TCR-mediated patch formation of LAT-GFP. (A) LAT-GFP transfectants were mixed at a 2:1 ratio with poly-l-lysine–, anti-CD28–, or anti-CD3–coated latex beads. After 20 min at 37°C, conjugates were fixed with formaldehyde and observed by confocal microscopy. In the left column are bright-field images, in the middle column are LAT-GFP fluorescence images, and the right column contains the merged image of left and middle. LAT-GFP patches at the contact site between the cell and the bead are indicated by arrows. (B) Patch formation of LAT-GFP is specifically induced by TCR/CD3 cross-linking. LAT-GFP transfectants were mixed for 20 min with beads coated with either poly-l-lysine or antibodies against CD3, CD28, LFA-1, or CD44. Each column represents the average of at least three individual experiments in which more than 100 conjugates were scored for patch formation. Cells that incorporated or attached to beads were scored as conjugates. Cells that showed at least one distinct patch at the bead contact site were scored as positive for patch formation. (C) LAT-GFP transfectants were mixed with anti-CD3 beads at 37°C for the times indicated. Each column represents the average of at least three individual experiments.

LAT-GFP localized in patches is less mobile than that in nonpatched regions of the plasma membrane. (A) After LAT-GFP transfectants were mixed with anti-CD3 beads for 20 min, a selected area (2-μm square) on the LAT-GFP patches or LAT-GFP in the plasma membrane (PM) was photobleached, and fluorescence recovery was monitored. Images at representative time points are shown. (B) Bleaching recovery kinetics is represented as the percentage of FRAP for LAT-GFP in patches and that in the plasma membrane. Data are representative of five individual experiments.

The effect of cholesterol depletion on the mobility of LAT-GFP in patches. (A) Cholesterol in Jurkat cells treated with or without 4 mM MβCD for 40 min was extracted, separated with HPTLC, and visualized with 3% cupric acetate/8% phosphoric acid. (B) Jurkat cells treated with or without MβCD were stimulated with OKT3 for the times indicated. Tyrosine phosphorylation of cellular proteins was analyzed by immunoblotting using anti-PY antibody. (C) Jurkat cells were transiently transfected with an NFAT-luc reporter plasmid, treated with MβCD for 40 min, washed, stimulated with OKT3 or PMA plus ionomycin (P+I) in conditioned culture medium without FCS for 6 h, and then tested for NFAT activity by NFAT reporter assay. (D) After LAT-GFP transfectants were pretreated for 40 min with 4 mM MβCD, the cells were stimulated with poly- l-lysine beads or anti-CD3 beads for 20 min in the presence of 4 mM MβCD. Conjugates were fixed with formaldehyde and observed by confocal microscopy. (E) A selected area (2-μm square) on the LAT-GFP patches in LAT-GFP transfectants treated with or without MβCD was photobleached, and fluorescence recovery was monitored. (F) Bleaching recovery kinetics is represented as the percentage of FRAP for LAT-GFP in patches in the presence or absence of MβCD. Data are representative of three individual experiments.

The PLCγ1 binding site of LAT is important for the reduced mobility of LAT in aggregated rafts. (A) Schematic representations of the LAT-GFP, LAT(Y136F)-GFP, and LAT(4YF)-GFP fusion proteins. A LAT-GFP chimera was constructed by attaching EGFP to the full length of mouse LAT. Y136 is a binding site for PLCγ1, and Y175, Y195, and Y235 are binding sites for Grb2/Gads in mouse LAT. Mutated amino acids are shown for each construct. (B) Association of LAT-GFP with PLCγ1 or Grb2. The LAT-GFP, LAT(Y136F)-GFP, and LAT(4YF)-GFP transfectants were stimulated with OKT3 for 5 min. Cell lysates were immunoprecipitated with either anti-PLCγ1 or anti-Grb2 antibody, and immunoprecipitates were analyzed by immunoblotting with anti-GFP and antibodies specific for the immunoprecipitated protein. (C) After the LAT-GFP, LAT(Y136F)-GFP, and LAT(4YF)-GFP transfectants were mixed with anti-CD3 beads for 20 min, a selected area (2-μm square) on the patches was photobleached, and fluorescence recovery was monitored. Images at representative time points are shown. (D) Bleaching recovery kinetics is represented as the percentage of FRAP in patches. Data are representative of three individual experiments.

Characterization of the LAT-GFP fusion protein used in this work. (A) Localization of LAT-GFP in rafts from LAT-GFP–transfected Jurkat cells. LAT-GFP transfectants were lysed with MBS containing 1% Triton X-100, and the lysates were subjected to equilibrium gradient centrifugation. An aliquot of each fraction was electrophoresed and immunoblotted with HRP-conjugated CTx-B and anti-GFP antibody to detect GM1 and LAT-GFP, respectively. Fractions 4 and 5 correspond to the raft fractions. (B) Tyrosine phosphorylation of LAT-GFP after TCR cross-linking. The LAT-GFP transfectants were stimulated with OKT3 for the times indicated. Cell lysates were immunoprecipitated with anti-LAT antibody, and immunoprecipitates were analyzed by immunoblotting with anti-PY and anti-LAT antibodies. The closed arrowhead indicates LAT-GFP, whereas open arrowheads indicate endogenous LAT. The band migrating at 55 kD in each lane is the heavy chain (H) of the antibody used for immunoprecipitation.

Colocalization of raft markers with LAT-GFP at the site of patch formation. LAT-GFP transfectants were mixed with anti-CD3 beads for 20 min. Conjugates were fixed with formaldehyde, permeabilized, and stained with biotinylated CTx-B to detect GM1 or biotinylated anti-Lck, followed by incubation with SA-TR or with phalloidin-TRITC to detect F-actin. With regard to the staining for CD43, CD45, and CD59, conjugates were stained with biotinylated antibodies against CD43, CD45, and CD59 without permeabilization, followed by incubation with SA-TR, and were then fixed. Conjugates were observed by confocal microscopy. Single confocal sections show fluorescence in GFP and Texas red channels.

The mobility of LAT-GFP in patches in primary activated T cells. Primary activated T cells transduced with LAT-GFP were stimulated with antibody-coated beads and LAT-GFP fluorescence was analyzed. (A and B) Primary activated T cells were stimulated with poly-l-lysine–, anti-CD28 (PV1)–, anti-TCRβ (H57)–, or anti-TCRβ plus anti-CD28 (H57+PV-1)–coated beads. After 20 min at 37°C, conjugates were fixed with formaldehyde and observed by confocal microscopy. In the left column are differential interference contrast (DIC) images, and in the left column are LAT-GFP fluorescence images. Each column represents the average of at least three individual experiments in which more than 100 conjugates were scored for patch formation. (C) A selected area (2-μm square) on the LAT-GFP patches or LAT-GFP in the plasma membrane (PM) was photobleached, and fluorescence recovery was monitored. Images at representative time points are shown. (D) Bleaching recovery kinetics is represented as the percentage of FRAP for LAT-GFP in patches and that in the plasma membrane. Data are representative of three individual experiments.

The mobility of LAT(TM)-GFP in patches after TCR stimulation. Jurkat-derived transfectants expressing LAT-GFP or LAT(TM)-GFP were mixed with anti-CD3 beads for 20 min. (A) Conjugates were fixed with formaldehyde and observed by confocal microscopy. In the left column are differential interference contrast (DIC) images, and in the left column are LAT-GFP fluorescence images. LAT-GFP and LAT(TM)-GFP patches were observed at the contact site between the cell and the bead. (B) Each column represents the average of at least three individual experiments in which more than 100 conjugates were scored for patch formation. (C) A selected area (2-μm square) on the LAT/GFP or LAT(TM)-GFP patches was photobleached, and fluorescence recovery was monitored. Images at representative time points are shown. (D) Bleaching recovery kinetics is represented as the percentage of FRAP for LAT-GFP and LAT(TM)-GFP in patches. Data are representative of five individual experiments.