Mechanical Regulation of Actin Networks

描述(申请人提供)：肌动蛋白细胞骨架的一个显著特征是在单个细胞内共存不同的肌动蛋白结构，含有不同的肌动蛋白结合蛋白，并表现出不同的形态、机械行为和周转率。肌动蛋白细胞骨架的错误调节和与细胞外基质的机械相互作用的改变与许多形式的癌症有关，并被认为是导致运动和形态与正常细胞不同的原因。最近的研究表明，机械刺激可以对细胞的运动性和细胞骨架形态产生很大影响，但力转化为生化信号的机制才刚刚开始被发现。我们建议研究机械力在肌动蛋白细胞骨架的调节中所起的作用。特别是，我们专注于稳定或破坏F-肌动蛋白稳定的两种蛋白质：原肌球蛋白和ADF/cofilin。这些蛋白质影响F-肌动蛋白的周转，F-肌动蛋白的周转决定了细胞骨架结构重塑的速度，它们还可能通过排除和包括其他蛋白，在决定网络结构和网络中肌动蛋白结合蛋白的特定补充方面发挥作用。我们假设，F-肌动蛋白的应激状态改变了调节蛋白与细丝侧面的结合。而不是具有 一组动力学参数，可能只取决于细丝的核苷酸状态和蛋白质本身的生化调节，我们假设蛋白质结合可能被细丝上的张力变化促进或抑制。这一想法的部分灵感来自我们最近的发现，即分支复合体Arp2/3更有可能与弯曲的细丝外部结合，而不是与直细丝结合。在R01的更新应用中，我们建议使用独特的力显微镜和荧光显微镜工具的组合来测试F-肌动蛋白上的力改变ADF/cofilin和原肌球蛋白在体外和活细胞中的结合和竞争的想法。这项研究的结果将为细胞如何利用机械刺激来调节肌动蛋白细胞骨架的周转和组织提供一个新的视角。

DESCRIPTION (provided by applicant): A remarkable feature of the actin cytoskeleton is the coexistence within a single cell of diverse actin structures containing different sets of actin-binding proteins and exhibiting diverse morphologies, mechanical behavior, and turnover rates. Misregulation of the actin cytoskeleton and altered mechanical interactions with the extracellular matrix are associated with many forms of cancer and believed to contribute to differences in motility and morphology from normal cells. Recent studies have shown that mechanical stimuli can have a large impact on the motility and cytoskeletal morphology of cells, but mechanisms by which forces are converted into biochemical signals are only beginning to be uncovered. We propose to investigate the role that mechanical forces play in the regulation of the actin cytoskeleton. In particular, we focus on two proteins that stabilize or destabilize F-actin: tropomyosin and ADF/cofilin. These proteins affect the turnover of F-actin, which determines how quickly a cytoskeletal structure can be remodeled, and they may also play a role in determining network architecture and the particular complement of actin-binding proteins in the network by excluding some and including others. We hypothesize that the stress state of F-actin alters the binding of regulatory proteins to the sides of the filament. Rather than proteins having a single set of kinetic parameters that may depend only on the nucleotide state of the filament and on biochemical regulation of the proteins themselves, we hypothesize that protein binding may be promoted or inhibited by changes in tension on the filament. This idea is inspired in part by our recent finding that the branching complex Arp2/3 is more likely to bind to the outside of bent filaments than to straight filaments. In this R01 renewal application, we propose to test the idea that force on F-actin alters the binding and competition of ADF/cofilin and tropomyosin, both in vitro and in live cells, using a combination of unique force microscopy and fluorescence microscopy tools. The results of this study will provide the foundation for a new perspective on how cells may use mechanical stimuli to regulate actin cytoskeleton turnover and organization.