F-actin assembly, Cdc42 activation levels, and neutrophil morphology during polarization. (A) Rac1 null neutrophils display double leading lamellas. F-actin staining is shown in chemotaxing neutrophils. Wild-type and Rac2 null cells present with a characteristic single prominent leading edge of polymerization, whereas the Rac1 null cells showed a significant proportion of neutrophils with 2 distinct leading edges. Double null cells showed no polarization. (B) Increased proportion of Rac1 null neutrophils display more than one leading edge compared with Rac2 null and wild-type neutrophils. The proportion of neutrophils for each genotype with 2 prominent leading edges was calculated. Rac1 null neutrophils demonstrated a significantly higher likelihood of having 2 leading edges (*P < .0001). (C) fMLP-mediated Cdc42 activation neutrophils required Rac2 and not Rac1. Using the p21 PBD assay,20 we demonstrate that fMLP-mediated Cdc42 activation increases in a similar pattern in wild-type and Rac1 null neutrophils but is severely perturbed in Rac2 null neutrophils. Rac2 is significantly different from wild-type and Rac1 (*P < .04). (D) Perturbed tail retraction in Rac1 null neutrophils is shown in this representative photomicrograph of neutrophils in an fMLP gradient. It is clearly noted that Rac1 null neutrophils displayed poor tail retraction compared with wild-type neutrophils. Error bars represent standard error of the means.
