Structural and Molecular Mechanisms of Stress Fiber Repair

项目摘要 为了使组织与其动态环境（“机械环境”）保持物理稳态平衡， 稳态”），个体细胞必须能够感知局部环境中的机械线索并做出反应 相应地机械稳态在形态发生中起着重要作用，其失调可导致 疾病状态，如高血压、纤维化和哮喘。虽然在以下方面取得了重大进展： 了解机械稳态和细胞机械感觉的生理意义， 蛋白质将机械刺激转化为生化信号的分子机制 （“机械传导”）的理解很少，阻碍了针对神经传导的靶向治疗的发展。 失调的机械转导及其疾病状态。 肌动蛋白细胞骨架在机械力传递中起着重要的作用，特别是肌动蛋白-肌球蛋白电缆 称为应力纤维（SF），其既主动产生收缩力又传递细胞外力 冲击细胞-细胞和细胞-基质粘附进入细胞质。SF组件的动态调节， 分解和收缩对于涉及细胞力学的许多生理过程是重要的， 动态细胞形状变化，如上皮组织稳态和形态发生。随机力学 SF的不平衡可导致机械诱导的破裂，称为应力纤维应变部位（SFSS）。虽然一些 SFSS发生灾难性的破坏，大部分由zyxin修复，zyxin是一种机械敏感的LIM（LIN- 11，Isl-l，& Mec-3）蛋白。Zyxin首先通过其三个C-末端串联LIM结构域定位于菌株位点， 然后通过其N-末端结构域募集交联蛋白辅肌动蛋白和聚合因子VASP 几分钟内就能修复旧金山虽然有证据表明，这一系列的事件，在细胞 在水平上，zyxin介导的SF修复的生物物理机制还不清楚。而且 由zyxin的LIM结构域识别的SFSS的结构特征是未知的。 在这里，我建议确定zyxin介导的SF修复的分子和结构机制。 通过生物物理重建和细胞分析，我将测试的假设，zyxin，α-辅肌动蛋白，和VASP 直接共组装以修复机械损伤的肌动蛋白丝并确定生物物理机制 zyxin介导的机械稳态（目的1）。然后我会用最先进的相关低温光电子 显微镜来测试zyxin结合力依赖性肌动蛋白构象的假设，我们已经观察到， 体外（目标2）。除了提供对SF的机械稳态的具体见解外，这些研究还 也可能揭示通过细胞骨架的机械转导的一般机制。从长远来看， 这项工作将指导针对失调的机械转导途径的治疗方法的开发。

PROJECT SUMMARY For tissues to maintain a physical steady-state equilibrium with its dynamic surroundings (“mechanical homeostasis”), individual cells must be able to perceive mechanical cues in their local environment and respond accordingly. Mechanical homeostasis plays an essential role in morphogenesis, and its dysregulation can lead to disease states such as hypertension, fibrosis, and asthma. While there has been significant progress in understanding the physiological significance of mechanical homeostasis and cellular mechanosensation, the molecular mechanisms by which proteins convert mechanical stimuli into biochemical signals (“mechanotransduction”) are poorly understood, impeding the development of targeted therapeutics for dysregulated mechanotransduction and its disease states. The actin cytoskeleton plays a prominent role in mechanotransduction, notably actin-myosin cables known as stress fibers (SFs) which both actively generate contractile forces and transmit extracellular forces impinging on cell-cell and cell-matrix adhesions into the cytoplasm. Dynamic regulation of SF assembly, disassembly, and contractility are important for many physiological processes involving cellular mechanics and dynamic cell shape changes, such as epithelial tissue homeostasis and morphogenesis. Stochastic mechanical imbalance in SFs can result in mechanically-induced ruptures, termed stress fiber strain site (SFSS). While some SFSS proceed towards catastrophic breakage, the majority are repaired by zyxin, a mechanosensitive LIM (LIN- 11, Isl-1, & Mec-3) protein. Zyxin first localizes to strain sites through its three C-terminal tandem LIM domains, then recruits the cross-linking protein ɑ-actinin and polymerization factor VASP through its N-terminal domains to mediate SF repair in a matter of minutes. While there is evidence for this sequence of events at the cellular level, the biophysical mechanism of zyxin-mediated SF repair is not well understood. Furthermore, the architectural features of a SFSS which are recognized by zyxin’s LIM domains are unknown. Here I propose to determine the molecular and structural mechanism of zyxin-mediated SF repair. Through biophysical reconstitution and cellular assays, I will test the hypothesis that zyxin, α-actinin, and VASP directly co-assemble to repair mechanically damaged actin filaments and determine the biophysical mechanism of zyxin-mediated mechanical homeostasis (Aim 1). I will then apply cutting-edge correlative cryo-light electron microscopy to test the hypothesis that zyxin binds to a force-dependent actin conformation we have observed in vitro (Aim 2). In addition to providing specific insights into mechanical homeostasis of SFs, these studies are also likely to reveal general mechanisms of mechanotransduction through the cytoskeleton. In the longer term, this work will guide the development of therapeutics against dysregulated mechanotransduction pathways.