PIP3-dependent synaptic remodeling is largely mediated by PI3Kδ. (A and B) OT-1 CTLs were pretreated with isoform-specific class IA PI3K inhibitors (alone or in combination) or vehicle controls (Veh) and then stimulated for the indicated times on lipid bilayers containing pMHC and ICAM-1. (A) P-AKT levels were assessed by immunoblot. High and low film exposures are shown. Numbers denote corrected, normalized amounts of P-AKT. (B) Cell spreading and F-actin clearance ratio were assessed after phalloidin-staining (n = 40 cells). Representative TIRF images are shown above, with quantification below. (C) OT-1 CTLs were pretreated with AS252424 (AS25) or vehicle control (Veh) and then stimulated and imaged as described in B. Quantification of cell spreading and F-actin clearance ratio are shown (n > 30 cells). (D) OT-1 CTLs expressing shRNA against p110α together with p110δ or nontargeting control shRNA (shNT) were stimulated and imaged as described in B. Left, transduced cells were probed for p110α and p110δ by immunoblot. Numbers denote corrected, normalized amounts of p110α and p110δ. Right, quantification of cell spreading and F-actin clearance ratio (n ≥ 33 cells). (E) Top, OT-1 CTLs expressing RFP-tagged CD3ζ together with the indicated GFP-labeled PI3K regulatory subunits were fixed on stimulatory bilayers. Top left, representative TIRF images are shown. Top right, linescans (derived from the white lines shown on the TIRF images) showing colocalization between CD3ζ and the PI3K regulatory subunits. Bottom, representative TIRF images of OT-1 CTLs expressing the indicated GFP-labeled PI3K regulatory subunits after fixation on stimulatory bilayers. (F) OT-1 CTLs expressing RFP-labeled CD3ζ together with GFP-labeled Grp1PH were imaged by TIRF microscopy on bilayers containing pMHC and ICAM-1. A representative time-lapse montage is shown (12-s intervals). 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. Data in A–D are representative of at least two independent experiments. Images in E and F are representative of at least 17 cells imaged over at least two independent experiments.
