Rac1 and RhoA regulate neutrophil chemotaxis. (A-B) Chemotaxis in human neutrophils expressing dominant-negative Rac1. Human neutrophils were treated with GTPase mutants at 9 μg/mL (dominant negative) or 12 μg/mL (constitutively active) in the presence of Bioporter reagent as described in “Materials and methods.” The cells were then stimulated with a point source of chemoattractant, and the movement of the cells was recorded at 30-second intervals for 15 minutes. At these relatively low levels of expression of the dominant-negative mutants, inhibition of either Rac1 or RhoA activity results in marked reduction in the ability of human neutrophils to chemotax toward a point source of fMLP (A). Positive values represent the percentage of cells that moved freely toward the point source over a 15-minute interval. This was associated with a marked defect in tail retraction (quantified in B). Cells that sensed the gradient but exhibited elongated tails (inset in panel C; bar = 10 μM) and the inability to move toward the chemoattractant point source over the course of the 15-minute period were counted. Cells expressing Rac1T17N that were treated with RhoAG14V were now able to chemotax effectively (A), associated with a restoration of the cell's ability to retract the uropod, quantified in panel B. Data are derived from 4 to 8 experiments, and for each treatment in each experiment the movement of 40 to 100 cells was tracked. (C) Cell morphology during chemotaxis following Rac inhibition in human neutrophils. Cell length measurements were made on live human neutrophils undergoing chemotaxis as described in “Materials and methods.” The values are derived from 4 to 7 experiments and are an average ± standard error from 150 to 350 cells per condition. Measurements were made at random times during the 15-minute duration of the experiment, and thus represent an “average” over this time frame. The differences in cell length were highly significant (P < .001). Inset images were visualized using a 60×/1.45 NA objective lens. (D) Cell morphology during chemotaxis in Rac-deficient mouse neutrophils. Quantification of perturbed tail retraction in Rac1-null mouse neutrophils, as shown in the representative photomicrograph of neutrophils in an fMLP gradient (inset). Rac1-null neutrophils display poor tail retraction compared with wild-type and Rac2-null neutrophils. The average Rac1-null neutrophil length in fMLP-stimulated cells (head to tail) is more than twice as long as in stimulated wild-type cells (mean of 50 cells per genotype). Bar = 10 μm.