Figure 2.
Figure 2. In vitro single-cell chemokinesis and chemotaxis analysis. (Ai) Normal chemokinesis in Rac1 null neutrophils but not in Rac2 null neutrophils. Chemokinesis mediated by fMLP (10-6 M) was assessed in a Zigmond chamber as described (see “Materials and methods”). There was no significant difference between Rac1 and wild-type neutrophil chemokinesis; however, Rac2 null neutrophils were significantly slower than either wild-type or Rac1 null cells (*P > .001), whereas Rac1/2 null cells were significantly slower than Rac2 null cells (**P < .01). (Aii) Chemokinesis concentration kinetics. (B) Abnormal chemotaxis speeds in both Rac2 and Rac1/2 null neutrophils. The absolute speed of neutrophils undergoing chemotaxis was calculated using the indicated chemoattractant in a Zigmond chamber. Rac2 null and Rac1/2 null cells were significantly slower than both Rac1 null and wild-type cells. (*P > .01;**P < .001). (C) Rac1 null neutrophils display a direction defect in chemotaxis plots. Plots of neutrophil chemotaxis migration in the Zigmond chamber assay are shown. Neutrophils undergoing chemotaxis in response to fMLP (10-6 M) were recorded using time-lapse imaging. Tracings were used to plot final positioning of cells after 17 minutes. Positive x values represent movement up the gradient. Note the random migration in the Rac1 null cells compared to both wild-type and Rac2 null cells. (D) Rac1 required for orientation toward chemoattractant source. Time-lapse images of chemotaxing neutrophils were used to quantify the proportion of neutrophils moving toward the chemoattractant source. Rac1 null neutrophils are unable to orient toward the source of a chemoattractant. The proportions of neutrophils exposed to a gradient of chemoattractant that are polarized toward the source (leading edge within 180° of the source) were counted for the 3 genotypes shown. A complete inability to polarize preferentially toward the source (random polarization) would be reflected with a 0.5 proportion (*P < .001, Rac1 null compared to wild-type). (E) F-actin assembly following fMLP stimulation requires Rac2. Impaired fMLP-mediated (10-6 M) actin polymerization (30 seconds after stimulation) is noted in neutrophils lacking Rac2 or both Rac1 and Rac2. Note the pronounced actin polymerization defect in Rac2 and Rac1/2 neutrophils along with the lower initial resting levels of F-actin (representative of 3 experiments; significantly different at 30 seconds between wild-type and Rac2 null, wild-type and Rac1/2 null, Rac1 null and Rac2 null, and Rac1 null and Rac1/2 null; *P < .01). Error bars represent standard error of the means.

In vitro single-cell chemokinesis and chemotaxis analysis. (Ai) Normal chemokinesis in Rac1 null neutrophils but not in Rac2 null neutrophils. Chemokinesis mediated by fMLP (10-6 M) was assessed in a Zigmond chamber as described (see “Materials and methods”). There was no significant difference between Rac1 and wild-type neutrophil chemokinesis; however, Rac2 null neutrophils were significantly slower than either wild-type or Rac1 null cells (*P > .001), whereas Rac1/2 null cells were significantly slower than Rac2 null cells (**P < .01). (Aii) Chemokinesis concentration kinetics. (B) Abnormal chemotaxis speeds in both Rac2 and Rac1/2 null neutrophils. The absolute speed of neutrophils undergoing chemotaxis was calculated using the indicated chemoattractant in a Zigmond chamber. Rac2 null and Rac1/2 null cells were significantly slower than both Rac1 null and wild-type cells. (*P > .01;**P < .001). (C) Rac1 null neutrophils display a direction defect in chemotaxis plots. Plots of neutrophil chemotaxis migration in the Zigmond chamber assay are shown. Neutrophils undergoing chemotaxis in response to fMLP (10-6 M) were recorded using time-lapse imaging. Tracings were used to plot final positioning of cells after 17 minutes. Positive x values represent movement up the gradient. Note the random migration in the Rac1 null cells compared to both wild-type and Rac2 null cells. (D) Rac1 required for orientation toward chemoattractant source. Time-lapse images of chemotaxing neutrophils were used to quantify the proportion of neutrophils moving toward the chemoattractant source. Rac1 null neutrophils are unable to orient toward the source of a chemoattractant. The proportions of neutrophils exposed to a gradient of chemoattractant that are polarized toward the source (leading edge within 180° of the source) were counted for the 3 genotypes shown. A complete inability to polarize preferentially toward the source (random polarization) would be reflected with a 0.5 proportion (*P < .001, Rac1 null compared to wild-type). (E) F-actin assembly following fMLP stimulation requires Rac2. Impaired fMLP-mediated (10-6 M) actin polymerization (30 seconds after stimulation) is noted in neutrophils lacking Rac2 or both Rac1 and Rac2. Note the pronounced actin polymerization defect in Rac2 and Rac1/2 neutrophils along with the lower initial resting levels of F-actin (representative of 3 experiments; significantly different at 30 seconds between wild-type and Rac2 null, wild-type and Rac1/2 null, Rac1 null and Rac2 null, and Rac1 null and Rac1/2 null; *P < .01). Error bars represent standard error of the means.

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