Structural and Molecular Mechanisms of Stress Fiber Repair

应力纤维修复的结构和分子机制

• 批准号: 10707029
• 负责人: Donovan Yong Zhi Phua
• 金额: $ 4.77万
• 依托单位: ROCKEFELLER UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-01 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10707029
• 关键词: Actins; Architecture; Asthma; Binding; Biochemical; Biological Assay; Biophysical Process; Biophysics; Blood Vessels; C-terminal; Cell Shape; Cell-Matrix Junction; Cells; Cellular Assay; Cryoelectron Microscopy; Cytoplasm; Cytoskeleton; Detection; Development; Disease; Electron Microscopy; Environment; Epithelium; Equilibrium; Event; F-Actin; Feedback; Fibrosis; Fluorescence Microscopy; Functional disorder; Gene Expression Regulation; Heart; Heart Diseases; Homeostasis; Hypertension; In Situ; In Vitro; Individual; Light; Lobular Neoplasia; Lung; Mechanics; Mediating; Methods; Microfilaments; Molecular; Molecular Conformation; Morphogenesis; Myosin ATPase; N-terminal; Pathway interactions; Physical condensation; Physical environment; Physiological; Physiological Processes; Play; Polymers; Process; Property; Protein Family; Proteins; Regulation; Role; Rupture; Signal Transduction; Site; Stress Fibers; Tertiary Protein Structure; Testing; Time; Tissues; Visualization; Work; ZYX gene; alpha Actinin; biophysical properties; crosslink; electron tomography; extracellular; fiber cell; insight; mechanical force; mechanical signal; mechanical stimulus; mechanotransduction; novel; polymerization; protein crosslink; reconstitution; recruit; repaired; structural determinants; targeted treatment; therapeutic development; therapy development; tool; transmission process; vasodilator-stimulated phosphoprotein

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.

项目摘要 为了使组织保持与动态环境的物理稳态相等（“机械） 稳态”），单个细胞必须能够在其本地环境中感知机械提示并做出回应 机械体内稳态在形态发生中起着至关重要的作用，其功能障碍可以引导 到诸如高血压，纤维化和哮喘等疾病状态。虽然取得了重大进展 了解机械稳态和细胞机理的身体意义， 蛋白质通过将机械刺激转化为生化信号的分子机制 （“机械转导”）对 机械转导及其疾病状态的失调。 肌动蛋白细胞骨架在机械转导，尤其是肌动蛋白肌球蛋白电缆中起着重要作用 称为应力纤维（SFS） 撞击细胞细胞和细胞矩阵粘合在细胞质中。 SF组装的动态调节， 拆卸和收缩力对于许多涉及蜂窝力学和的物理过程很重要 动态细胞形状的变化，例如上皮组织稳态和形态发生。随机机械 SF中的不平衡会导致机械诱导的破裂，称为应力纤维应变位点（SFSS）。而有些 SFSS朝着灾难性的破裂进行，大多数是由Zyxin（一种机械敏感的LIM）修复的（lin- 11，ISL-1和MEC-3）蛋白质。 Zyxin首先将位点定位于其三个C末端串联lim结构域， 然后，通过其N末端结构域募集交联蛋白质 - 肌动蛋白和聚合因子VASP 在短短几分钟内调节SF维修。虽然有证据表明该细胞处的事件序列 水平，Zyxin介导的SF修复的生物物理机制尚不清楚。此外， Zyxin的Lim域认可的SFS的建筑特征尚不清楚。 在这里，我建议确定Zyxin介导的SF修复的分子和结构机制。 通过生物物理重构和细胞测定，我将检验Zyxin，α-肌动蛋白和VASP的假设 直接共同组装以修复机械损坏的肌动蛋白丝并确定生物物理机制 Zyxin介导的机械稳态（AIM 1）。然后，我将应用尖端正确的冷冻光电子 显微镜检查了Zyxin与我们在中的力依赖性肌动蛋白构象结合的假设。 体外（AIM 2）。除了对SF的机械稳态提供特定的见解外，这些研究是 还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 这项工作将指导理论的发展，反对失调的机械转导途径。