Analyses of activation mechanisms of cell mechanosensor at nano- and microlevels.

几乎每个细胞都能对机械刺激做出反应，机械刺激在调节细胞体积、细胞迁移和心血管系统以及重力感觉等多种生物功能中发挥着关键作用。然而，由于细胞机械传感器的分子实体的模糊性，这一功能背后的分子机制被称为细胞机械感觉，长期以来一直不清楚。机械敏感(MS)通道的发现在很大程度上改善了这种情况，并在这一领域取得了很大进展。基于这种背景，我们确定了本研究的目标是：1)MS通道激活的机制；2)细胞骨架的作用；3)粘着斑细胞在细胞机械感觉中的作用。取得的主要成果如下：1.研究成果。MS通道的激活机制：利用已知的三维结构的MS通道，细菌MSCL，我们试图在纳米水平上解析其激活机制。通过分子生物学、膜片钳和分子动力学模拟的结合，我们成功地确定了其位于膜外小叶表面附近的机械传感位置。我们还澄清了，当感觉到机械传感部位的膜张力时，通道通过跨膜螺旋的倾斜和滑动打开它的孔。细胞骨架在机械传感中的作用：使用半完整的培养内皮细胞模型，我们发现应力纤维的主要结构成分肌动蛋白纤维可以通过其去多聚来响应肌动蛋白纤维张力的降低而作为一种新型的机械传感器工作。焦点粘连在机械感觉中的作用：使用半完整的培养上皮细胞系模型，我们发现焦点蛋白Zysin在机械刺激下肌动蛋白纤维的发育和焦点粘连的形成中起着关键作用。

Virtually every cell can respond to mechanical stimuli, which plays a critical role in a variety of biofunctions including the regulations of cell volume, cell migration and cardiovascular system, and gravisensing. The molecular mechanisms underlying this function termed cell mechanosensing, however, had been unknown for a long time due to the ambiguity of the molecular entity of cell mechanosensors. Such situation was largely improved by the discovery of mechanosensitive (MS) channels, by which great progresses have been made in this field.Based on this background, we set the aim of the present study to resolve, 1) the mechanisms of MS channel activation, and the role of 2) cytoskeleton and 3) focal adhesion in cell mechanosensing. The obtained major results are as follows.1. Activation mechanism of MS channels: Using the best studied MS channel with known 3D structure, bacterial MscL, we tried to resolve its activation mechanism at the nanoscale level. We succeeded in identifying its mechanosensing site that resides near the surface of the outer leaflet of the membrane, by the combination of molecular biology, patch clamping, and molecular dynamics simulation. We also clarified that the channel opens its pore thorough the tilting and sliding of the transmemebrane helices upon sensing of membrane tension at the mechanosensing site.2. Role of cytoskeleton in mechanosensing: Using a semi-intact model of cultured endothelial cells, we found that the major structural component of stress fiber, actin fiber, can work as a new type of mechanosensor through its depolymelization in response to the tension decrease in the actin fiber.3. Role of focal adhesion in mechanosensing: Using a semi-intact model of cultured epithelial cell line, we found that the focal protein zyxin plays a critical role in the development of actin fibers and focal adhesions by mechanical stimuli.