Structural and Molecular Mechanisms of Stress Fiber Repair

应力纤维修复的结构和分子机制

基本信息

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
项目类别：
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

项目摘要

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.

项目概要让组织与其动态环境保持物理稳态平衡（“机械稳态”），单个细胞必须能够感知局部环境中的机械信号并做出反应机械稳态在形态发生中起着至关重要的作用，其失调可能会导致相应的结果。高血压、纤维化和哮喘等疾病的治疗已取得重大进展。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（“机械转导”）知之甚少，阻碍了靶向治疗的发展机械转导失调及其疾病状态。肌动蛋白细胞骨架在力传导中发挥着重要作用，尤其是肌动蛋白-肌球蛋白电缆被称为应力纤维（SF），它既能主动产生收缩力又能传递细胞外力影响 SF 组装的细胞-细胞和细胞-基质粘附，分解和收缩性对于涉及细胞力学和细胞力学的许多生理过程很重要动态细胞形状变化，例如上皮组织稳态和形态发生。 SF 的不平衡会导致机械引起的断裂，称为应力纤维应变位点 (SFSS)。 SFSS 走向灾难性的断裂，大部分由 zyxin 修复，zyxin 是一种机械敏感的 LIM（LIN- 11、Isl-1 和 Mec-3) 蛋白首先通过其三个 C 端串联 LIM 结构域定位到菌株位点。然后通过其 N 端结构域招募交联蛋白 ɑ-肌动蛋白和聚合因子 VASP 在几分钟内介导 SF 修复，尽管有证据表明细胞内发生了这一系列事件。此外，zyxin 介导的 SF 修复的生物物理机制尚不清楚。 zyxin 的 LIM 域识别的 SFSS 的架构特征尚不清楚。在这里，我建议确定 zyxin 介导的 SF 修复的分子和结构机制。通过生物物理重建和细胞测定，我将检验 zyxin、α-肌动蛋白和 VASP 的假设直接共组装修复机械损伤的肌动蛋白丝并确定生物物理机制然后我将应用尖端相关的低温光电子。显微镜来检验 zyxin 与我们观察到的力依赖性肌动蛋白构象结合的假设除了提供对 SF 机械稳态的具体见解外（目标 2）。从长远来看，也可能揭示通过细胞骨架的机械传导的一般机制。这项工作将指导针对机械转导途径失调的治疗方法的开发。