Rac1 and Rac2 are essential for synaptic F-actin remodeling. (A) OT-1 CTLs were stimulated for the indicated times with beads coated with pMHC and ICAM-1. Top, active Rac was isolated using GST-PAK1-PBD and visualized by immunoblot. Bottom, quantification of activated Rac from the blot shown above, corrected using total Rac expression and normalized to the first time point. (B) OT-1 CTLs were transduced with nontargeting control shRNA (shNT) or shRNA specific for Rac1 and/or Rac2. Cells were then stimulated on lipid bilayers containing pMHC and ICAM-1, fixed, and stained with fluorescently labeled phalloidin. Representative TIRF images are shown (left). Cell spreading (top graph) and F-actin clearance ratio (bottom graph) were quantified for each condition (n = 20 cells). (C) shRNA-transduced OT-1 CTLs were probed for Rac1 and Rac2 by immunoblot. Numbers denote corrected, normalized amounts of Rac1 and Rac2 in each lane (see Materials and methods). (D) OT-1 CTLs were transduced with the indicated shRNAs together with shRNA-resistant, GFP-labeled Rac1 or Rac2 as shown. Transduced cells were stimulated as described in B, and F-actin was visualized by phalloidin staining. Quantification of cell spreading and F-actin clearance ratio is shown (n ≥ 37 cells). (E) OT-1 CTLs expressing GFP-labeled V12Rac1 or GFP alone were stimulated as described in B, and F-actin was analyzed by phalloidin staining. Representative TIRF images are shown (left). Right, quantification of cell spreading and F-actin clearance ratio (n = 30 cells). Bars, 5 µm. In scatter plots, red lines and error bars denote the mean and SEM, respectively. P-values were calculated using the Mann-Whitney test (two-tailed). ***, P < 0.001, **, P < 0.01, *, P ≤ 0.05, and ns, P > 0.05. All data are representative of at least two independent experiments.