Decoding mechanotransduction mechanisms of cell-surface receptors

解码细胞表面受体的机械转导机制

• 批准号: 9897757
• 负责人: WENDY RYAN GORDON
• 金额: $ 7.4万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-07-20 至 2021-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9897757
• 关键词: Adaptor Signaling Protein; Biological Assay; Biophysics; Birds; Cell Surface Proteins; Cell Surface Receptors; Cell membrane; Cell surface; Cells; Cues; Cytoskeleton; DNA; Diagnosis; Diagnostic; Disease; Disease Progression; Environment; Explosion; Genetic Transcription; Guanosine Triphosphate Phosphohydrolases; Heart Diseases; Homeostasis; Hybrids; Image; Immobilization; Magnetism; Malignant Neoplasms; Measures; Mechanics; Mission; Molecular; Molecular Conformation; Muscular Dystrophies; Mutation; Nanostructures; National Institute of General Medical Sciences; Pathogenesis; Phosphotransferases; Physiological; Polycystic Kidney Diseases; Positioning Attribute; Proteins; Proteolysis; Proteome; Role; Signal Pathway; Spectrum Analysis; Structure; Surface; Technology; Testing; Tissues; X-Ray Crystallography; base; cell behavior; improved; insight; magnetic beads; mechanical force; mechanotransduction; notch protein; novel diagnostics; novel therapeutics; protein function; receptor; response; sensor; single molecule; synergism; tool

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.

项目概要 最近大量的研究表明，细胞微环境中的机械力，或 它的“机械部分”是疾病中一个潜在的可目标和可量化的因素，就像基因的变化一样。 对细胞和组织水平的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其宏观刚度来实现，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的变化。 疾病的僵硬伴随着细胞张力稳态的大量分子变化，其中 “机械传导”信号通路在张力感应的中心被异常激活。 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代谢 我们的总体假设是，研究来自细胞膜外部、内部和内部的机械线索。 细胞表面受体如何改变构象来感知和响应力将导致严重的不足 在分子水平上研究细胞的机械微环境，从而产生新的治疗方法 以及许多疾病的诊断工具，而先进的单分子光谱工具可用于探测。 在分子水平上力诱导构象变化，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 需要 NIGMS 任务中“细胞生物物理学”和“结构功能”方法之间的协同作用。 应对测量细胞感知的分子水平力以识别细胞表面的挑战 机械传感器，定义生理力的大小，并测量疾病期间力的变化 新的混合荧光分子张力传感器的进展将被设计出来，结合当前的优点 使用新的融合标签技术租用基因编码和固定的基于 DNA 的传感器，该技术允许 DNA 纳米结构与细胞中基因编码蛋白质的共价附着 测量对特定细胞表面受体施加力的下游细胞效应，改进版本 为研究 Notch 受体的机械转导而开发的高通量磁镊测定法将 使用，它将皮牛顿力施加到拴在特定受体上的磁珠上，并测量 使用成像和基于细胞裂解物的读数进行下游反应，例如转录、定位 接头蛋白、细胞骨架动力学以及相关激酶和 GTP 酶活性来应对挑战。 受体用来感知和响应力、X 射线晶体学和免疫学的解码机制 已证明的单分子蛋白水解将用于检验力诱导蛋白水解测定是一种假设 一般的机械传感机制，正如最近通过表征 Notch 受体发现的那样。 细胞“机械体”在分子水平上，这些研究有可能确定新的治疗方法 分子和诊断工具，并普遍阐明机械力在疾病发病机制中的作用。