The loss of Rac1 activity leads to defective RhoA subcellular localization. (A) Effect of ROCK inhibition on F-actin structure of WT neutrophils. WT cells, pretreated with the ROCK inhibitor Y-27632, were stimulated and stained with rhodamine-phalloidin and mounted with reagent containing DAPI for nucleus, as in Figure 2, and F-actin structure (in red) was compared with WT and Rac1^−/− neutrophils. The nucleus is visualized in blue. The arrows point out the leading edge. The arrowheads point out the uropod in WT cells. Note the presence of narrow lamellipodia and uropod formation in WT cells, whereas cells treated with Y-27632 or Rac1^−/− cells display a large lamellipodia without uropod formation. (B) Spreading area. (C) Width of the uropod. (D) WT, Rac1^−/−, and Rac1^−/− (wtRac1) polymorphonuclear neutrophils (PMNs) expressing EGFP-RhoA (in green) were stimulated and stained as above. Arrows point out EGFP-RhoA. Arrowheads point out the tail. Note the presence of RhoA in the uropod of WT cells and Rac1^−/− (wtRac1) cells, whereas RhoA remained in a perinuclear domain in Rac1^−/− neutrophils. (E) EGFP-RhoA in the tail. Fluorescent intensity of EGFP-RhoA in the tail was measured in arbitrary units. Representative images from at least 2 independent experiments, scale bar = 5 μm. Results show mean ± SEM.