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Dynamic changes in the mobility of LAT in aggregated lipid rafts upon T cell activation.

Tanimura N, Nagafuku M, Minaki Y, Umeda Y, Hayashi F, Sakakura J, Kato A, Liddicoat DR, Ogata M, Hamaoka T, Kosugi A - J. Cell Biol. (2003)

Bottom Line: Photobleaching experiments using live cells revealed that LAT-GFP in patches was markedly less mobile than that in nonpatched regions.The decreased mobility in patches was dependent on raft organization supported by membrane cholesterol and signaling molecule binding sites, especially the phospholipase C gamma 1 binding site in the cytoplasmic domain of LAT.Thus, although LAT normally moves rapidly at the plasma membrane, it loses its mobility and becomes stably associated with aggregated rafts to ensure organized and sustained signal transduction required for T cell activation.

View Article: PubMed Central - PubMed

Affiliation: School of Allied Health Sciences, Faculty of Medicine, Osaka University, Suita, Japan.

ABSTRACT
Lipid rafts are known to aggregate in response to various stimuli. By way of raft aggregation after stimulation, signaling molecules in rafts accumulate and interact so that the signal received at a given membrane receptor is amplified efficiently from the site of aggregation. To elucidate the process of lipid raft aggregation during T cell activation, we analyzed the dynamic changes of a raft-associated protein, linker for activation of T cells (LAT), on T cell receptor stimulation using LAT fused to GFP (LAT-GFP). When transfectants expressing LAT-GFP were stimulated with anti-CD3-coated beads, LAT-GFP aggregated and formed patches at the area of bead contact. Photobleaching experiments using live cells revealed that LAT-GFP in patches was markedly less mobile than that in nonpatched regions. The decreased mobility in patches was dependent on raft organization supported by membrane cholesterol and signaling molecule binding sites, especially the phospholipase C gamma 1 binding site in the cytoplasmic domain of LAT. Thus, although LAT normally moves rapidly at the plasma membrane, it loses its mobility and becomes stably associated with aggregated rafts to ensure organized and sustained signal transduction required for T cell activation.

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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.
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fig5: 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.

Mentions: Because dynamics of LAT after T cell activation could be different in primary T cells compared with that in Jurkat cells, we next investigated the mobility of LAT-GFP in primary activated T cells. C57BL/6 T cells were activated with anti-CD3 plus anti-CD28 and transduced with retrovirus encoding LAT-GFP. These cells were stimulated with antibody-coated beads and LAT-GFP fluorescence was analyzed. Because a previous report demonstrated that raft aggregation was greatly influenced by costimulatory signal in primary resting T cells (Viola et al., 1999), we compared the efficiency of patch formation in cells stimulated with beads coated with anti-TCR alone or with anti-TCR plus anti-CD28. As shown in Fig. 5 (A and B), LAT-GFP patches similar to that observed in Jurkat-derived transfectants were clearly detectable when cells were stimulated with anti-TCR plus anti-CD28 beads. Although stimulation for primary T cells with anti-TCR beads alone was able to induce LAT-GFP patches qualitatively similar to those observed in anti-TCR plus anti-CD28 bead stimulation, the frequency of patch formation was clearly reduced (Fig. 5 B). This suggests that the requirements for activation of raft aggregation are different in Jurkat and primary activated T cells.


Dynamic changes in the mobility of LAT in aggregated lipid rafts upon T cell activation.

Tanimura N, Nagafuku M, Minaki Y, Umeda Y, Hayashi F, Sakakura J, Kato A, Liddicoat DR, Ogata M, Hamaoka T, Kosugi A - J. Cell Biol. (2003)

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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172749&req=5

fig5: 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.
Mentions: Because dynamics of LAT after T cell activation could be different in primary T cells compared with that in Jurkat cells, we next investigated the mobility of LAT-GFP in primary activated T cells. C57BL/6 T cells were activated with anti-CD3 plus anti-CD28 and transduced with retrovirus encoding LAT-GFP. These cells were stimulated with antibody-coated beads and LAT-GFP fluorescence was analyzed. Because a previous report demonstrated that raft aggregation was greatly influenced by costimulatory signal in primary resting T cells (Viola et al., 1999), we compared the efficiency of patch formation in cells stimulated with beads coated with anti-TCR alone or with anti-TCR plus anti-CD28. As shown in Fig. 5 (A and B), LAT-GFP patches similar to that observed in Jurkat-derived transfectants were clearly detectable when cells were stimulated with anti-TCR plus anti-CD28 beads. Although stimulation for primary T cells with anti-TCR beads alone was able to induce LAT-GFP patches qualitatively similar to those observed in anti-TCR plus anti-CD28 bead stimulation, the frequency of patch formation was clearly reduced (Fig. 5 B). This suggests that the requirements for activation of raft aggregation are different in Jurkat and primary activated T cells.

Bottom Line: Photobleaching experiments using live cells revealed that LAT-GFP in patches was markedly less mobile than that in nonpatched regions.The decreased mobility in patches was dependent on raft organization supported by membrane cholesterol and signaling molecule binding sites, especially the phospholipase C gamma 1 binding site in the cytoplasmic domain of LAT.Thus, although LAT normally moves rapidly at the plasma membrane, it loses its mobility and becomes stably associated with aggregated rafts to ensure organized and sustained signal transduction required for T cell activation.

View Article: PubMed Central - PubMed

Affiliation: School of Allied Health Sciences, Faculty of Medicine, Osaka University, Suita, Japan.

ABSTRACT
Lipid rafts are known to aggregate in response to various stimuli. By way of raft aggregation after stimulation, signaling molecules in rafts accumulate and interact so that the signal received at a given membrane receptor is amplified efficiently from the site of aggregation. To elucidate the process of lipid raft aggregation during T cell activation, we analyzed the dynamic changes of a raft-associated protein, linker for activation of T cells (LAT), on T cell receptor stimulation using LAT fused to GFP (LAT-GFP). When transfectants expressing LAT-GFP were stimulated with anti-CD3-coated beads, LAT-GFP aggregated and formed patches at the area of bead contact. Photobleaching experiments using live cells revealed that LAT-GFP in patches was markedly less mobile than that in nonpatched regions. The decreased mobility in patches was dependent on raft organization supported by membrane cholesterol and signaling molecule binding sites, especially the phospholipase C gamma 1 binding site in the cytoplasmic domain of LAT. Thus, although LAT normally moves rapidly at the plasma membrane, it loses its mobility and becomes stably associated with aggregated rafts to ensure organized and sustained signal transduction required for T cell activation.

Show MeSH