Dock2/Elmo and PIP3 localization at the IS. (A) OT-1 CTLs expressing RFP-labeled Lifeact together with GFP-labeled Elmo1 were analyzed by TIRF microscopy on lipid bilayers containing pMHC and ICAM-1. Left, representative time-lapse montages (9-s intervals). Right, linescans (derived from the white line in the left panel, time = 27 s) showing F-actin and Elmo1 accumulation. (B) OT-1 CTLs expressing GFP-fusions of either Dock2 or a Dock2 mutant lacking the DHR-1 domain (ΔDHR1) were imaged on stimulatory bilayers after fixation and staining with phalloidin. Left, representative TIRF images are shown. Right, linescans (derived from the white lines in the images to the left) showing F-actin and Dock2 accumulation at the IS. (C and D) OT-1 CTLs expressing RFP-labeled Lifeact together with GFP-labeled Grp1PH (C) or DynPH (D) were analyzed by TIRF microscopy on lipid bilayers containing pMHC and ICAM-1. Left, representative time-lapse montages (9-s intervals). Right, linescans (derived from the white line in the left panel, time = 27 s) showing F-actin and Grp1PH/Dyn1PH accumulation. Bars, 5 µm. Images in A, C, and D are representative of at least 80 cells imaged over at least four independent experiments. Images in B are representative of at least 12 cells imaged over two independent experiments.
