Design and Implementation of Genetically Encoded Myosin Based Force Sensors

基于基因编码肌球蛋白的力传感器的设计与实现

• 批准号: 8320666
• 负责人: DANIEL PETER KIEHART
• 金额: $ 21.88万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-04-01 至 2014-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8320666
• 关键词: ATP phosphohydrolase; Actinin; Acute; Address; Atomic Force Microscopy; Azides; Biological; Biological Assay; Biology; Biophysics; Biosensor; Calibration; Cell Line; Cell Shape; Cells; Cellular biology; Cultured Cells; Cytokinesis; Cytoskeleton; Development; Developmental Biology; Dorsal; Drosophila genus; Environment; Family; Fluorescence; Fluorescence Resonance Energy Transfer; Gene Expression; Goals; Health; Homeostasis; Human; In Vitro; Interdisciplinary Study; Laser Surgery; Life; Location; Maps; Measurement; Measures; Mechanics; Microscopy; Monitor; Morphogenesis; Motor; Myosin ATPase; Myosin Type II; N-terminal; Neck; Output; Pharmaceutical Preparations; Play; Positioning Attribute; Process; Production; Proteins; RNA Interference; Reporting; Research; Resolution; Role; Scanning Probe Microscopes; Shapes; Signal Transduction; Solutions; Specimen; Spectrum Analysis; Stretching; Tail; Testing; Traction; Transgenic Organisms; Vinculin; Work; base; cell motility; design; design and construction; driving force; experience; expression vector; flexibility; fly; frontier; improved; in vitro Assay; in vivo; knock-down; non-muscle myosin; protein purification; research study; sensor; single molecule; small molecule

DESCRIPTION (provided by applicant): Understanding the mechanisms by which cells produce, transmit and sense forces constitutes a next frontier in cellular and developmental biology. Major advances have improved our understanding of the biophysics of chemomechanical force production by motor proteins in vitro. Nevertheless, how such proteins collaborate with the dynamic cytoskeleton in vivo to produce forces that drive cell shape changes for morphogenesis, cytokinesis and cell locomotion is unclear. Moreover, how such forces cause changes in gene expression and signaling also remain challenging unanswered research questions. We propose to construct and characterize a family of genetically encoded myosin force sensors designed to quantitatively map forces in vivo with subcellular resolution. Our approach is based on successful force sensors in vinculin and ¿-actinin. Our focus is on the forces produced by ensembles of nonmuscle myosin II in Drosophila, which are essential for cytokinesis and morphogenesis. Specific Aim 1: We will construct genetically encoded Tension Sensing Modules (TSMs) based on Fluorescence Resonance Energy Transfer (FRET) pairs separated by protein springs. We will systematically test a) different FRET pairs; b) different protein springs; and c) different locations in the myosin motors. These myosin force sensors will be expressed in both fly cell lines and transgenic flies. Specific Aim 2: We will characterize the myosin force sensors (MFSs) in fly cell lines. We will evaluate their ability a) to localize with endogenous myosin; b) to sense tension, using force traction microscopy on flexible substrates; and c) to respond to acute changes in tension induced by drugs or laser surgery. Appropriate control constructs will verify that changes in FRET are due to changes in force across the sensor. Specific Aim 3: We will characterize the myosin force sensors in transgenic flies. We will evaluate their ability a) to rescue myosin function in specimens depleted of endogenous myosin; b) to sense tension in dorsal closure (myosin II) and c) to report acute changes in tension induced by drugs or laser surgery. Specific Aim 4: We will calibrate the myosin force sensors that work in cultured cells and in fly specimens using single-molecule and/or solution strategies in vitro. Our experiments will deliver optimized myosin force sensors and the first measurements of the forces produced by ensembles of myosin in vivo. They will provide new information about the biophysics of cell shape changes in cytokinesis and morphogenesis. Because myosins play key roles in a variety of processes fundamental to human homeostasis and development, we expect these force sensors to have broad impact. Moreover, they will contribute to the resolution of several existing controversies regarding the role of myosins in biology. PUBLIC HEALTH RELEVANCE: Living cells are the fundamental building blocks of all life and respond to and influence their environments in a variety of ways. Here, we focus on how cells generate and respond to mechanical forces by proposing to design and implement genetically encoded tension sensing modules that will allow us monitor when and where cells generate forces as they grow, divide, crawl and change shape. This multidisciplinary research will impact human health because it addresses the basic biological problem of how myosin motor proteins produce forces for development and homeostasis.

描述(由申请人提供)：了解细胞产生、传递和感知力的机制是细胞和发育生物学的下一个前沿。主要的进展提高了我们对体外运动蛋白产生化学机械力的生物物理学的理解。然而，这些蛋白质如何与体内动态的细胞骨架合作，产生驱动形态发生、胞质分裂和细胞运动的细胞形状变化的力量尚不清楚。此外，这些力量如何导致基因表达和信号的变化也仍然是具有挑战性的尚未回答的研究问题。我们建议构建和表征一系列基因编码的肌球蛋白力传感器，旨在以亚细胞分辨率定量绘制体内的力图。我们的方法是基于纽蛋白和肌动蛋白中成功的力传感器。我们的重点是果蝇中非肌肉肌球蛋白II的集合产生的力量，这对细胞质分裂和形态发生是必不可少的。具体目标1：我们将构建基于蛋白质弹簧分隔的荧光共振能量转移(FRET)对的基因编码的张力传感模块(TSM)。我们将系统地测试a)不同的FRET对；b)不同的蛋白质弹簧；以及c)肌球蛋白马达中的不同位置。这些肌球蛋白力传感器将在果蝇细胞系和转基因果蝇中表达。具体目标2：我们将对飞行细胞系中的肌球蛋白力传感器(MFSS)进行表征。我们将评估它们a)用内源性肌球蛋白定位的能力；b)在柔性底物上使用力牵引显微镜来感知张力的能力；c)对药物或激光手术引起的张力的急剧变化做出反应的能力。适当的控制结构将验证FRET的变化是由于整个传感器的力的变化所致。具体目标3：我们将对转基因果蝇中的肌球蛋白力传感器进行表征。我们将评估他们的能力，a)在内源性肌球蛋白耗尽的标本中拯救肌球蛋白功能；b)感觉背部闭合时的张力(肌球蛋白II)；c)报告药物或激光手术引起的张力的急性变化。具体目标4：我们将在体外使用单分子和/或溶液策略来校准在培养细胞和苍蝇标本中工作的肌球蛋白力传感器。我们的实验将提供优化的肌球蛋白力传感器，并首次测量肌球蛋白整体在体内产生的力。它们将提供关于细胞质分裂和形态发生中细胞形状变化的生物物理学的新信息。由于肌球蛋白在人类体内平衡和发育的各种基本过程中发挥着关键作用，我们预计这些力传感器将产生广泛的影响。此外，它们将有助于解决有关肌球蛋白在生物学中的作用的几个现有争议。 与公共卫生相关：活细胞是所有生命的基本构件，以各种方式响应和影响环境。在这里，我们通过提议设计和实现遗传编码的张力传感模块来关注细胞如何产生和响应机械力，该模块将允许我们监控细胞在生长、分裂、爬行和改变形状时何时何地产生力。这项多学科的研究将影响人类健康，因为它解决了肌球蛋白马达蛋白如何产生发育和动态平衡的力量这一基本生物学问题。