利用超分辨显微镜研究压力调控Hippo信号通路的机制

Hippo signaling pathway participates in cell growth and differentiation, tissue and organ formation, adult stem cell maintenance and homeostasis. It also has a close relationship with tumor initiation and progression. Previous studies of Hippo pathway are mostly based on gene expression and protein-protein interaction. However, for the issue how mechanical signals regulate Hippo pathway and determine cell fate, we only know that cytoskeleton and stress fiber are implicated in this process. The detailed regulatory mechanism remains to be explored. Moreover, the first receiver of mechanical signals is cell membrane. Whether cell membrane participates in the regulation and how it works is also a problem to be solved. Therefore, we choose press as a mechanical signal, which is very similar with extrusion between cells in tissues. Firstly, we will detect the role of press in Hippo pathway, and then reveal the press-controlled differences of YAP localization and spatial clustering in normal and cancer cells by super-resolution fluorescence microscopy, which will provide good support for targeting Hippo pathway to treat tumor. By combined dual-color dSTORM imaging with biochemical techniques, we will illustrate the potential mechanism of press-regulated Hippo pathway in terms of cytoskeleton tension variation and cell membrane deformation.

Hippo信号通路参与细胞的生长分化、组织器官形成以及成体干细胞的维持和自稳态的保持等，同时与肿瘤的发生、发展也密不可分。以往对Hippo通路的研究多从基因表达和蛋白质相互作用的角度出发，而对于机械信号是如何调控Hippo通路进而决定细胞命运的，目前仅知道细胞骨架和应力纤维有参与其中，但具体的调控机制还尚待完善。另外，作为最先接收机械信号的细胞膜是否参与调控以及是通过何种方式进行的，也是目前亟需解决的问题。因此，本项目将从压力这一与组织中细胞承受的挤压最为相似的机械信号入手，首先考察压力对Hippo信号通路的调控作用；然后利用超分辨荧光显微镜揭示压力对正常细胞和癌细胞内YAP的定位和空间集簇程度调控的差异性，为探索以Hippo通路作为治疗肿瘤的靶点提供支撑；最后通过双色dSTORM成像和生物化学手段相结合，从细胞骨架张力变化和细胞膜形变两个角度阐明压力调控Hippo信号通路的可能机制。

Hippo信号通路在包括机械传导在内的许多生物过程中起着关键作用。这一途径的关键激活因子YAP被认为是机械传导信号的中心成分，可以感知细胞外微环境的变化，如不同的细胞密度、组织结构和基质硬度。虽然已经有大量的研究表明YAP参与了这些过程，但机械力诱导YAP调节的机制尚不清楚。本项目中，我们使用PDMS薄片对细胞表面施加压力，并利用超分辨率成像技术dSTORM结合生物化学方法，研究了YAP如何感知细胞外机械力的变化。压力能够促使YAP向胞质定位且聚集性降低。通过双色超分辨成像和细胞松弛素D处理，我们证明了压力能导致F-actin的解聚。并且蛋白免疫印迹实验显示，肌动蛋白丝组装和功能的关键调控因子RhoA以及YAP的下游蛋白Cyr61在压力作用下都有明显的下调，再次证实了F-actin在压力调控YAP中的作用。另外，我们还发现与RhoA相关联的细胞膜上的一种Gα12/13偶联受体LPAR1在压力作用下其聚集体被削弱，表明LPAR1的激活受到压力的抑制。综上，我们证明了压力通过促使F-actin解聚、下调RhoA和使LPAR1失活，从而导致YAP出核和聚集减少。我们的工作揭示了压力调控Hippo信号通路的一条完整的信号轴线，即压力-GPCR-RhoA-F-actin-YAP，为Hippo信号通路的机械调节机制提供了新的见解，也为开发以Hippo通路作为肿瘤治疗靶点的药物提供新的依据。