Mechanotransduction in Fibroblast

描述(申请人提供)：所有细胞都能感知机械力并对其做出反应。与我们所知道的化学信号的感知和传递相比，细胞如何感知和传递机械力还知之甚少。这项建议的目的是确定成纤维细胞中机械转导单元的分子组成。在可电兴奋的感觉细胞中，机械转导单元包括连接到细胞外和细胞内系绳的质膜离子传输蛋白。在不可兴奋的细胞中，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，但尚未得到实验证实。此外，与细胞外和细胞内连接的离子转运蛋白的机械转导单位在成纤维细胞中是否保守也是未知的。这一建议探讨了一种假设，即普遍表达的质膜Na-H交换器NHE1锚定在肌动蛋白细胞骨架上，是成纤维细胞机械转导单位的重要组成部分。机械力增加了NHE1的活性和磷酸化，并增加了细胞内NHE1依赖的pH。此外，作为对机械力的响应，NHE1是增加粘着斑激酶FAK的活性、将FAK招募到焦点接触者以及F-肌动蛋白组装所必需的。我们设计了两个特定的目标来研究NHE1在机械转导中的作用。目标1重点介绍NHE1的机械传感。将确定NHE1活性对机械力的响应动力学，并将测试激活是否依赖于NHE1的磷酸化或肌动蛋白锚定、去新生F-肌动蛋白组装或整合素参与。目的2重点研究NHE1的机械转导及其离子转运和肌动蛋白锚定是否对肌动蛋白细丝组装和局部黏附重塑的机械敏感性反应是必需的。NHE1如何调节肌动蛋白细丝对机械力的响应，将通过检测依赖于pH的cofilin细丝切割活性、Arp2/3复合体的成核活性以及荧光散斑显微镜确定的肌动蛋白细丝的周转和移动来确定。NHE1如何调节局部粘连的动态重塑，将通过成像局部粘连蛋白的空间和时间招募以及询问pH依赖的肌动蛋白结合蛋白是否决定局部粘连的稳定性来确定。 机械力在形态发生、细胞增殖、决定恶性转化等方面起着重要作用。机械力还调节结缔组织重塑和修复反应，这突显了了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.