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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 actin depolymerization on membrane structure at the TCR activation site. Laurdan-labeled JCaM2 cells expressing WT LAT, mutant All Y-F LAT, or LAT- cells (JCaM2) were conjugated with α-CD3–coated (A–E) or TfR-coated (F) beads and were incubated for 7 min at 37°C (A and B) followed by the addition of 12.5 μM latrunculin B and incubation for a further 3 min at 37°C (C–E). (A and C) GP images were obtained and pseudocolored as described in Fig. 1. Insets show DIC images. (B and D) F-actin staining with phalloidin–Alexia 637. Note that no F-actin was detected in latrunculin B–treated cells (D). Bars, 5 μm. (E and F) GP values at contact sites between α-CD3–coated (E) or TfR-coated (F) beads and cells after latrunculin B treatment. Means (indicated by horizontal lines) and SDs are given in Table I. For all comparisons within E or F, P > 0.05. For all cell types, no statistically significant difference (P > 0.05) was found between means of α-CD3– and TfR-coated beads.
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fig4: Effect of actin depolymerization on membrane structure at the TCR activation site. Laurdan-labeled JCaM2 cells expressing WT LAT, mutant All Y-F LAT, or LAT- cells (JCaM2) were conjugated with α-CD3–coated (A–E) or TfR-coated (F) beads and were incubated for 7 min at 37°C (A and B) followed by the addition of 12.5 μM latrunculin B and incubation for a further 3 min at 37°C (C–E). (A and C) GP images were obtained and pseudocolored as described in Fig. 1. Insets show DIC images. (B and D) F-actin staining with phalloidin–Alexia 637. Note that no F-actin was detected in latrunculin B–treated cells (D). Bars, 5 μm. (E and F) GP values at contact sites between α-CD3–coated (E) or TfR-coated (F) beads and cells after latrunculin B treatment. Means (indicated by horizontal lines) and SDs are given in Table I. For all comparisons within E or F, P > 0.05. For all cell types, no statistically significant difference (P > 0.05) was found between means of α-CD3– and TfR-coated beads.

Mentions: Means ± SDs are given for JCaM2 cells, PBLs, and synapses between naive T cells and DCs. For comparisons between WT LAT, All Y-F LAT, and JCaM2 cells, see Figs. 2–4. Superscripts indicate pairs of GP means, which were significantly different with P < 0.001 in JCaM2 cells (a–o), PBLs (e–g), and in naive T cells (a–e) and with P < 0.05 in PBLs (a–d).


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 actin depolymerization on membrane structure at the TCR activation site. Laurdan-labeled JCaM2 cells expressing WT LAT, mutant All Y-F LAT, or LAT- cells (JCaM2) were conjugated with α-CD3–coated (A–E) or TfR-coated (F) beads and were incubated for 7 min at 37°C (A and B) followed by the addition of 12.5 μM latrunculin B and incubation for a further 3 min at 37°C (C–E). (A and C) GP images were obtained and pseudocolored as described in Fig. 1. Insets show DIC images. (B and D) F-actin staining with phalloidin–Alexia 637. Note that no F-actin was detected in latrunculin B–treated cells (D). Bars, 5 μm. (E and F) GP values at contact sites between α-CD3–coated (E) or TfR-coated (F) beads and cells after latrunculin B treatment. Means (indicated by horizontal lines) and SDs are given in Table I. For all comparisons within E or F, P > 0.05. For all cell types, no statistically significant difference (P > 0.05) was found between means of α-CD3– and TfR-coated beads.
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Related In: Results  -  Collection

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fig4: Effect of actin depolymerization on membrane structure at the TCR activation site. Laurdan-labeled JCaM2 cells expressing WT LAT, mutant All Y-F LAT, or LAT- cells (JCaM2) were conjugated with α-CD3–coated (A–E) or TfR-coated (F) beads and were incubated for 7 min at 37°C (A and B) followed by the addition of 12.5 μM latrunculin B and incubation for a further 3 min at 37°C (C–E). (A and C) GP images were obtained and pseudocolored as described in Fig. 1. Insets show DIC images. (B and D) F-actin staining with phalloidin–Alexia 637. Note that no F-actin was detected in latrunculin B–treated cells (D). Bars, 5 μm. (E and F) GP values at contact sites between α-CD3–coated (E) or TfR-coated (F) beads and cells after latrunculin B treatment. Means (indicated by horizontal lines) and SDs are given in Table I. For all comparisons within E or F, P > 0.05. For all cell types, no statistically significant difference (P > 0.05) was found between means of α-CD3– and TfR-coated beads.
Mentions: Means ± SDs are given for JCaM2 cells, PBLs, and synapses between naive T cells and DCs. For comparisons between WT LAT, All Y-F LAT, and JCaM2 cells, see Figs. 2–4. Superscripts indicate pairs of GP means, which were significantly different with P < 0.001 in JCaM2 cells (a–o), PBLs (e–g), and in naive T cells (a–e) and with P < 0.05 in PBLs (a–d).

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