Figure 3.
Figure 3. Dock2 drives F-actin dynamics at the IS through Rac. (A) 2B4 T cell blasts from Dock2+/+ (WT) and Dock2−/− mice were stimulated for the indicated times with beads coated with pMHC and ICAM-1. Cell extracts were incubated with GST-PAK1-PBD, and activation of Rac analyzed by immunoblot. Numbers denote corrected, normalized amounts of activated Rac in each lane, quantified from high and low film exposures, as indicated. (B) WT and Dock2−/− 2B4 T cells were stimulated on bilayers containing pMHC and ICAM-1, fixed, and stained with fluorescently labeled phalloidin. Representative TIRF images are shown (top). Bottom, quantification of cell spreading and F-actin clearance ratio (n = 30 cells). (C) Dock2−/− 2B4 T cells expressing GFP-labeled Dock2 or GFP alone were stained with phalloidin after spreading on stimulatory bilayers. Representative TIRF images are shown (top). Bottom, quantification of cell spreading (n ≥ 30 cells). (D and E) OT-1 CTLs were transduced with nontargeting control shRNA (shNT) or shRNA specific for Dock2. (D) Transduced cells were stimulated with beads coated with pMHC and ICAM-1, and Rac activation was analyzed by pulldown assay using GST-PAK1-PBD. Numbers denote corrected, normalized amounts of activated Rac. (E) Transduced cells were stimulated on lipid bilayers containing pMHC and ICAM-1, fixed, and stained with fluorescently labeled phalloidin. Representative TIRF images are shown (left). Middle, quantification of cell spreading and F-actin clearance ratio (n ≥ 30 cells). Right, transduced cells were probed for Dock2 by immunoblot. Numbers denote corrected, normalized amounts of Dock2. (F and G) Dock2-deficient 2B4 T cells (F) and shDock2-expressing OT-1 CTLs (G) were transduced with GFP-labeled V12Rac1 or GFP alone, stimulated as described in B, and imaged after phalloidin staining. Quantification of cell spreading and F-actin clearance ratio are shown (n > 30 cells for Dock2−/− experiment; n ≥ 28 cells for shDock2 experiment). 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.

Dock2 drives F-actin dynamics at the IS through Rac. (A) 2B4 T cell blasts from Dock2+/+ (WT) and Dock2−/− mice were stimulated for the indicated times with beads coated with pMHC and ICAM-1. Cell extracts were incubated with GST-PAK1-PBD, and activation of Rac analyzed by immunoblot. Numbers denote corrected, normalized amounts of activated Rac in each lane, quantified from high and low film exposures, as indicated. (B) WT and Dock2−/− 2B4 T cells were stimulated on bilayers containing pMHC and ICAM-1, fixed, and stained with fluorescently labeled phalloidin. Representative TIRF images are shown (top). Bottom, quantification of cell spreading and F-actin clearance ratio (n = 30 cells). (C) Dock2−/− 2B4 T cells expressing GFP-labeled Dock2 or GFP alone were stained with phalloidin after spreading on stimulatory bilayers. Representative TIRF images are shown (top). Bottom, quantification of cell spreading (n ≥ 30 cells). (D and E) OT-1 CTLs were transduced with nontargeting control shRNA (shNT) or shRNA specific for Dock2. (D) Transduced cells were stimulated with beads coated with pMHC and ICAM-1, and Rac activation was analyzed by pulldown assay using GST-PAK1-PBD. Numbers denote corrected, normalized amounts of activated Rac. (E) Transduced cells were stimulated on lipid bilayers containing pMHC and ICAM-1, fixed, and stained with fluorescently labeled phalloidin. Representative TIRF images are shown (left). Middle, quantification of cell spreading and F-actin clearance ratio (n ≥ 30 cells). Right, transduced cells were probed for Dock2 by immunoblot. Numbers denote corrected, normalized amounts of Dock2. (F and G) Dock2-deficient 2B4 T cells (F) and shDock2-expressing OT-1 CTLs (G) were transduced with GFP-labeled V12Rac1 or GFP alone, stimulated as described in B, and imaged after phalloidin staining. Quantification of cell spreading and F-actin clearance ratio are shown (n > 30 cells for Dock2−/− experiment; n ≥ 28 cells for shDock2 experiment). 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.

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