1. The absence of the centrosome promotes small GTPase Rac1 activation via acentrosomal microtubules.
The absence of the centrosome promotes small GTPase Rac1 activation via acentrosomal microtubules. (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 <
Hung-Wei Cheng et al. LSA 2019;2:e
© 2019 Cheng et al.