Focal Adhesion Kinase Dynamics under Shear Stress in Live Endothelial Cells Studied with a FRET Biosensor

Abstract

Focal adhesion kinase (FAK) plays an important role in integrin‐mediated mechanotransduction signaling events. We transfected endothelial cells (ECs) with a FRET‐based FAK biosensor capable of specifically detecting the phosphorylation of FAK at Tyr‐397, which is reflected in ECFP/YPet emission ratio. The effects of shear stress on FAK activity in live ECs were tracked with high temporal and spatial resolution by recording the FRET responses with a live cell imaging system under flow. The results showed that FAK activity was induced preferentially on the perinuclear and nuclear regions in ECs. To investigate the roles of microtubules and actin filaments in the dynamics of FAK activity, we examined the FRET response in ECs treated with cytochalasin D (CD) or colchicine, respectively. The analysis of FRET responses showed that CD caused an inhibition of FAK activity. In contrast, chochicine caused an increase in FAK activity, particularly in the perinuclear and nuclear regions. These results indicate that the spatial distribution of FAK activity is dependent on the integrity and re‐organization of actin networks, as well as the dynamics of microtubules. The findings suggest that FAK activity may play a significant role in regulating FA formation and cell adhesion to resist cell detachment in ECs subjected to shear stress. This work was supported by NHLBI Research Grants HL‐080518 and HL‐085159 (S.C.).