Mechanotransduction in Fibroblast

描述（由申请人提供）：所有细胞都能感知并响应机械力。与我们所知道的化学信号的感知和传导相比，细胞如何感知和消除机械力的理解很少。本提案的目的是确定成纤维细胞中机械转导单位的分子组成。在电可兴奋的感觉细胞中，机械转导单元包括连接到细胞外和细胞内系链的质膜离子转运蛋白。在不可兴奋的细胞，如成纤维细胞，质膜离子转运蛋白在机械转导中的关键作用已被提出，但没有实验证实。此外，连接到细胞外和细胞内系链的离子转运蛋白的机械转导单元是否在成纤维细胞中是保守的尚不清楚。该提案调查的假设，即普遍表达的质膜钠-氢交换NHE 1，这是锚定的肌动蛋白细胞骨架，是一个机械转导单位在成纤维细胞的重要组成部分。机械力增加NHE 1的活性和磷酸化，和NHE 1依赖性增加细胞内的pH值。此外，在响应机械力NHE 1是必要的增加活性的粘着斑激酶FAK，为招聘FAK的焦点接触，并为F-肌动蛋白组装。两个具体的目标是设计调查的作用NHE 1在机械转导。目标1重点关注NHE 1的机械传感。将确定NHE 1活性响应于机械力的动力学，并将测试活化是否依赖于NHE 1磷酸化或肌动蛋白锚定、从头F-肌动蛋白组装或整联蛋白接合。目的2着重于NHE 1的机械转导，以及其离子移位和肌动蛋白锚定是否是肌动蛋白丝组装和粘着斑重塑的机械敏感反应所必需的。NHE 1如何调节肌动蛋白丝动力学响应机械力将通过测试pH依赖性丝切割活性的cofilin，Arp 2/3复合物的成核活性，和肌动蛋白丝的周转和运动，如通过荧光斑点显微镜测定。NHE 1如何调节动态重塑的粘着斑将确定成像的空间和时间募集的粘着斑蛋白，并询问是否pH依赖性肌动蛋白结合蛋白决定粘着斑的稳定性。 机械力在形态发生、细胞增殖和恶性转化中起重要作用。机械力还调节结缔组织重塑和修复反应，这强调了理解成纤维细胞如何感知机械力以及它们如何将机械信号转化为驱动细胞和组织反应的生化事件的重要性。

DESCRIPTION (provided by applicant): All cells sense and respond to mechanical forces. Compared with what we know about the sensing and transduction of chemical signals, how cells sense and transduce mechanical force is poorly understood. The objective of this proposal is to determine molecular components of a mechanotransduction unit in fibroblasts. In electrically excitable sensory cells, mechanotransduction units include a plasma membrane ion transport protein linked to extracellular and intracellular tethers. In non-excitable cells, such as fibroblasts, a critical role for plasma membrane ion transport proteins in mechanotransduction has been proposed but not experimentally confirmed. Also, whether a mechanotransduction unit of an ion transport protein linked to extracellular and intracellular tethers is conserved in fibroblasts is unknown. This proposal investigates the hypothesis that the ubiquitously expressed plasma membrane Na-H exchanger NHE1, which is anchored to the actin cytoskeleton, is an essential component of a mechanotransduction unit in fibroblasts. Mechanical force increases NHE1 activity and phosphorylation, and an NHE1-dependent increase in intracellular pH. Additionally, in response to mechanical force NHE1 is necessary for increased activity of the focal adhesion kinase FAK, for recruitment of FAK to focal contacts, and for F-actin assembly. Two specific aims are designed to investigate the role of NHE1 in mechanostransduction. Aim 1 focuses on mechanosensing by NHE1. The kinetics of NHE1 activity in response to mechanical force will be determined, and whether activation is dependent on NHE1 phosphoryation or actin anchoring, de-novo F-actin assembly, or integrin engagement will be tested. Aim 2 focuses on mechanotransduction by NHE1 and whether its ion translocation and actin anchoring are necessary for the mechanosensitive responses of actin filament assembly and focal adhesion remodeling. How NHE1 regulates actin filament dynamics in response to mechanical force will be determined by testing pH-dependent filament severing activity of cofilin, nucleating activity of the Arp2/3 complex, and the turnover and movement of actin filaments, as determined by fluorescence speckle microscopy. How NHE1 regulates dynamic remodeling of focal adhesions will be determined by imaging the spatial and temporal recruitment of focal adhesion proteins and by asking whether pH-dependent actin binding proteins determine focal adhesion stability. Mechanical forces play important roles morphogenesis, cell proliferation, and determining malignant transformation. Mechanical forces also regulate connective tissue remodeling and reparative responses, which underscores the significance of understanding how fibroblasts sense mechanical force and how they transduce mechanical signals into biochemical events that drive cell and tissue responses.