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

项目摘要 本提案的目的是了解肌动蛋白解聚因子的两个不同成员 肌动蛋白调节蛋白的同源（ADF-H）家族，Cofilin和Abp 1，相互影响彼此的相互作用， 以及对肌动蛋白的功能性影响。Cofilin和Abp 1在体内是高度丰富和普遍存在的，并且它们都结合 直接与肌动蛋白丝使用结构相关的ADF-H结构域，但这两种蛋白质有惊人的 对肌动蛋白丝动力学的不同影响。切丝蛋白促进蛋白质的切断、解聚和去支化。 而Abp 1则通过Arp 2/3复合物促进细丝的组装，并保护（而不是 破坏）丝分支连接。这就提出了一个关键问题：Cofilin和Abp 1是如何共存于同一个细胞中的？ 细胞中的丝状肌动蛋白结构，以及它们如何影响彼此在这些位置的功能？基于 根据我收集的初步数据，我的工作假设是细胞表达一定水平的Abp 1， 在不干扰Cofilin在肌动蛋白中的功能的情况下， 拆卸此外，我假设这种平衡的实现部分是因为绑定中的关键差异 Cofilin和Abp 1在肌动蛋白丝上的动力学。然而，我的初步数据也表明， 生理浓度的Abp 1强烈干扰Cofilin介导的细丝去支化。我 假设这是因为Abp 1稳定地结合于分支连接并竞争性地阻断Cofilin结合 Arp 2/3复合物。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。 单分子成像与遗传学和活细胞成像在S。酿酒。建议的目的是：（1）在 Abp 1对BMP 1介导的肌动蛋白分解和去支化的影响的体外分析，以及（2）体内 分析Abp 1和Cofilin在调节肌动蛋白动力学中的关系。

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.