FAK and paxillin dynamics at focal adhesions in the protrusions of migrating 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 coordination between FA and actin cytoskeleton, which is essential for cell migration. We studied the spatiotemporal relationship between the dynamics of focal adhesion kinase (FAK) and paxillin at FAs in the protrusion of living endothelial cells. Concurrent dual-color imaging showed that FAK was assembled at FA first, which was followed by paxillin recruitment to the FA. By tracking and quantifying FAK and paxillin in migrating cells, the normalized FAK/Paxillin fluorescence intensity (FI) ratio is > 1 (≈ 4 fold) at cell front, ≈ 1 at cell center, and < 1 at cell rear. The significantly higher FAK FI than paxillin FI at cell front indicates that the assembly of FAK-FAs occurs ahead of paxillin at cell front. To determine the time difference between the assemblies of FAK and paxillin at nascent FAs, FAs containing both FAK and paxillin were quantified by image analysis and time correlation. The results show that FAK assembles at the nascent FAs earlier than paxillin in the protrusions at cell front.

Figure 1. Dynamics of FAK and paxillin in a live EC transfected with GFP-FAK (green) and mCherry–paxillin (red).

(A) FAK and paxillin in the cell. The three boxed regions are cell front (B), center (C) and rear (D), with enlarged views shown in panels (B), (C) and (D), respectively. B–D show the results for FAK, paxillin, and FAK + Paxillin at 0, 30 and 60 min. In these ratio photos, superimposition of GFP-FAK (green) and mCherry-paxillin (red) images appears as yellow. During the 60-min of cell migration, there are many more GFP-FAK clusters than mCherry-paxillin at cell front (B), but they are more similar in numbers at cell center (C), and rear (D). The corresponding Supplementary Movies are S4–6 for B, S7 for C, and S8 D. Scale bars: A = 20 μm, B = 15 μm, C = 4.5 μm and D = 12 μm. (E) FI ratio images of FAK/paxillin in the same cell as 1A at 0 (left) and 60 min (right). The cell contour at 60 min is outlined in solid white, whereas the initial cell contour (0 min) is outlined with dashed white. Pseudo-color for the FAK/paxillin FI ratio value ranges from low (blue) to high (red) (ROIs in both panels are cyan for cell front, green for center, and pink for rear) (Supplementary Movie S9). Scale bar = 18 μm. (F) Time courses (over 60-min) of FAK/paxillin FI ratio at the three ROIs as shown in Figs. 1E. FI ratios of FAK/paxillin at cell front, center and rear were obtained from eleven migrating cells, each with seven time points. The bar graphs represent mean ± s.e.m. (F1) ROIs moving with the cell migration. (F2) ROIs staying at fixed positions (0-time).

Figure 2. Dual-color images showing the dynamic motion of FAK and paxillin at the protrusion front of a live EC transfected with GFP-FAK (green) and mCherry–paxillin (red).

(A) An intact cell image showing (from left to right) FAK (green) + Paxillin (red), FAK (green), paxillin (red), and FAK/Paxillin FI ratio (pseudo-color). In FAK+Paxillin, the superimposition of FAK and Paxillin yield yellow (Supplementary Movie S10). (B) Enlarged images from the boxed regions in A row. During the 60-min time course, there were more FAK clusters (arrows) than paxillin at the protrusions of cell front (Supplementary Movies S11–13). (C) FAK/paxillin FI ratio image, pseudo-color ranges from low (blue) to high (red). Enlarged images were from the boxed region in A-“FAK/Pax Ratio”. FAK-FAs are more apparent at the protrusion front than paxillin during 60-min time course (Supplementary Movies 14–15). Scale bars: A = 20 μm; B and C = 7 μm.

Figure 3. Image analysis of dynamics of FAK and paxillin at FAs at the protrusion front of an EC transfected with GFP-FAK and mCherry-paxillin.

(A) Co-localization of FAK (green) and paxillin (red) yielded a yellow color. Boxed region was selected as regions of interest shown in B. (B) Identified FAs were labeled with numbers. The pairs of FAK (left) and paxillin (right) at the same FAs were selected for quantification analysis. (C) The time courses of normalized intensities (y-axis) of FAK (green) and paxillin (red) in four FAs (No. 4, 5, 16 and 20, arrowed and labeled in B) are plotted. (D) The values of the time correlation for all 22 FAs in the same cell (including the four FAs in C) are plotted as a function of time shift between FAK and paxillin at FAs. Also plotted is the mean curve for all 22 FAs in this cell. The results of time correlation analysis of this cell (22 individual FAs) yielded a time shift of 4.21 ± 0.87 min (mean ± s.e.m.). The value is significantly different from 0 (p < 0.001) indicates the time shift at dynamic FAs.

Figure 4. The results of time correlation analysis of independent experiments on thirteen migrating cells (206 individual FAs) yielded a time shift of 2.62 ± 0.27 min (mean ± s.e.m.) between GFP-FAK and mCherry-paxillin, which is significantly different from 0 (n = 206, p < 0.001).

Figure 5. Dual-color images show the dynamic motion of FAK and actin filaments in protrusion of cell front in live EC transfected with GFP-FAK and RFP-actin.

(A-left) Cell images show the superimposition (“FAK/Actin”; yellow) of FAK (green) and actin (red). Scale bar = 20 µm. (A-right) The enlarged images for actin and FAK show the boxed region in A-left. Scale bar = 8 µm. FAK clusters are associated with actin filaments (arrows). (B) The dynamics of FAK and actin filaments in the box region (A-left). The superimposition (FAK/Actin; yellow) of FAK (green) and actin (red) show FAK associated with actin filaments at the leading edge of the protrusion front during the 60-min time course (arrows) (Supplementary Movie 16). Scale bar = 12.5 µm.

Figure 6. The immunostaining images of an EC stained with FAK, paxillin and actin filaments.

(A) The fluorescence photomicrographs show FAK immunostained with anti-FAK (left column), F-actin stained with rhodamine-phalloidin (middle column), and merging of FAK (green) and F-actin (red) images, with a yellow color for co-localization of FAK and actin filaments (right column). FAK and actin filaments are shown to be co-localized. FAK plaques are present at the points where stress fibers end and some FAK plaques are present at cell periphery (arrows). Actin filaments show staining weakly (up arrow). Scale bar = 6 μm. (B) Fluorescence photomicrographs show FAK (left column) and paxillin (middle column) immunfluorescence stained with anti-FAK and anti-paxillin antibodies, respectively. Merging of FAK (green) and paxillin (red) images (right column), with a yellow color for co-localization of FAK and paxillin. At the cell periphery, there are many more FAK plaques than paxillin plaques (arrows). Scale bar = 10 μm.