Fig. 4.

EphA2 signaling increases FA dynamics. (A) FAs and ephrinA1:EphA2 clusters are spatially separated in the hybrid substrate. (B) Immunofluorescence images of paxillin and F-actin of cells fixed after 90 min of spreading. (C and D) Quantification of FA distribution from cell edge to center (normalized by the highest probability in each group) (C) and FA numbers per cell, counted from n = 47 cells in RGD-only sample, and n = 49 cells in RGD+EphrinA1 sample (D). ***P < 0.001 for Student’s t test. (E) Lifetime color-coded trajectories of paxillin-GFP of cells spreading on indicated substrates. TIRF images were taken after 50 min of spreading, at a rate of 1 frame per minute for a total of 92 min. Lifetime color-coded paxillin adhesion centroids were plotted and assembled from all images to reflect FA trajectories. Adhesions are blue when they appear, and over time they gradually change toward red until disappearing. (F–H) Quantifications of FA lifetime and formation rate. (F) Distribution of the lifetime of all individual FAs in the two cells tracked in E. Lifetime distribution was fitted with second-order exponential decay function to get characteristic time constant τ2. (G) τ2 was compared in a group of cells. Data are presented as (τ2 ± fitting error) for individual cells, and (mean ± SD) for average values. (H) FA formation rate of cells on the two substrates. Data are presented as (mean ± SD) for both individual cells and average values.