Fig. 5. LAT is required for its own TCR-induced recruitment to the immune synapse. (A) Transiently transfected LATWT-, LATCt- and LATY/F-GFP Jurkat cells were added to CellTracker^TM Orange-labelled APCs for 3 minutes. Cells were then fixed and analysed by confocal microscopy. Histograms depict the percentage of T cells, after 3 minutes or 7 minutes of contact with SEE-pulsed APCs, showing either a clear clustering of LATWT-GFP (left panel) or a polarization of the intracellular LAT-containing compartment towards the APC (right panel). Quantification was performed on more than 35 conjugates for each LAT construct. Results of one representative experiment out of two are shown. Bar, 5 µm. (B) GFP fluorescence of LATWT-, LATCt-, LATY/F- and LAT32-104-GFP in JCAM2.5 cells interacting with SEE-pulsed Raji B cells. JCAM2.5 transiently reconstituted with the four LAT constructs were added to CMTMR-labelled (red) Raji B cells and shortly centrifuged to synchronize the contacts. After 8 minutes, cells were fixed and analysed by confocal microscopy. White arrowheads point to the site of initial contact and white stars show the LAT-containing intracellular compartment when visible. (C) Histograms depict the percentage of T cells showing either a clear clustering of LATWT-GFP (grey) or a polarization of the intracellular LAT-containing compartment towards the APC (black). Quantification was performed blindly on 15 conjugates for each LAT construct. Black star indicates the weak intracellular expression of LAT32-104-GFP, such that polarization could not be properly assessed. Bar, 5 µm.