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: 10536382
• 负责人: Donovan Yong Zhi Phua
• 金额: $ 4.68万
• 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/10536382
• 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; Epithelial; Equilibrium; Event; F-Actin; Feedback; Fibrosis; Fluorescence Microscopy; Functional disorder; Gene Expression Regulation; Heart; Heart Diseases; Homeostasis; Hypertension; In Situ; In Vitro; Individual; Lead; Light; Lobular Neoplasia; Longitudinal Studies; 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; Work; ZYX gene; alpha Actinin; crosslink; electron tomography; extracellular; fiber cell; insight; link protein; mechanical force; mechanical signal; mechanical stimulus; mechanotransduction; novel; polymerization; reconstitution; recruit; repaired; targeted treatment; therapeutic development; therapy development; tool; 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 为了使组织保持物理稳态状态平衡，并具有动态环境 稳态），单个细胞必须能够在其本地环境中感知机械提示并响应 因此。 疾病状态，例如高血压，纤维化和哮喘。 了解机械稳态和细胞机械的生理意义， 蛋白质通过将机械刺激转化为生化信号的分子机制 （“机械转移”）知之甚少，阻碍了针对性的治疗剂的发展 失调的机械转移及其疾病状态。 肌动蛋白细胞骨架在机械转换中起着重要作用，尤其是肌动蛋白肌球蛋白电缆 称为应力纤维（SFS） 将细胞细胞 - 矩阵粘附到细胞质中。 拆卸和侵入性对于许多涉及细胞力学和的生理过程很重要 动态细胞形状的变化，例如上皮组织稳态和形态发生。 SF中的不平衡会导致机械诱导的折扣，称为应力纤维应变位点（SFSS） SFSS进行灾难性破裂，大多数是通过机械敏感的LIM（LIM-）修复的。 11，ISL-1和MEC-3）蛋白质。 然后，通过使用N末端结构域募集交联蛋白质和聚合因子VASP 在几分钟内介导SF修复。 水平，Zyxin介导的SF修复的生物物理机制尚不清楚。 由Zyxin的LIM域识别的SFSS的建筑特征未知。 在这里，我建议确定Zyxin介导的SF修复的分子和结构机制。 通过生物物理重新构造和细胞测定，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）。 显微镜检查了Zyxin与我们具有{在IN中观察到的力的肌动蛋白置信度结合的假设！ 体外（AIM 2）。 还可以揭示机械转移的一般机制，从而在长期内通过细胞骨架。 这项工作将指导针对失调的机械转导途径的疗法的开发。