Effect of Focal Adhesion Proteins on Endothelial Cell Adhesion, Motility and Orientation Response to Cyclic Strain
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Focal adhesion proteins link cell surface integrins and intracellular actin stress fibers and therefore play an important role in mechanotransduction and cell motility. When endothelial cells are subjected to cyclic mechanical strain, time-lapse imaging revealed that cells underwent significant morphological changes with their resultant long axes aligned away from the strain direction. To explore how this response is regulated by focal adhesion-associated proteins the expression levels of paxillin, focal adhesion kinase (FAK), and zyxin were knocked down using gene silencing techniques. In addition, rescue of endogenous and two mutant zyxins were used to investigate the specific role of zyxin interactions. Cells with decreased zyxin expression levels and rescue with the mutant lacking zyxin/α-actinin binding exhibited lower orientation angles after comparable times of stretching as compared to normal and control cells. However, knockdown of the expression levels of paxillin and FAK and rescue with the mutant lacking zyxin/VASP (vasodilator-stimulated phosphoprotein) binding did not significantly affect the degree of cell orientation. In addition, wound closure speed and cell–substratum adhesive strength were observed to be significantly reduced only for cells with zyxin depletion and the mutation lacking zyxin/α-actinin binding. These results suggest that zyxin and its interaction with α-actinin are important in the regulation of endothelial cell adhesive strength, motility and orientation response to mechanical stretching.
KeywordsCell stretching Cell orientation Gene silencing Zyxin Focal adhesion Time-lapse imaging
This research was supported by a grant-in-aid from the American Heart Association (PI: FCPY).
Representative time-lapse movie (5 min increments) of wildtype cells during six-hours of cyclic uniaxial (horizontal direction) stretching. These are expanded views of the upper right-hand corner of the wide field shown in supplementary movie 2 and correspond to the images in Fig. 2a (AVI 21660 kb)
Representative time-lapse movie (5 min increments) of wildtype cells during six-hours of cyclic uniaxial (horizontal direction) stretching (AVI 77901 kb)
Representative time-lapse movie (5 min increments) of zyxin knockdown (Zn-sh1) cells during six-hours of cyclic uniaxial (horizontal direction) stretching (AVI 74883 kb)
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