JCS00968 Supplemental Data

Files in this Data Supplement:

• Figure 1 -

  Fig. S1. Intracellular localization of GM-1 and signaling molecules in comparison to LAT distribution. Wild-type resting Jurkat T cells were fixed with PFA, permeabilized and processed for immunofluorescence labeling (see Materials and Methods). Co-labelings were performed with anti-LAT Abs and either (A) FITC-conjugated cholera toxin to detect the GM-1 ganglioside or (B) a mouse anti-p56Lck mAb, or (C) a mouse anti-PKC-q mAb or (D) a mouse anti-ZAP-70 mAb. Rabbit anti-LAT Abs and the mouse mAbs were detected respectively by Cy3-conjugated anti-rabbit and Alexa488-conjugated anti-mouse Abs. Images were acquired on a confocal microscope. Absence of cross-reactivity of the secondary Abs was carefully controlled and bleedthrough was avoided by sequential scanning of the samples. Insets show the 3-fold magnified region comprised in the white box. Although the expression pattern of LAT, GM-1 and Lck were similar (intracellular compartment and plasma membrane), the co-distribution was only partial. White arrows point to co-localization areas whereas white arrowheads show vesicles where only LAT is found. Co-localization of LAT and PKC-q or ZAP-70 is clearly not detectable since both these signaling effectors are cytosolic in resting cells. Scale bar : 10 mm.

• Movie 1A
• Movie 1B -

  Movie 1. Jurkat T cells transiently transfected either with the LATwt-GFP (Movie 1A) or the LATY/F-GFP (Movie 1B) plasmids were plated onto poly-lysine-coated coverslips and placed at 37°C in the chamber of a videomicroscope. Raji B cells, labeled with 0.5 mM of Cell Tracker Orange (CMTMR) and pulsed with 5 mg/ml SEE for 20 minutes were added at a 1:1 ratio. Every 15 seconds, one image with the rhodamine filter set and a stack of 8 z-planes (step : 1 mm) with the GFP filter set were acquired. Movies show the overlay of the CMTMR labeling in red and the projection of the GFP sections in green. Time in minutes and seconds is indicated, films are accelerated 75´.