Figure 4.
Figure 4. PIP3-Dock2 signaling is required for F-actin accumulation in CTL–target cell conjugates. (A) OT-1 CTLs expressing nontargeting (NT) control shRNA or shRNA against Dock2 or PTEN were mixed with OVA-pulsed RMA-s cells and the conjugates imaged by confocal microscopy after fixation and staining with fluorescently labeled phalloidin. Representative images are shown on the left. Right, quantification of IS size and synaptic F-actin enrichment (see Materials and methods). (B) Fura-2AM–loaded WT and Dock2−/− 2B4 T cells were imaged on lipid bilayers containing pMHC and ICAM-1. Mean, background-corrected Fura-2AM ratios are plotted versus time for each condition (n = 12 cells). (C) Immunoblot analysis of phospho-Erk1/2 (P-Erk1/2) and phospho-AKT (P-AKT) in WT and Dock2−/− 2B4 T cells stimulated on lipid bilayers for the indicated times. Numbers denote corrected, normalized amounts of P-Erk1/2 and P-AKT. (D) WT and Dock2−/− 2B4 T cells expressing GFP-labeled tubulin were attached to glass surfaces containing immobilized, photoactivatable pMHC. Localized UV irradiation was then used to activate TCRs in a small region of membrane, and the position of the MTOC monitored by epifluorescence microscopy. The mean distance between the MTOC and the irradiated region is plotted against time (n > 20 cells), with UV irradiation indicated by the green line. Error bars denote SEM. (E) Left, representative images of phalloidin-stained conjugates formed by OVA-pulsed RMA-s cells and OT-1 CTLs expressing GFP-labeled Dock2 either in the presence of wortmannin or vehicle control (Veh). Right, quantification of synaptic Dock2 enrichment. (F) Left, representative images of phalloidin-stained conjugates formed by OVA-pulsed RMA-s cells and OT-1 CTLs in the presence of wortmannin (Wort) or vehicle control (Veh). Right, quantification of IS size and synaptic F-actin enrichment. In all images, T cells and APCs are indicated with white text. Bars, 3 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 ≤ 0.05, and ns, P > 0.05. Data in A, E, and F were pooled from two independent experiments. Data in B–D are representative of at least two independent experiments.

PIP3-Dock2 signaling is required for F-actin accumulation in CTL–target cell conjugates. (A) OT-1 CTLs expressing nontargeting (NT) control shRNA or shRNA against Dock2 or PTEN were mixed with OVA-pulsed RMA-s cells and the conjugates imaged by confocal microscopy after fixation and staining with fluorescently labeled phalloidin. Representative images are shown on the left. Right, quantification of IS size and synaptic F-actin enrichment (see Materials and methods). (B) Fura-2AM–loaded WT and Dock2−/− 2B4 T cells were imaged on lipid bilayers containing pMHC and ICAM-1. Mean, background-corrected Fura-2AM ratios are plotted versus time for each condition (n = 12 cells). (C) Immunoblot analysis of phospho-Erk1/2 (P-Erk1/2) and phospho-AKT (P-AKT) in WT and Dock2−/− 2B4 T cells stimulated on lipid bilayers for the indicated times. Numbers denote corrected, normalized amounts of P-Erk1/2 and P-AKT. (D) WT and Dock2−/− 2B4 T cells expressing GFP-labeled tubulin were attached to glass surfaces containing immobilized, photoactivatable pMHC. Localized UV irradiation was then used to activate TCRs in a small region of membrane, and the position of the MTOC monitored by epifluorescence microscopy. The mean distance between the MTOC and the irradiated region is plotted against time (n > 20 cells), with UV irradiation indicated by the green line. Error bars denote SEM. (E) Left, representative images of phalloidin-stained conjugates formed by OVA-pulsed RMA-s cells and OT-1 CTLs expressing GFP-labeled Dock2 either in the presence of wortmannin or vehicle control (Veh). Right, quantification of synaptic Dock2 enrichment. (F) Left, representative images of phalloidin-stained conjugates formed by OVA-pulsed RMA-s cells and OT-1 CTLs in the presence of wortmannin (Wort) or vehicle control (Veh). Right, quantification of IS size and synaptic F-actin enrichment. In all images, T cells and APCs are indicated with white text. Bars, 3 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 ≤ 0.05, and ns, P > 0.05. Data in A, E, and F were pooled from two independent experiments. Data in B–D are representative of at least two independent experiments.

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