Mechanical dynamics of focal adhesions of vascular endothelial cells using nano-imaging

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The goal of this project is to study dynamics of FAT domain in focal adhesions and associated actin filaments in order to understand remodeling process of adherent cells in response to external mechanical stimuli. In particular, newly developed experimental techniques here include a time-course observation of dynamics of FAT domain and actin filaments under application of fluid flow and a stretching system using an elastic membrane with nonuniform surface structure. First, we have developed an experimental system which enables to observe dynamics of cells using RFP-conjugated FAT-domain vector and GFP-conjugated actin vector. The result showed that actin filament structure was reconfigured in a close association with appearance/disappearance of FAT domain. Second, PDMS membrane with an array of micropillars has been fabricated by using soft lithography technique to locally apply stretching between focal adhesions. Endothelial cells formed focal adhesion complexes at peripheries of the top of the micropillars and developed stress fibers, thick actin bundles, perpendicular to the direction of stretch. Finally, we have developed a micro-tensile tester to measure mechanical properties of stress fibers, which would be critical for understanding of intracellular stress transfer. Tensile tests were, for the first time, carried out on stress fibers chemically isolated from endothelial cells, providing a load-displacement relationship. It was found that the load-displacement curve was nonlinear with high extensibility. We have successfully studied important roles of cytoskeletons and focal adhesions of mechanically stimulated cells in intracellular force transmission mechanism.