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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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Characterization of the LAT-GFP fusion protein used in this work. (A) Localization of LAT-GFP in rafts from LAT-GFP–transfected Jurkat cells. LAT-GFP transfectants were lysed with MBS containing 1% Triton X-100, and the lysates were subjected to equilibrium gradient centrifugation. An aliquot of each fraction was electrophoresed and immunoblotted with HRP-conjugated CTx-B and anti-GFP antibody to detect GM1 and LAT-GFP, respectively. Fractions 4 and 5 correspond to the raft fractions. (B) Tyrosine phosphorylation of LAT-GFP after TCR cross-linking. The LAT-GFP transfectants were stimulated with OKT3 for the times indicated. Cell lysates were immunoprecipitated with anti-LAT antibody, and immunoprecipitates were analyzed by immunoblotting with anti-PY and anti-LAT antibodies. The closed arrowhead indicates LAT-GFP, whereas open arrowheads indicate endogenous LAT. The band migrating at 55 kD in each lane is the heavy chain (H) of the antibody used for immunoprecipitation.
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fig1: Characterization of the LAT-GFP fusion protein used in this work. (A) Localization of LAT-GFP in rafts from LAT-GFP–transfected Jurkat cells. LAT-GFP transfectants were lysed with MBS containing 1% Triton X-100, and the lysates were subjected to equilibrium gradient centrifugation. An aliquot of each fraction was electrophoresed and immunoblotted with HRP-conjugated CTx-B and anti-GFP antibody to detect GM1 and LAT-GFP, respectively. Fractions 4 and 5 correspond to the raft fractions. (B) Tyrosine phosphorylation of LAT-GFP after TCR cross-linking. The LAT-GFP transfectants were stimulated with OKT3 for the times indicated. Cell lysates were immunoprecipitated with anti-LAT antibody, and immunoprecipitates were analyzed by immunoblotting with anti-PY and anti-LAT antibodies. The closed arrowhead indicates LAT-GFP, whereas open arrowheads indicate endogenous LAT. The band migrating at 55 kD in each lane is the heavy chain (H) of the antibody used for immunoprecipitation.

Mentions: A fusion gene was constructed consisting of GFP linked to the COOH-terminal of the entire LAT gene coding sequence. The LAT-GFP fusion gene was then transfected into Jurkat cells and stable transfectants were established. Previously, it has been shown that LAT localizes to rafts by way of palmitoylation of two Cys residues in its juxtamembrane region (Zhang et al., 1998b). To investigate whether LAT-GFP localized to rafts as per endogenous LAT, LAT-GFP transfectants were solubilized and the raft fraction was purified using a sucrose gradient. As shown in Fig. 1 A, LAT-GFP predominantly exists in rafts like endogenous LAT, suggesting that the fusion of GFP to LAT does not affect the localization of LAT in the plasma membrane.


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)

Characterization of the LAT-GFP fusion protein used in this work. (A) Localization of LAT-GFP in rafts from LAT-GFP–transfected Jurkat cells. LAT-GFP transfectants were lysed with MBS containing 1% Triton X-100, and the lysates were subjected to equilibrium gradient centrifugation. An aliquot of each fraction was electrophoresed and immunoblotted with HRP-conjugated CTx-B and anti-GFP antibody to detect GM1 and LAT-GFP, respectively. Fractions 4 and 5 correspond to the raft fractions. (B) Tyrosine phosphorylation of LAT-GFP after TCR cross-linking. The LAT-GFP transfectants were stimulated with OKT3 for the times indicated. Cell lysates were immunoprecipitated with anti-LAT antibody, and immunoprecipitates were analyzed by immunoblotting with anti-PY and anti-LAT antibodies. The closed arrowhead indicates LAT-GFP, whereas open arrowheads indicate endogenous LAT. The band migrating at 55 kD in each lane is the heavy chain (H) of the antibody used for immunoprecipitation.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172749&req=5

fig1: Characterization of the LAT-GFP fusion protein used in this work. (A) Localization of LAT-GFP in rafts from LAT-GFP–transfected Jurkat cells. LAT-GFP transfectants were lysed with MBS containing 1% Triton X-100, and the lysates were subjected to equilibrium gradient centrifugation. An aliquot of each fraction was electrophoresed and immunoblotted with HRP-conjugated CTx-B and anti-GFP antibody to detect GM1 and LAT-GFP, respectively. Fractions 4 and 5 correspond to the raft fractions. (B) Tyrosine phosphorylation of LAT-GFP after TCR cross-linking. The LAT-GFP transfectants were stimulated with OKT3 for the times indicated. Cell lysates were immunoprecipitated with anti-LAT antibody, and immunoprecipitates were analyzed by immunoblotting with anti-PY and anti-LAT antibodies. The closed arrowhead indicates LAT-GFP, whereas open arrowheads indicate endogenous LAT. The band migrating at 55 kD in each lane is the heavy chain (H) of the antibody used for immunoprecipitation.
Mentions: A fusion gene was constructed consisting of GFP linked to the COOH-terminal of the entire LAT gene coding sequence. The LAT-GFP fusion gene was then transfected into Jurkat cells and stable transfectants were established. Previously, it has been shown that LAT localizes to rafts by way of palmitoylation of two Cys residues in its juxtamembrane region (Zhang et al., 1998b). To investigate whether LAT-GFP localized to rafts as per endogenous LAT, LAT-GFP transfectants were solubilized and the raft fraction was purified using a sucrose gradient. As shown in Fig. 1 A, LAT-GFP predominantly exists in rafts like endogenous LAT, suggesting that the fusion of GFP to LAT does not affect the localization of LAT in the plasma membrane.

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