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Condensation of the plasma membrane at the site of T lymphocyte activation.

Gaus K, Chklovskaia E, Fazekas de St Groth B, Jessup W, Harder T - J. Cell Biol. (2005)

Bottom Line: The formation of ordered domains depends on the presence of the transmembrane protein linker for the activation of T cells and Src kinase activity.Moreover, these ordered domains are stabilized by the actin cytoskeleton.The formation of condensed membrane domains at T cell activation sites biophysically reflects membrane raft accumulation, which has potential implications for signaling at ISs.

View Article: PubMed Central - PubMed

Affiliation: Centre for Vascular Research at the School of Medical Sciences, University of New South Wales, Sydney 2052 NSW, Australia. k.gaus@unsw.edu.au

ABSTRACT
After activation, T lymphocytes restructure their cell surface to form membrane domains at T cell receptor (TCR)-signaling foci and immunological synapses (ISs). To address whether these rearrangements involve alteration in the structure of the plasma membrane bilayer, we used the fluorescent probe Laurdan to visualize its lipid order. We observed a condensation of the plasma membrane at TCR activation sites. The formation of ordered domains depends on the presence of the transmembrane protein linker for the activation of T cells and Src kinase activity. Moreover, these ordered domains are stabilized by the actin cytoskeleton. Membrane condensation occurs upon TCR stimulation alone but is prolonged by CD28 costimulation with TCR. In ISs, which are formed by conjugates of TCR transgenic T lymphocytes and cognate antigen-presenting cells, similar condensed membrane phases form first in central regions and later at the periphery of synapses. The formation of condensed membrane domains at T cell activation sites biophysically reflects membrane raft accumulation, which has potential implications for signaling at ISs.

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Effect of Src kinase inhibitors on membrane structure at TCR activation sites. Laurdan-labeled JCaM2 cells expressing WT LAT, mutant All Y-F LAT, or LAT- parent cells were conjugated with α-CD3–coated beads and were incubated for 7 min at 37°C. Cells were preincubated for 1 h at 37°C with 10 μM PP2 (A) or the nonactive control PP3 (B). Inhibitors were also present during the incubation period after conjugation. Cells were imaged, and GP values of bead–cell contact sites were determined as described in Fig. 1. Means (indicated by horizontal lines) and SDs are given in Table I. For all comparisons within A and B, P > 0.05 and P < 0.001, respectively.
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fig3: Effect of Src kinase inhibitors on membrane structure at TCR activation sites. Laurdan-labeled JCaM2 cells expressing WT LAT, mutant All Y-F LAT, or LAT- parent cells were conjugated with α-CD3–coated beads and were incubated for 7 min at 37°C. Cells were preincubated for 1 h at 37°C with 10 μM PP2 (A) or the nonactive control PP3 (B). Inhibitors were also present during the incubation period after conjugation. Cells were imaged, and GP values of bead–cell contact sites were determined as described in Fig. 1. Means (indicated by horizontal lines) and SDs are given in Table I. For all comparisons within A and B, P > 0.05 and P < 0.001, respectively.

Mentions: Next, we tested the role of Lck and Fyn activation in the condensation of TCR-signaling domains by using the inhibitor for Src-related kinases PP2 (Fig. 3 A) or the inactive control PP3 (Fig. 3 B). TCR-signaling domains in JCaM2 WT LAT cells that were treated with PP2 (0.289 ± 0.104) exhibited GP values that were as equally low as those of All Y-F LAT JCaM2 (0.265 ± 0.056) and the parental JCaM2 cell line (Table I; 0.265 ± 0.087). The noninhibitory PP2 analogue PP3 had no effects on the LAT-dependent increase in GP value at anti-CD3 bead contact sites (Fig. 3 B). These results show that Src kinase activity is required for membrane condensation in TCR-signaling domains.


Condensation of the plasma membrane at the site of T lymphocyte activation.

Gaus K, Chklovskaia E, Fazekas de St Groth B, Jessup W, Harder T - J. Cell Biol. (2005)

Effect of Src kinase inhibitors on membrane structure at TCR activation sites. Laurdan-labeled JCaM2 cells expressing WT LAT, mutant All Y-F LAT, or LAT- parent cells were conjugated with α-CD3–coated beads and were incubated for 7 min at 37°C. Cells were preincubated for 1 h at 37°C with 10 μM PP2 (A) or the nonactive control PP3 (B). Inhibitors were also present during the incubation period after conjugation. Cells were imaged, and GP values of bead–cell contact sites were determined as described in Fig. 1. Means (indicated by horizontal lines) and SDs are given in Table I. For all comparisons within A and B, P > 0.05 and P < 0.001, respectively.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2171224&req=5

fig3: Effect of Src kinase inhibitors on membrane structure at TCR activation sites. Laurdan-labeled JCaM2 cells expressing WT LAT, mutant All Y-F LAT, or LAT- parent cells were conjugated with α-CD3–coated beads and were incubated for 7 min at 37°C. Cells were preincubated for 1 h at 37°C with 10 μM PP2 (A) or the nonactive control PP3 (B). Inhibitors were also present during the incubation period after conjugation. Cells were imaged, and GP values of bead–cell contact sites were determined as described in Fig. 1. Means (indicated by horizontal lines) and SDs are given in Table I. For all comparisons within A and B, P > 0.05 and P < 0.001, respectively.
Mentions: Next, we tested the role of Lck and Fyn activation in the condensation of TCR-signaling domains by using the inhibitor for Src-related kinases PP2 (Fig. 3 A) or the inactive control PP3 (Fig. 3 B). TCR-signaling domains in JCaM2 WT LAT cells that were treated with PP2 (0.289 ± 0.104) exhibited GP values that were as equally low as those of All Y-F LAT JCaM2 (0.265 ± 0.056) and the parental JCaM2 cell line (Table I; 0.265 ± 0.087). The noninhibitory PP2 analogue PP3 had no effects on the LAT-dependent increase in GP value at anti-CD3 bead contact sites (Fig. 3 B). These results show that Src kinase activity is required for membrane condensation in TCR-signaling domains.

Bottom Line: The formation of ordered domains depends on the presence of the transmembrane protein linker for the activation of T cells and Src kinase activity.Moreover, these ordered domains are stabilized by the actin cytoskeleton.The formation of condensed membrane domains at T cell activation sites biophysically reflects membrane raft accumulation, which has potential implications for signaling at ISs.

View Article: PubMed Central - PubMed

Affiliation: Centre for Vascular Research at the School of Medical Sciences, University of New South Wales, Sydney 2052 NSW, Australia. k.gaus@unsw.edu.au

ABSTRACT
After activation, T lymphocytes restructure their cell surface to form membrane domains at T cell receptor (TCR)-signaling foci and immunological synapses (ISs). To address whether these rearrangements involve alteration in the structure of the plasma membrane bilayer, we used the fluorescent probe Laurdan to visualize its lipid order. We observed a condensation of the plasma membrane at TCR activation sites. The formation of ordered domains depends on the presence of the transmembrane protein linker for the activation of T cells and Src kinase activity. Moreover, these ordered domains are stabilized by the actin cytoskeleton. Membrane condensation occurs upon TCR stimulation alone but is prolonged by CD28 costimulation with TCR. In ISs, which are formed by conjugates of TCR transgenic T lymphocytes and cognate antigen-presenting cells, similar condensed membrane phases form first in central regions and later at the periphery of synapses. The formation of condensed membrane domains at T cell activation sites biophysically reflects membrane raft accumulation, which has potential implications for signaling at ISs.

Show MeSH
Related in: MedlinePlus