Figure 3

Phosphorylation of paxillin S273, PAK1 kinase activity, βPIX GEF activity and βPIX binding to PAK1 regulate paxillin binding at sites of adhesion. (A) Average paxillin binding rates across the cell (lower bars) for paxillin variants (dark blue bars), PAK1 variants (red bars) and βPIX variants (green bars). Paxillin binding rates at protrusive (cyan bars) and retractive (orange bars) edges of the cell (upper bars). Error bars represent SEM. One Star (*) corresponds to p < 0.01, two stars **p < 0.001, three stars ***p < 0.0001. N = 30 cells from three independent experiments. n = 10–20 adhesions in each cell identified by thresholding and analyzed. (B) Spatial maps of paxillin binding dynamics across cells expressing paxillin-WT (left), paxillin-S273A (middle), and paxillin-S273D (right). The protrusive edges are indicated with a cyan line, retractive edges are indicated with an orange line. Regions of short binding times (12–20 s⁻¹) shown in cyan, intermediate binding times (9–12 s⁻¹) in yellow and slow binding times (<9 s⁻¹) in magenta. Zoomed image insert represents long-binding time small adhesions in the paxillin-S273D expressing cells. Scale bars are 5 μm. (C) Percentage of pixels across multiple cells with binding dynamics in defined binding time bins as in B. (D) Representative spatial dynamic maps of paxillin binding to adhesions in three different cells expressing paxillin-WT at protrusive (cyan lines) and retractive (orange lines) edges. Scale bars are 2 μm.