Focal adhesion kinase leads paxillin in the assembly of nascent focal adhesions in lamellipodial protrusions of migrating endothelial cells

Abstract

Cell migration requires the fine spatiotemporal integration of many proteins that regulate the fundamental processes that drive cell movement. Focal adhesion (FA) dynamics is a continuous process involving the coordination between FA and actin cytoskeleton that is essential for cell migration. We studied the spatiotemporal dynamics of FAK and paxillin by using time-lapse dual-color imaging to monitor concurrently the two molecules in live endothelial cells (ECs) co-transfected with GFP-FAK and mCherry-paxillin. The fluorescence intensities (FI) of paxillin and FAK at FAs were determined in cell front, center and rear. The mean FAK/Paxillin FI ratio is highest at cell front (mean±SEM: 3.73±0.15), near 1 at cell center (0.88±0.04), and lowest at cell rear (0.56±0.02) (P<0.01). Determination of the time difference between the assemblies of FAK and paxillin at FAs in lamellipodial protrusion (LP) showed that the assembly of FAK occurs ahead of that of paxillin, leading by 2.62±0.27 min (mean±SEM), after using analysis on thirteen cells with a total 206 FAs. The results show that, for these two molecules that modulate FA dynamics during cell migration, FAK assembles at the nascent FAs earlier than paxillin in the protrusions at the cell front. This work was supported by NHLBI Research Grants HL-104402 and HL-106579 (S.C.).