Decoding mechanotransduction mechanisms of cell-surface receptors

项目摘要 最近的大量研究表明，改变细胞微环境中的机械力，或 它的“机械体”，是疾病中一个潜在的可靶向和可量化的因素，就像GE的变化一样. Nome或蛋白质组。在细胞和组织水平上对机械微环境的有价值的见解 是通过测量细胞施加在可变形表面上的力或其宏观硬度来实现的，但 在很大程度上忽略了细胞如何在分子水平上感知和对力做出反应。宏观上的变化 疾病中的僵硬伴随着细胞张力稳态的大量分子变化，其中 “机械转导”信号通路被异常激活。处于张力传感中心的是 跨膜细胞表面受体，其独特的位置可以感知和整合所有细胞内的蛋白质和蛋白质。 从细胞膜的外部、内部和内部发出的机械信号。我们的总体假设是，学习 细胞表面受体如何改变构象以感知和响应力，将导致关键的欠... 在分子水平上站在细胞机械微环境的立场，从而导致新的治疗方法 以及多种疾病的诊断工具。虽然先进的单分子光谱学工具可以用来探测 在分子水平上，力诱导的构象变化，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，并重新- 在NIGMS任务中寻求“细胞-生物物理学”和“结构-功能”方法之间的协同。 为了解决测量细胞感受到的分子水平的力以识别细胞表面的挑战 机械传感器，定义生理力的大小，并测量力在疾病过程中的变化 随着时间的推移，新型混合型荧光分子张力传感器将结合CURR-C的优点而被设计出来。 使用一种新的融合标签技术租用基于基因编码和固定的DNA传感器，该技术可以 DNA纳米结构与细胞中遗传编码的蛋白质的共价连接。应对……的挑战 测量向特定细胞表面受体施加力的下游细胞效应，一种改进版本 为研究Notch受体Will的机械转导而开发的高通量磁钳实验 对系在特定受体上的磁珠施加皮牛顿力，并采取措施 使用成像和基于细胞裂解物的读数的下游反应，如转录、定位 接头蛋白，细胞骨架动力学，以及相关的激酶和GTP酶活性。应对……的挑战 受体用来感知和响应力的解码机制，X射线结晶学和免疫... 已证实的单分子蛋白分解试验将用于检验强迫诱导的蛋白分解是一种 Notch受体的一般机械传感机制，就像最近发现的那样。通过将 在分子水平上，这些研究有可能发现新的治疗方法-- NUE和诊断工具，并一般阐明机械力在疾病发病机制中的作用。

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.