Mechanical Regulation of Actin Networks

描述（由申请人提供）：肌动蛋白细胞骨架的一个显著特征是在单个细胞内共存多种肌动蛋白结构，其中包含不同的肌动蛋白结合蛋白集，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机械相互作用的改变与多种形式的癌症有关，并被认为是导致正常细胞运动和形态差异的原因。最近的研究表明，机械刺激可以对细胞的运动和细胞骨架形态产生很大的影响，但如何将力转化为生化信号的机制才刚刚开始被揭示。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。我们特别关注两种稳定或破坏f -肌动蛋白的蛋白：原肌球蛋白和ADF/cofilin。这些蛋白质影响f -肌动蛋白的周转，这决定了细胞骨架结构的重塑速度，它们也可能在决定网络结构和网络中肌动蛋白结合蛋白的特定补体中发挥作用，通过排除和包括一些。我们假设f -肌动蛋白的应激状态改变了调控蛋白与丝侧的结合。而不是蛋白质

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.