Design and Implementation of Genetically Encoded Myosin Based Force Sensors

描述（由申请人提供）：了解细胞产生、传递和感知力的机制构成了细胞和发育生物学的下一个前沿。重大进展提高了我们的理解的生物物理学的化学机械力生产的马达蛋白在体外。然而，这些蛋白质如何在体内与动态细胞骨架合作以产生驱动细胞形状变化以进行形态发生、胞质分裂和细胞运动的力尚不清楚。此外，这些力量如何引起基因表达和信号传导的变化也仍然是具有挑战性的未回答的研究问题。我们建议构建和表征一个家族的基因编码肌球蛋白力传感器，旨在定量映射力在体内亚细胞分辨率。我们的方法是基于黏着斑蛋白和辅肌动蛋白中成功的力传感器。我们的重点是在果蝇的非肌肉肌球蛋白II，这是必不可少的胞质分裂和形态发生的合奏所产生的力量。具体目标1：我们将构建基于蛋白质弹簧分离的荧光共振能量转移（FRET）对的遗传编码张力传感模块（TSM）。我们将系统地测试a）不同的FRET对; B）不同的蛋白质弹簧;和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：我们将在苍蝇细胞系中表征肌球蛋白力传感器（MFS）。我们将评估它们的能力a）与内源性肌球蛋白定位; B）感知张力，使用力牵引显微镜在柔性基板上;和c）对药物或激光手术引起的张力急性变化的反应。适当的对照结构将验证FRET的变化是由于传感器上的力的变化。具体目标3：我们将在转基因苍蝇的肌球蛋白力传感器的特点。我们将评估它们的能力：a）在内源性肌球蛋白耗竭的标本中挽救肌球蛋白功能; B）感知背侧闭合（肌球蛋白II）中的张力; c）报告药物或激光手术诱导的张力急性变化。具体目标4：我们将校准肌球蛋白力传感器，在培养的细胞和苍蝇标本中使用单分子和/或解决方案的策略在体外工作。我们的实验将提供优化的肌球蛋白力传感器和肌球蛋白在体内的合奏所产生的力量的第一次测量。它们将提供有关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过程中起着关键作用，我们希望这些力传感器具有广泛的影响。此外，他们将有助于解决现有的几个争议的生物学中的肌球蛋白的作用。

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.