Focal adhesion kinase leads paxillin in focal adhesion assembly at lamellipodial protrusion of migrating endothelial cells

Abstract

The dynamics of focal adhesions (FAs) can regulate cell migration. Focal adhesion kinase (FAK) and paxillin are involved in FA disassembly and formation. We used double‐color imaging to monitor concurrently the spatial‐temporal dynamics of FAK and paxillin in live endothelial cells (ECs) co‐transfected with GFP‐FAK and DsRed‐paxillin. The fluorescence intensities (FI) of paxillin‐FAs and FAK‐FAs were determined in cell front, center and rear. The mean FAK/Paxillin FI ratio (n=5) is highest at cell front (mean±SEM: 4.73±0.48), not significantly different from 1 at cell center (0.95±0.11), and lowest at cell rear (0.64±0.05). In ECs co‐transfected with RFP‐actin and GFP‐FAK, FAK is found to be associated with actin filaments at cell leading edge. Determination of the time difference between the assemblies of FAK and paxillin at nascent FA of lamellipodial protrusion (LP) by computational analysis indicates that FAK reaches its maximum intensity earlier than paxillin (mean±SEM: 3.63±0.68 min). Our results suggest that FAK is assembled in FAs at LP earlier than paxillin. While both FAK and paxillin play important roles in modulating FA dynamics, FAK promotes FA formation ahead of paxillin at the cell leading edge. This work was supported by NHLBI Research Grants HL‐104402 and HL‐106579 (S.C.).