(A) The effects of the centrosome on Rac1 activation. The level of GTP-bound Rac1 isolated from lysates of RPEp53−/−, RPEp53−/−SAS6−/−, and RPEp53−/−STIL−/−cells by GST-PAK-CRIB pull-down and the amount Rac1 protein (total Rac) present in the input lysate were detected by Western blotting (top). Fold changes in the level of GTP-Rac relative to total Rac determined by Western blotting is indicated below the blots. Data are mean ± SEM (n = 8 independent experiments). **P < 0.01, compared with RPEp53−/−. (B) Rac1 FRET/ECFP ratio images using the Rac1 biosensor (pTriEx4-Rac1-2G) in RPEp53−/−, RPEp53−/−SAS6−/−, and RPEp53−/−STIL−/−cells. The cold and hot colors in the color bar represent low and high Rac1 activity levels, respectively. Bar, 20 μm. (C) Images of YFP-CAAX and a cell protrusion/retraction (white/black, respectively) dynamics map of migrating RPEp53−/−, RPEp53−/−SAS6−/−, and RPEp53−/−STIL−/−cells. Bar, 20 μm. (D) Effects of growing microtubules on Rac1 activation. The level of GTP-bound Rac1 isolated from lysates of RPEp53−/−, RPEp53−/−SAS6−/−, and RPEp53−/−STIL−/−cells treated with DMSO (control) or nocodazole (10 μM, 16 h) by GST-PAK-CRIB pull-down and the protein level of Rac1 (Total Rac) in the input lysate were detected by Western blotting (top). Fold changes in the levels of GTP-Rac relative to total Rac determined by Western blotting is indicated below the blots. Data are mean ± SEM (n = 5 independent experiments). *P < 0.05; **P < 0.01; ***P < 0.001.