FIGURE 3.
Recruitment of THEMIS to the IS is mediated via GRB2. (A) GRB2 association to murine THEMIS wt and PRR1 mutant (P557/560A) GFP constructs in HEK293 cells. Data are representative of three independent experiments. (B and C) Live-cell imaging of conjugates between 5C.C7 T cells (MCC82-103 in context of I-Ek) transduced with muTHEMIS-GFP (B) or muTHEMIS-PRR1-GFP (C) with MCC-pulsed (10 μM) CH27 B cells. Time point zero marks the formation of a tight cell contact. Differential interference contrast images are shown in the top rows, with top-down, maximum projections of three-dimensional fluorescence data in the bottom rows (intensities in rainbow-like false-color scale). The graphs display the accumulation of GFP sensors with the indicated patterns expressed as percentages of the total number of tight cell couples formed over time (see Supplemental Fig. 2 for description of the individual patterns). Forty-three and 54 couples were analyzed for THEMIS-wt-GFP and THEMIS-PRR1-GFP, respectively.