PIP3 controls Rac-dependent F-actin dynamics through Dock2/Elmo. (A) OT-1 CTLs expressing GFP-labeled Elmo1 were pretreated with wortmannin (Wort) or vehicle control (Veh) and imaged on stimulatory lipid bilayers after fixation. Left, representative TIRF images are shown. Right, quantification of Elmo1 clearance ratio (n > 40 cells). (B) Fura-2AM–loaded OT-1 CTLs pretreated with wortmannin or vehicle were imaged on lipid bilayers containing pMHC and ICAM-1. Mean, background-corrected Fura-2AM ratios are plotted versus time for each condition (n = 20 cells). Error bars denote SEM. (C) WT and Dock2−/− 2B4 T cells expressing GFP-labeled Grp1PH were imaged on lipid bilayers containing pMHC and ICAM-1 after phalloidin staining. Left, representative TIRF images are shown. Right, quantification of Grp1PH clearance ratio (n = 15 cells). (D) OT-1 CTLs were treated with wortmannin as indicated, stimulated on bilayers containing pMHC and ICAM-1, and stained with fluorescently labeled phalloidin. Left, representative TIRF images are shown. Right, quantification of cell spreading and F-actin clearance ratio (n ≥ 30 cells). (E) OT-1 CTLs pretreated with wortmannin as indicated were stimulated with beads coated with pMHC and ICAM-1. Cell extracts were incubated with GST-PAK1-PBD and activation of Rac was determined by immunoblot. Numbers denote corrected, normalized amounts of activated Rac. (F) CTLs expressing GFP-labeled V12Rac1 or GFP alone were treated with wortmannin as indicated and then stimulated and imaged as described in D. Quantification of cell spreading is shown (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. ***, P < 0.001, **, P < 0.01, *, P ≤ 0.05, and ns, P > 0.05. All data are representative of at least two independent experiments.
