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Selective accumulation of raft-associated membrane protein LAT in T cell receptor signaling assemblies.

Harder T, Kuhn M - J. Cell Biol. (2000)

Bottom Line: We found that the raft transmembrane protein linker for activation of T cells (LAT), but not a palmitoylation-deficient non-raft LAT mutant, strongly accumulated in TCR-enriched immunoisolates in a tyrosine phosphorylation-dependent manner.In contrast, other raft-associated molecules, including protein tyrosine kinases Lck and Fyn, GM1, and cholesterol, were not highly concentrated in TCR-enriched plasma membrane immunoisolates.Our results indicate that TCR signaling assemblies in plasma membrane subdomains, rather than generally concentrating raft-associated membrane proteins and lipids, form by a selective protein-mediated anchoring of the raft membrane protein LAT in vicinity of TCR.

View Article: PubMed Central - PubMed

Affiliation: Basel Institute for Immunology, CH-4005, Basel, Switzerland.

ABSTRACT
Activation of T cell antigen receptor (TCR) induces tyrosine phosphorylations that mediate the assembly of signaling protein complexes. Moreover, cholesterol-sphingolipid raft membrane domains have been implicated to play a role in TCR signal transduction. Here, we studied the assembly of TCR with signal transduction proteins and raft markers in plasma membrane subdomains of Jurkat T leukemic cells. We employed a novel method to immunoisolate plasma membrane subfragments that were highly concentrated in activated TCR-CD3 complexes and associated signaling proteins. We found that the raft transmembrane protein linker for activation of T cells (LAT), but not a palmitoylation-deficient non-raft LAT mutant, strongly accumulated in TCR-enriched immunoisolates in a tyrosine phosphorylation-dependent manner. In contrast, other raft-associated molecules, including protein tyrosine kinases Lck and Fyn, GM1, and cholesterol, were not highly concentrated in TCR-enriched plasma membrane immunoisolates. Many downstream signaling proteins coisolated with the TCR/LAT-enriched plasma membrane fragments, suggesting that LAT/TCR assemblies form a structural scaffold for TCR signal transduction proteins. Our results indicate that TCR signaling assemblies in plasma membrane subdomains, rather than generally concentrating raft-associated membrane proteins and lipids, form by a selective protein-mediated anchoring of the raft membrane protein LAT in vicinity of TCR.

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Fluorescence microscopy of Jurkat cells. Jurkat cells were conjugated with α-CD3–coated (A, C, and E) and α-TfR–coated (B, D, and F) beads (marked by x) and incubated for 7 min at 37°C. A and B show immunofluorescence staining of LAT. Arrows indicate LAT accumulating at α-CD3–coated beads. C and D show distribution of Lck. Arrows in C indicate membrane ruffles and microvilli stained with α-Lck antibodies. In E–H, Jurkat cells were stained with the lipid dye DiIC16 before conjugate formation. Insets in E and F show DiIC16-stained PM fragments after the immunoisolation procedure. Exposure times of DiIC16-labeled immunoisolates were 10 times longer than DiIC16-labeled whole cells. G shows distribution of α-CD3 patches visualized by FITC-conjugated secondary antibodies; H shows distribution of lipid dye DiIC16. Bar, 10 μM.
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Figure 3: Fluorescence microscopy of Jurkat cells. Jurkat cells were conjugated with α-CD3–coated (A, C, and E) and α-TfR–coated (B, D, and F) beads (marked by x) and incubated for 7 min at 37°C. A and B show immunofluorescence staining of LAT. Arrows indicate LAT accumulating at α-CD3–coated beads. C and D show distribution of Lck. Arrows in C indicate membrane ruffles and microvilli stained with α-Lck antibodies. In E–H, Jurkat cells were stained with the lipid dye DiIC16 before conjugate formation. Insets in E and F show DiIC16-stained PM fragments after the immunoisolation procedure. Exposure times of DiIC16-labeled immunoisolates were 10 times longer than DiIC16-labeled whole cells. G shows distribution of α-CD3 patches visualized by FITC-conjugated secondary antibodies; H shows distribution of lipid dye DiIC16. Bar, 10 μM.

Mentions: The biochemical analysis was complemented by fluorescence microscopy of bead–cell conjugates (Fig. 3). Immunofluorescence microscopy showed a specific LAT accumulation at the α-CD3 bead–cell contact area (Fig. 3 A, arrows) but not at the α-TfR control beads. In contrast, Lck was not specifically concentrated in the PM region contacting α-CD3 beads (Fig. 3 C). Increased Lck staining was visible in membrane ruffles and microvilli forming at the cell pole facing the α-CD3 bead (Fig. 3 C, arrows). This strongly supports our biochemical data that Lck is, if at all, only weakly concentrated in the PM region contacting the TCR activating beads.


Selective accumulation of raft-associated membrane protein LAT in T cell receptor signaling assemblies.

Harder T, Kuhn M - J. Cell Biol. (2000)

Fluorescence microscopy of Jurkat cells. Jurkat cells were conjugated with α-CD3–coated (A, C, and E) and α-TfR–coated (B, D, and F) beads (marked by x) and incubated for 7 min at 37°C. A and B show immunofluorescence staining of LAT. Arrows indicate LAT accumulating at α-CD3–coated beads. C and D show distribution of Lck. Arrows in C indicate membrane ruffles and microvilli stained with α-Lck antibodies. In E–H, Jurkat cells were stained with the lipid dye DiIC16 before conjugate formation. Insets in E and F show DiIC16-stained PM fragments after the immunoisolation procedure. Exposure times of DiIC16-labeled immunoisolates were 10 times longer than DiIC16-labeled whole cells. G shows distribution of α-CD3 patches visualized by FITC-conjugated secondary antibodies; H shows distribution of lipid dye DiIC16. Bar, 10 μM.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2192654&req=5

Figure 3: Fluorescence microscopy of Jurkat cells. Jurkat cells were conjugated with α-CD3–coated (A, C, and E) and α-TfR–coated (B, D, and F) beads (marked by x) and incubated for 7 min at 37°C. A and B show immunofluorescence staining of LAT. Arrows indicate LAT accumulating at α-CD3–coated beads. C and D show distribution of Lck. Arrows in C indicate membrane ruffles and microvilli stained with α-Lck antibodies. In E–H, Jurkat cells were stained with the lipid dye DiIC16 before conjugate formation. Insets in E and F show DiIC16-stained PM fragments after the immunoisolation procedure. Exposure times of DiIC16-labeled immunoisolates were 10 times longer than DiIC16-labeled whole cells. G shows distribution of α-CD3 patches visualized by FITC-conjugated secondary antibodies; H shows distribution of lipid dye DiIC16. Bar, 10 μM.
Mentions: The biochemical analysis was complemented by fluorescence microscopy of bead–cell conjugates (Fig. 3). Immunofluorescence microscopy showed a specific LAT accumulation at the α-CD3 bead–cell contact area (Fig. 3 A, arrows) but not at the α-TfR control beads. In contrast, Lck was not specifically concentrated in the PM region contacting α-CD3 beads (Fig. 3 C). Increased Lck staining was visible in membrane ruffles and microvilli forming at the cell pole facing the α-CD3 bead (Fig. 3 C, arrows). This strongly supports our biochemical data that Lck is, if at all, only weakly concentrated in the PM region contacting the TCR activating beads.

Bottom Line: We found that the raft transmembrane protein linker for activation of T cells (LAT), but not a palmitoylation-deficient non-raft LAT mutant, strongly accumulated in TCR-enriched immunoisolates in a tyrosine phosphorylation-dependent manner.In contrast, other raft-associated molecules, including protein tyrosine kinases Lck and Fyn, GM1, and cholesterol, were not highly concentrated in TCR-enriched plasma membrane immunoisolates.Our results indicate that TCR signaling assemblies in plasma membrane subdomains, rather than generally concentrating raft-associated membrane proteins and lipids, form by a selective protein-mediated anchoring of the raft membrane protein LAT in vicinity of TCR.

View Article: PubMed Central - PubMed

Affiliation: Basel Institute for Immunology, CH-4005, Basel, Switzerland.

ABSTRACT
Activation of T cell antigen receptor (TCR) induces tyrosine phosphorylations that mediate the assembly of signaling protein complexes. Moreover, cholesterol-sphingolipid raft membrane domains have been implicated to play a role in TCR signal transduction. Here, we studied the assembly of TCR with signal transduction proteins and raft markers in plasma membrane subdomains of Jurkat T leukemic cells. We employed a novel method to immunoisolate plasma membrane subfragments that were highly concentrated in activated TCR-CD3 complexes and associated signaling proteins. We found that the raft transmembrane protein linker for activation of T cells (LAT), but not a palmitoylation-deficient non-raft LAT mutant, strongly accumulated in TCR-enriched immunoisolates in a tyrosine phosphorylation-dependent manner. In contrast, other raft-associated molecules, including protein tyrosine kinases Lck and Fyn, GM1, and cholesterol, were not highly concentrated in TCR-enriched plasma membrane immunoisolates. Many downstream signaling proteins coisolated with the TCR/LAT-enriched plasma membrane fragments, suggesting that LAT/TCR assemblies form a structural scaffold for TCR signal transduction proteins. Our results indicate that TCR signaling assemblies in plasma membrane subdomains, rather than generally concentrating raft-associated membrane proteins and lipids, form by a selective protein-mediated anchoring of the raft membrane protein LAT in vicinity of TCR.

Show MeSH
Related in: MedlinePlus