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

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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.