Limits...
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.

Show MeSH
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.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2172749&req=5

fig2: 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.

Mentions: Next, we investigated changes in the localization of LAT-GFP after TCR stimulation using confocal microscopy. LAT-GFP transfectants were stimulated with anti-CD3 antibody–coated latex beads, which have previously been reported to act as artificial antigen-presenting cells owing to their ability to induce cell polarization and reorganization of the cytoskeleton in Jurkat cells (Lowin-Kropf et al., 1998). After mixing LAT-GFP transfectants and the beads, these conjugates were fixed and LAT-GFP fluorescence was analyzed microscopically. Although LAT-GFP was homogeneously distributed at the plasma membrane in unstimulated cells, it became concentrated in distinct patches at the cell–bead interface in a large proportion of cells stimulated by the anti-CD3–coated beads (Fig. 2, A and B). When LAT-GFP transfectants were stimulated with poly-l-lysine–coated beads as a negative control, no patch formation was observed at the cell–bead interface.


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)

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.
© Copyright Policy
Related In: Results  -  Collection

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

fig2: 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.
Mentions: Next, we investigated changes in the localization of LAT-GFP after TCR stimulation using confocal microscopy. LAT-GFP transfectants were stimulated with anti-CD3 antibody–coated latex beads, which have previously been reported to act as artificial antigen-presenting cells owing to their ability to induce cell polarization and reorganization of the cytoskeleton in Jurkat cells (Lowin-Kropf et al., 1998). After mixing LAT-GFP transfectants and the beads, these conjugates were fixed and LAT-GFP fluorescence was analyzed microscopically. Although LAT-GFP was homogeneously distributed at the plasma membrane in unstimulated cells, it became concentrated in distinct patches at the cell–bead interface in a large proportion of cells stimulated by the anti-CD3–coated beads (Fig. 2, A and B). When LAT-GFP transfectants were stimulated with poly-l-lysine–coated beads as a negative control, no patch formation was observed at the cell–bead interface.

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