Figure 5

PAK1 and βPIX proteins are less dynamic in adhesions and have a higher immobile fraction. Binding rates at adhesions depend on paxillin pS273. (A) Images of cells and FRAP image time series of cells co-expressing paxillin-WT-EGFP and PAK1-WT or βPIX-WT mCherry fusions. Adhesions were identified by paxillin-EGFP localization and mCherry fusions were bleached with a 561 nm laser. Scale bars are 5 μm. (B) The recovery curves of PAK1-WT (top) and βPIX-WT (bottom) in adhesions and the non-adhesion regions are shown. Each curve was averaged over multiple adhesions (n = 60 adhesion or non-adhesion ROIs), multiple cells (n = 10–15) and 3 independent experiments. The first time point plotted after bleaching for each curve was obtained from FRAP experiments done on fixed regions since the proteins were already recovered significantly in live cell experiments. (C) Half-time of fluorescence recovery and the (D) immobile fraction were determined in both adhesion and non-adhesion regions. (E) ICM data showing average protein binding rates for PAK1-WT or βPIX-WT in cells expressing with paxillin variants in protrusive (cyan) and retractive (orange) edges of the cell. n = 10–20 cells. All error bars are SEM. For D one star exceptionally corresponds to P < 0.05. In all other cases, one Star (*) corresponds to p < 0.01, two stars **p < 0.001, three stars ***p < 0.0001.