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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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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.
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fig3: 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.

Mentions: To determine whether sites of LAT-GFP patch formation corresponded with sites of raft aggregation, colocalization of molecules previously reported as raft markers to LAT-GFP patches was examined. Cholera toxin B (CTx-B) specifically binds to glycosphingolipids with a strong affinity for ganglioside GM1 (GM1), which is enriched in membrane rafts. Hence, CTx-B was used to test whether GM1 colocalized with LAT-GFP patch sites. In addition to GM1, the expression pattern of CD59, CD45, CD43, Lck, and F-actin was also investigated. As shown in Fig. 3, GM1 and Lck were found to clearly colocalize with LAT-GFP patches. In addition, we observed an accumulation of F-actin in LAT-GFP patches. Previously, it was reported that raft patches formed by CD59 and GM1 accumulate F-actin (Harder and Simons, 1999). In contrast, CD43 and CD45, large glycoproteins that have been shown to be excluded from rafts (Rodgers and Rose, 1996; Janes et al., 1999; Allenspach et al., 2001), were mostly not colocalized with LAT-GFP patches. Although CD43 was distributed away from the cell–bead contact site, CD45 was partially expressed on the plasma membrane surrounding the beads. CD59, a glycosylphosphatidylinositol-anchored protein, could be used as another raft marker. CD59 formed patches not only at the bead interface, but also at the plasma membrane outside the cell–bead contact site in response to stimulation with anti-CD3–coated beads. In most cells, CD59 patches at the bead interface were colocalized with LAT-GFP patches as shown in Fig. 3, but there existed a certain number of cells that did not accumulate CD59 at the site of stimulation (unpublished data). Together, these results indicate that LAT-GFP patches have characteristics specific for aggregated lipid rafts.


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)

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.
© Copyright Policy
Related In: Results  -  Collection

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

fig3: 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.
Mentions: To determine whether sites of LAT-GFP patch formation corresponded with sites of raft aggregation, colocalization of molecules previously reported as raft markers to LAT-GFP patches was examined. Cholera toxin B (CTx-B) specifically binds to glycosphingolipids with a strong affinity for ganglioside GM1 (GM1), which is enriched in membrane rafts. Hence, CTx-B was used to test whether GM1 colocalized with LAT-GFP patch sites. In addition to GM1, the expression pattern of CD59, CD45, CD43, Lck, and F-actin was also investigated. As shown in Fig. 3, GM1 and Lck were found to clearly colocalize with LAT-GFP patches. In addition, we observed an accumulation of F-actin in LAT-GFP patches. Previously, it was reported that raft patches formed by CD59 and GM1 accumulate F-actin (Harder and Simons, 1999). In contrast, CD43 and CD45, large glycoproteins that have been shown to be excluded from rafts (Rodgers and Rose, 1996; Janes et al., 1999; Allenspach et al., 2001), were mostly not colocalized with LAT-GFP patches. Although CD43 was distributed away from the cell–bead contact site, CD45 was partially expressed on the plasma membrane surrounding the beads. CD59, a glycosylphosphatidylinositol-anchored protein, could be used as another raft marker. CD59 formed patches not only at the bead interface, but also at the plasma membrane outside the cell–bead contact site in response to stimulation with anti-CD3–coated beads. In most cells, CD59 patches at the bead interface were colocalized with LAT-GFP patches as shown in Fig. 3, but there existed a certain number of cells that did not accumulate CD59 at the site of stimulation (unpublished data). Together, these results indicate that LAT-GFP patches have characteristics specific for aggregated lipid rafts.

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