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

本项目的目的是研究局灶性粘连和相关肌动蛋白细丝中脂肪结构域的动力学，以了解粘连细胞对外界机械刺激的重塑过程。特别是，新开发的实验技术包括在流体作用下对脂肪域和肌动蛋白细丝动力学的时程观察，以及使用具有非均匀表面结构的弹性膜的拉伸系统。首先，我们开发了一个实验系统，可以使用RFP结合的脂肪结构域载体和GFP结合的肌动蛋白载体来观察细胞的动力学。结果表明，肌动蛋白细丝结构的重构与脂肪结构域的出现和消失密切相关。其次，利用软光刻技术在焦点粘结处局部施加拉伸，制备了具有微柱阵列的PDMS膜。内皮细胞在微柱顶端周围形成局灶性黏附复合体，并形成与拉伸方向垂直的应力纤维、粗大的肌动蛋白束。最后，我们开发了一种微拉伸测试仪来测量应力纤维的机械性能，这将是理解细胞内应力传递的关键。第一次对从内皮细胞化学分离出来的应力纤维进行了拉伸测试，提供了载荷-位移关系。结果表明，荷载-位移曲线呈非线性，具有较高的延伸性。我们已经成功地研究了机械刺激细胞的细胞骨架和焦点粘连在细胞内力传递机制中的重要作用。

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.