Decoding mechanotransduction mechanisms of cell-surface receptors

项目摘要 最近的大量研究表明，细胞微环境中机械力的改变， 它的“机械体”，是疾病中潜在的可靶向和可量化的因素，就像基因的变化一样， nome或proteome。在细胞和组织水平上对机械微环境的有价值的见解， 通过测量细胞施加在可变形表面上的力或它们的宏观刚度来实现，但是 在很大程度上忽略了细胞如何在分子水平上感知和响应力。宏观变化 疾病中的僵硬伴随着细胞张力稳态中的大量分子变化， “机械传导”信号通路被异常激活。在紧张感测的中心是 跨膜细胞表面受体，这是独特的定位，以感测和整合所有细胞的ME， 来自细胞外、细胞内和细胞膜内的化学信号。我们的总体假设是， 细胞表面受体如何改变构象以感知和响应力将导致一个关键的不足， 在分子水平上建立细胞的机械微环境，从而导致新的治疗方法 以及许多疾病的诊断工具虽然先进的单分子光谱学工具可以探测 力诱导的构象变化在分子水平上，解码机械转导机制， 由于缺乏工具来测量细胞如何在分子水平上感知和响应力， 要求在NIGMS使命中“细胞生物物理学”和“结构功能”方法之间的协同作用。 为了解决测量细胞感受到的分子水平力的挑战， 机械传感器，定义生理力的大小，并测量疾病期间力的变化 发展，新的混合荧光分子张力传感器将被设计，结合电流的优点， 使用一种新的融合标签技术， DNA纳米结构与细胞中遗传编码蛋白质的共价连接。为了应对…的挑战 测量对特定细胞表面受体施加力的下游细胞效应， 一个高通量磁镊分析开发研究机械转导的Notch受体将 它将皮牛顿力施加到拴在特定受体上的磁珠上， 使用成像和基于细胞裂解物的读数，如转录、细胞定位、 衔接蛋白、细胞骨架动力学和相关激酶和GT3活性。为了应对…的挑战 受体用来感知和响应力的解码机制，X射线晶体学和免疫组织化学， 已证实的单分子蛋白水解试验将用于检验力诱导的蛋白水解是一种 一般的机械感应机制，如最近发现的Notch受体。通过表征 细胞“机械体”在分子水平上，这些研究有可能确定新的治疗ave， 核和诊断工具，并一般阐明机械力在疾病发病机理中的作用。

Project Summary An explosion of recent studies has indicated that altered mechanical forces in the microenvironment of cells, or its “mechano-some”, is a potentially targetable and quantifiable factor in disease, much like changes in the ge- nome or proteome. Valuable insights into the mechanical microenvironment at the cell and tissue level have been achieved by measuring forces that cells exert on deformable surfaces or their macroscopic stiffness, but have largely ignored how cells sense and respond to force at the molecular level. Changes in macroscopic stiffness in disease are accompanied by a wealth of molecular changes in a cell's tensional homeostasis where “mechanotransduction” signaling pathways are aberrantly activated. At the epicenter of tension sensing are transmembrane cell-surface receptors, which are uniquely positioned to sense and integrate all cellular me- chanical cues from outside, inside, and within the membrane of the cell. Our overall hypothesis is that studying how cell-surface receptors change conformation to sense and respond to force will lead to a critical under- standing of the mechanical microenvironment of cells at a molecular level thus leading to novel therapeutics and diagnostic tools for many diseases. While advanced single molecule spectroscopy tools exist to probe force-induced conformational changes at a molecular level, decoding mechanotransduction mechanisms has been crippled by a lack of tools to measure how cells sense and respond to force at a molecular level and re- quires synergy between “cellular-biophysics” and “structure-function” approaches within the NIGMS mission. To tackle the challenge of measuring molecular-level forces that cells sense in order to identify cell-surface mechanosensors, define magnitudes of physiologic forces, and measure how force changes during disease progression, new hybrid fluorescent molecular tension sensors will be devised that marry advantages of cur- rent genetically-encoded and immobilized DNA-based sensors using a new fusion-tag technology that allows covalent attachment of DNA nanostructures to genetically-encoded proteins in cells. To tackle the challenge of measuring downstream cellular effects of applying force to specific cell-surface receptors, an improved version of a high-throughput magnetic tweezers assay developed to study mechanotransduction of Notch receptors will be used, which applies piconewton forces to magnetic beads tethered to specific receptors, and measures downstream responses using imaging and cell-lysate based readouts such as transcription, localization of adaptor proteins, cytoskeleton dynamics, and relevant kinase and GTPase activity. To tackle the challenge of decoding mechanisms that receptors use to sense and respond to force, x-ray crystallography and an im- proved single molecule proteolysis assay will be used to test the hypothesis that force-induced proteolysis is a general mechanosensing mechanism, as was recently discovered for Notch receptors. By characterizing the cellular “mechano-some” at a molecular level, these studies have the potential to identify new therapeutic ave- nues and diagnostic tools, and generally elucidate the role of mechanical forces in disease pathogenesis.