Probing the logic of Hippo-based mechanotransduction with optogenetics

物理力对细胞的增殖、分化和迁移等决策过程起着至关重要的作用。细胞力学的扰动会导致发育障碍和包括癌症在内的疾病。因此，了解物理力和细胞力换能器之间的相互调节对细胞生理学和病理生理学具有重要意义。由于肌动蛋白细胞骨架内的各个子过程的紧密耦合以及与其他机械感觉结构的联系，解决这个问题变得复杂起来。它需要工具来测量和操纵特定的、空间和时间上定义的细胞的物理和生化特性。为了实现这一点，我建议使用光遗传学来探索肌动蛋白依赖的机械转导的逻辑，通过河马路径，一个重要的细胞机械传感器。总的来说，我追求三个目标。首先，通过控制机械输入的大小和动力学来揭示河马通路信号接收和传输的逻辑。第二，询问机械和YAP激活在控制细胞命运中的相对贡献。第三，确定河马通路感受到的肌动蛋白细胞骨架的特征。结合使用共聚焦显微镜和原子力显微镜，将能够进行精确的光遗传操作，并同时分析细胞力学和河马信号。为了探索这些发现在一个需要河马信号作为其生理基础的系统中的意义，这项研究将在小鼠胚胎干细胞中进行。从结果中，我期待着对细胞力学和行为的相互调节的基本见解。

Physical forces play a fundamental role for cellular decision-making processes such as proliferation, differentiation, and migration. Perturbation of cellular mechanics causes both developmental disorders and diseases including cancer. Understanding the reciprocal regulation between physical forces and cellular mechanotransducers is therefore important for cell physiology and pathophysiology. Addressing this problem is complicated by the tight coupling of individual sub-processes both within the actin cytoskeleton and in connection with other mechanosensory structures. It requires tools for measuring and manipulating specific, spatially and temporally defined, physical and biochemical properties of the cell. To achieve this, I propose to use optogenetics to probe the logic of actin-dependent mechanotransduction through the Hippo pathway, an important cellular mechanosensor. Overall, I pursue three goals. First, to reveal the logic of signal reception and transmission of the Hippo pathway by controlling the magnitude and dynamics of the mechanical input. Second, to interrogate the relative contributions of mechanics and YAP activation in control of cell fate. And third, to identify the features of the actin cytoskeleton that are sensed by the Hippo pathway. Using a combined confocal and atomic force microscope will allow precise optogenetic manipulations and simultaneous analysis of cell mechanics and Hippo signaling. To probe the significance of the findings in a system that requires Hippo signaling for its physiology, the study will be performed in mouse embryonic stem cells. From the results, I expect fundamental insights into the reciprocal regulation of cell mechanics and behavior.