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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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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.
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fig4: 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.

Mentions: Next, we analyzed the change in mobility of LAT-GFP in live cells using the FRAP technique. After stimulation of LAT-GFP transfectants with anti-CD3–coated beads, a single photobleach was applied to areas of patch formation and the fluorescence recovery into these bleached regions was monitored. As shown in Fig. 4 A, fluorescence in a LAT-GFP patch was completely lost immediately after photobleaching, and little recovery was observed even 5 min after photobleaching. It should be noted that another LAT-GFP patch just below the bleached area was constantly observed during this experiment. This excludes the possibility that lack of fluorescence recovery in the bleached region was due to a movement of the cell, by itself. In contrast to LAT-GFP in patch areas, fluorescence of LAT-GFP in the plasma membrane outside the cell–bead interface was quickly recovered (Fig. 4 A). Whether we photobleached patch areas or the plasma membrane (nonpatch) areas, fluorescence recovered to a plateau level between 1 to 2 min after the initial bleach. However, although LAT-GFP fluorescence in the nonpatch area returned to ∼70% of total fluorescence, recovery in patch areas was limited to only 20% (Fig. 4 B). The recovery of LAT-GFP fluorescence in the plasma membrane from cells without stimulation was almost equivalent to that seen for bleaching of the plasma membrane from cells with bead stimulation (unpublished data). These results suggest that LAT localized in areas of raft aggregation has a remarkably low mobility compared with that in the plasma membrane outside aggregated rafts. LAT that does not accumulate in aggregated rafts maintains its rapid mobility irrespective of 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)

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.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172749&req=5

fig4: 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.
Mentions: Next, we analyzed the change in mobility of LAT-GFP in live cells using the FRAP technique. After stimulation of LAT-GFP transfectants with anti-CD3–coated beads, a single photobleach was applied to areas of patch formation and the fluorescence recovery into these bleached regions was monitored. As shown in Fig. 4 A, fluorescence in a LAT-GFP patch was completely lost immediately after photobleaching, and little recovery was observed even 5 min after photobleaching. It should be noted that another LAT-GFP patch just below the bleached area was constantly observed during this experiment. This excludes the possibility that lack of fluorescence recovery in the bleached region was due to a movement of the cell, by itself. In contrast to LAT-GFP in patch areas, fluorescence of LAT-GFP in the plasma membrane outside the cell–bead interface was quickly recovered (Fig. 4 A). Whether we photobleached patch areas or the plasma membrane (nonpatch) areas, fluorescence recovered to a plateau level between 1 to 2 min after the initial bleach. However, although LAT-GFP fluorescence in the nonpatch area returned to ∼70% of total fluorescence, recovery in patch areas was limited to only 20% (Fig. 4 B). The recovery of LAT-GFP fluorescence in the plasma membrane from cells without stimulation was almost equivalent to that seen for bleaching of the plasma membrane from cells with bead stimulation (unpublished data). These results suggest that LAT localized in areas of raft aggregation has a remarkably low mobility compared with that in the plasma membrane outside aggregated rafts. LAT that does not accumulate in aggregated rafts maintains its rapid mobility irrespective of 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