Figure 2.
Figure 2. Rac1 regulates Rho activation. (A) Rac1 increases RhoA activity in human neutrophils. Constitutively active or dominant-negative Rac1 mutants were expressed in human neutrophils, stimulated with 1 μM fMLP, fixed, and stained with phalloidin (green) and the Rhotekin RBD (red), as described in “Materials and methods.” Rhotekin RBD mutated at R37,39A,D40A was used as an inactive control and showed only background staining (not shown). Fluorescence intensities of more than 300 cells from at least 3 samples were measured for each treatment. Error bars show standard error, and significant differences (at least P < .05) are indicated by asterisks. Cells were treated with 20μM aluminum fluoride (AIF4) for 15 minutes as a positive control for Rho activation. (B) Distribution of active Rho from front to back in human neutrophils. Rhotekin RBD staining was mostly confined to the uropod region in control cells following fMLP stimulation. The intensity of Rho activation was greater in the uropod of cells treated with Rac1G12V, and substantially less intense in Rac1T17N-treated cells. Linescans of control (vector treated), Rac1G12V-, and Rac1T17N-expressing cells reveal significant differences in levels of active Rho along the length of these cells, particularly at the rear, compared with controls. Fluorescence intensities were measured from the leading edge to the tail, with cell lengths normalized to 100% to allow comparison. Results shown are the mean ± standard error of at least 30 cells per condition. (C) Rac1-null mouse neutrophils fail to activate Rho GTPase following fMLP stimulation. FMLP-mediated Rho activation in murine neutrophils requires Rac1 but not Rac2. Using the Rhotekin assay (“Materials and methods”), we demonstrate that in cells stimulated globally with 1 μM fMLP, RhoA activation increases in a similar pattern in wild-type and Rac2-null neutrophils over 600 seconds, but is severely perturbed in Rac1-null neutrophils. Rac1 is significantly different from wild-type and Rac2 for all time points greater than 100 seconds (P < .01).

Rac1 regulates Rho activation. (A) Rac1 increases RhoA activity in human neutrophils. Constitutively active or dominant-negative Rac1 mutants were expressed in human neutrophils, stimulated with 1 μM fMLP, fixed, and stained with phalloidin (green) and the Rhotekin RBD (red), as described in “Materials and methods.” Rhotekin RBD mutated at R37,39A,D40A was used as an inactive control and showed only background staining (not shown). Fluorescence intensities of more than 300 cells from at least 3 samples were measured for each treatment. Error bars show standard error, and significant differences (at least P < .05) are indicated by asterisks. Cells were treated with 20μM aluminum fluoride (AIF4) for 15 minutes as a positive control for Rho activation. (B) Distribution of active Rho from front to back in human neutrophils. Rhotekin RBD staining was mostly confined to the uropod region in control cells following fMLP stimulation. The intensity of Rho activation was greater in the uropod of cells treated with Rac1G12V, and substantially less intense in Rac1T17N-treated cells. Linescans of control (vector treated), Rac1G12V-, and Rac1T17N-expressing cells reveal significant differences in levels of active Rho along the length of these cells, particularly at the rear, compared with controls. Fluorescence intensities were measured from the leading edge to the tail, with cell lengths normalized to 100% to allow comparison. Results shown are the mean ± standard error of at least 30 cells per condition. (C) Rac1-null mouse neutrophils fail to activate Rho GTPase following fMLP stimulation. FMLP-mediated Rho activation in murine neutrophils requires Rac1 but not Rac2. Using the Rhotekin assay (“Materials and methods”), we demonstrate that in cells stimulated globally with 1 μM fMLP, RhoA activation increases in a similar pattern in wild-type and Rac2-null neutrophils over 600 seconds, but is severely perturbed in Rac1-null neutrophils. Rac1 is significantly different from wild-type and Rac2 for all time points greater than 100 seconds (P < .01).

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