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: 2020-07-16 至 2022-07-15
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10212353
• 关键词: Actin-Binding Protein; Actins; Address; Affect; Affinity; Binding; Biochemical; Biochemistry; Biological Assay; Biological Process; Cardiovascular Diseases; Cell division; Cells; Competitive Binding; Complex; Cytoskeleton; Data; Defect; Dissociation; Endocytosis; Equilibrium; F-Actin; Family; Family member; Filament; Fluorescence Anisotropy; Fluorescence Microscopy; Genetic; Glia Maturation Factor; Goals; Human Pathology; Image; Impairment; In Vitro; Kinetics; Label; Location; Mammals; Mediating; Microfilaments; Modeling; Morphogenesis; Mutation; Neoplasm Metastasis; Nerve Degeneration; Phagocytosis; Physiological; Play; Process; Protein Family; Proteins; Role; Saccharomyces cerevisiae; Side; Site; Structure; Temperature; Tertiary Protein Structure; Testing; Tissues; Total Internal Reflection Fluorescent; Work; Yeasts; actin 2; actin depolymerizing factor; base; cell growth regulation; cell motility; cofilin; depolymerization; genetic approach; genetic regulatory protein; imaging approach; in vivo; insight; live cell imaging; member; molecular imaging; mutant; overexpression; predictive modeling; recruit; single molecule

Project Summary The goal of this proposal is to understand how two different members of the Actin Depolymerizing Factor Homology (ADF-H) family of actin-regulatory proteins, Cofilin and Abp1, influence each other’s interactions with and functional effects on actin. Cofilin and Abp1 are highly abundant and ubiquitous in vivo, and they both bind directly to actin filaments using structurally related ADF-H domains, yet these two proteins have strikingly different effects on actin filament dynamics. Cofilin promotes severing, depolymerization, and debranching of filaments, whereas Abp1 instead promotes assembly of filaments by Arp2/3 complex and protects (rather than destabilizes) filament branch junctions. This raises a key question: how do Cofilin and Abp1 co-exist on the same filamentous actin structures in cells, and how do they affect each other’s functions at these locations? Based on preliminary data that I have gathered, my working hypothesis is that cells express a level of Abp1 that allows it to carry out its functions in promoting actin assembly without interfering with Cofilin’s functions in actin disassembly. Further, I hypothesize that this balance is achieved in part because of key differences in the binding kinetics of Cofilin and Abp1 on actin filaments. However, my preliminary data also suggest that the same physiological concentrations of Abp1 strongly interfere with Cofilin-mediated debranching of filaments. I hypothesize that this is because Abp1 stably binds to branch junctions and competitively blocks Cofilin binding to Arp2/3 complex. To test these models, I will use an integrated approach combining biochemistry and in vitro single molecule imaging with genetics and live-cell imaging in S. cerevisiae. The aims of the proposal are: (1) In vitro analysis of Abp1 effects on Cof1-mediated actin disassembly and debranching, and (2) In vivo analysis of the relationship between Abp1 and Cofilin in regulating actin dynamics.

项目摘要 这项提议的目的是了解肌动蛋白解聚因子的两个不同成员是如何 同源(ADF-H)肌动蛋白调节蛋白家族，cofilin和Abp1，相互影响彼此之间的相互作用 以及对肌动蛋白的功能影响。Cofilin和Abp1在体内高度丰富和普遍存在，它们都结合在一起 使用结构上相关的ADF-H结构域直接到肌动蛋白细丝，但这两种蛋白质具有惊人的 对肌动蛋白细丝动力学的不同影响。Cofilin促进血管的切断、解聚和分枝 而Abp1则通过Arp2/3复合体促进细丝的组装，并保护(而不是 破坏稳定)灯丝分支连接。这就提出了一个关键问题：Cofilin和Abp1如何在同一 细胞中的丝状肌动蛋白结构，以及它们如何影响这些位置上彼此的功能？基于 我收集的初步数据，我的工作假设是，细胞表达的Abp1水平允许它 在不干扰肌动蛋白中cofilin功能的情况下发挥其促进肌动蛋白组装的功能 拆卸。此外，我假设实现这种平衡的部分原因是绑定中的关键差异 Cofilin和Abp1在肌动蛋白细丝上的动力学。然而，我的初步数据也表明， 生理浓度的Abp1强烈干扰Cofilin介导的细丝脱支。我 假设这是因为Abp1稳定地与分支连接结合，并竞争性地阻止Cofilin结合 至Arp2/3复合体。为了测试这些模型，我将使用生物化学和体外实验相结合的综合方法。 酿酒酵母中的单分子成像与遗传学和活细胞成像。建议的目的是：(1) Abp1对Cof1介导的肌动蛋白解离和分支的影响的体外分析，以及(2)体内 Abp1和Cofilin在肌动蛋白动态调控中的关系分析。