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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(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.
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fig7: 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.

Mentions: The cytoplasmic domain of LAT has previously been reported to be essential for the interaction of LAT with other signaling molecules (Zhang et al., 2000). To investigate whether LAT-GFP patch formation after TCR stimulation is dependent on the cytoplasmic domain of LAT, and hence, interactions between LAT and other signaling molecules, Jurkat-derived transfectants expressing a fusion protein consisting of the 36 amino acid NH2-terminal transmembrane domain of LAT fused to GFP, LAT(TM)-GFP, were established. Although this fusion protein contains the two Cys residues required for LAT palmitoylation, it does not contain multiple tyrosine residues that are indispensable for interaction with other proteins. After stimulation by anti-CD3–coated beads, LAT(TM)-GFP transfectants were observed by confocal microscopy and scored for patch formation. The extent of patch formation at the cell–bead interface in LAT(TM)-GFP transfectants was comparable to that in LAT-GFP transfectants (Fig. 7, A and B). Moreover, the size of patches was similar in both LAT- and LAT(TM)-GFP transfectants. Thus, these results clearly demonstrate that the presence of transmembrane domain of LAT is sufficient for accumulation of LAT into aggregated rafts in response to TCR stimulation.


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(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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172749&req=5

fig7: 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.
Mentions: The cytoplasmic domain of LAT has previously been reported to be essential for the interaction of LAT with other signaling molecules (Zhang et al., 2000). To investigate whether LAT-GFP patch formation after TCR stimulation is dependent on the cytoplasmic domain of LAT, and hence, interactions between LAT and other signaling molecules, Jurkat-derived transfectants expressing a fusion protein consisting of the 36 amino acid NH2-terminal transmembrane domain of LAT fused to GFP, LAT(TM)-GFP, were established. Although this fusion protein contains the two Cys residues required for LAT palmitoylation, it does not contain multiple tyrosine residues that are indispensable for interaction with other proteins. After stimulation by anti-CD3–coated beads, LAT(TM)-GFP transfectants were observed by confocal microscopy and scored for patch formation. The extent of patch formation at the cell–bead interface in LAT(TM)-GFP transfectants was comparable to that in LAT-GFP transfectants (Fig. 7, A and B). Moreover, the size of patches was similar in both LAT- and LAT(TM)-GFP transfectants. Thus, these results clearly demonstrate that the presence of transmembrane domain of LAT is sufficient for accumulation of LAT into aggregated rafts in response to TCR stimulation.

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