利用力学调控和成像技术对力学敏感信号通路调控的研究

Mechanical signals play an important role in the normal physiological process of cells, but the effect of mechanical regulation signals on cell signaling pathways is not yet clear. This project takes YAP in Mechanical-sensitive Hippo pathway as the research object, investigates its distribution under macro/micro mechanical regulation, and discusses the effect of mechanical regulation on signaling pathway. The rapid development of single molecule technology in recent years has provided technical support for the study of YAP. Atomic force microscopy (AFM) can achieve mechanical regulation at the level of pico-Newton (pN); Fluorescence microscopy can observe the distribution of specific proteins in cells in real time. The combination of the two technologies provides high resolution in time and space to observe the distribution of YAP and the binding of YAP-TEADs while applying mechanical regulation signals. These results would help to investigate the effects of mechanical regulation on signaling pathways and the fate of cells after mechanical regulation. In addition, we assume GPCR—F-actin—YAP as a possible pathway for the transduction of mechanical regulation signal from cell membrane surface to intracellular matrix, and designed experiments to verify it. This project will reveal the molecular mechanism of mechanical regulation of YAP-related signaling pathways, and provide a theoretical basis for the diagnosis and treatment of YAP-related diseases.

机械力信号在细胞正常生理过程中起着至关重要的作用，而这种力学调控信号对细胞信号通路的影响尚未清楚。本项目以力学敏感的Hippo信号通路中的YAP为研究对象，考察其在宏观/微观力学调控下的分布情况，讨论力学调控对信号通路的影响。近年来迅速发展起来的单分子技术为YAP的研究提供了技术支持，原子力显微镜可以实现皮牛顿级别的力学调控，荧光显微镜可以实时观测细胞内部特定蛋白的分布情况，二者联用技术在时间和空间上的高分辨率可以实现在施加力学调控信号的同时实时观察活细胞内部YAP的分布情况以及YAP-TEADs的结合情况，考察力学调控对信号通路的影响以及调控后细胞的命运。此外，我们设定了GPCR—F-肌动蛋白—YAP为力学调控信号从细胞膜表面向内传导的可能的信号通路，并设计实验进行验证。本项目将揭示力学调控YAP相关信号通路的分子机制，对YAP相关疾病的诊疗提供了理论依据。

机械力信号帮助细胞了解自身所处的环境和位置，促进其完成增殖、修复等生理功能，在细胞的生命过程中起着至关重要的作用。对力学敏感的Hippo信号通路中的Yes-associated protein（YAP）的研究可以揭示机械力信号对细胞命运的调控作用。本项目利用原子力-超分辨联用显微镜在宏观和微观层面上对垂直压力调控Hippo信号通路的机制进行了深入系统地研究。从细胞在组织中垂直方向的宏观作用力入手，在纳米尺度上研究了压力最有可能影响的F-actin为起始，到合成F-actin所需的RhoA以及上游细胞膜上压力的感受器GPCR，揭示了压力调控Hippo信号通路的一条完整的信号轴线，即压力-GPCR-RhoA-F-actin-YAP，为阐明机械力控制的YAP机制提供理论支持。进一步的，量化了单细胞层面上诱导癌细胞中YAP蛋白的空间分布和集簇程度差异的机械刺激的大小、作用位置、作用时间。通过考察对机械调控下破坏微丝、微管、核转运蛋白和渗透压对YAP分布的影响，总结出作用于细胞膜的机械信号借由微丝、微管的介导传递至细胞核，并在核转运蛋白协助下诱导YAP核质分布异位的结论，揭示了机械刺激从细胞膜传递至细胞核的简单通路，加深了我们对机械力调控细胞命运的理解。在此期间，项目课题组还完成了联用显微镜从搭建、配套软件编写、应用、改进、到商品化的蜕变，为后续国家自然科学基金面上项目的开展奠定了硬件基础。