Mechanotransduction in Fibroblast

描述（由申请人提供）：所有细胞感知并响应机械力。与我们对化学信号的感知和转导的了解相比，我们对细胞如何感知和转导机械力知之甚少。本提案的目的是确定成纤维细胞中机械转导单元的分子成分。在可电兴奋的感觉细胞中，机械转导单元包括与细胞外和细胞内系链相连的质膜离子转运蛋白。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，与细胞外和细胞内系链相关的离子转运蛋白的机械转导单元在成纤维细胞中是否保守尚不清楚。本研究研究了一种假设，即普遍表达的质膜Na-H交换器NHE1是成纤维细胞机械转导单元的重要组成部分，它锚定在肌动蛋白细胞骨架上。机械力增加NHE1活性和磷酸化，以及NHE1依赖性细胞内ph的增加。此外，在机械力的作用下，NHE1对于局灶粘附激酶FAK活性的增加、FAK向局灶接触的募集以及f -肌动蛋白的组装都是必需的。设计了两个特定的目的来研究NHE1在机械转导中的作用。目的1侧重于NHE1的机械传感。NHE1活性响应机械力的动力学将被确定，并且激活是否依赖于NHE1磷酸化或肌动蛋白锚定、去novo f -肌动蛋白组装或整合素接合将被测试。目的2关注NHE1的机械转导，以及它的离子易位和肌动蛋白锚定是否对肌动蛋白丝组装和局灶粘连重塑的机械敏感反应是必要的。NHE1如何调节肌动蛋白丝在机械力作用下的动态，将通过检测ph依赖性的cofilin断丝活性、Arp2/3复合物成核活性以及荧光斑点显微镜测定的肌动蛋白丝的转换和运动来确定。NHE1如何调节局灶黏附的动态重塑，将通过成像局灶黏附蛋白的时空募集，以及询问ph依赖性肌动蛋白结合蛋白是否决定局灶黏附稳定性来确定。

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.