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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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Membrane structure at PBL activation sites. Laurdan-labeled PBLs were conjugated with beads coated with α-CD3 mAb (A), a combination of α-CD3 and CD28 mAb (B), or CD28 mAb alone (C) and were incubated for 0–60 min at 37°C. Imaging and GP quantification at the contact site were performed as described for Fig. 1. (A) All datasets are significantly different to each other (P < 0.001) with the exception of 23 and 60 min, for which P > 0.05. (B) The mean of 0-min incubation is significantly different from 7, 23, and 60 min (P < 0.001), and differences between 23 and 60 min were also significant (P < 0.05); all other sets of data were not significantly different. (C) Only the means of 0 min is significantly different from 7, 23, and 60 min (P < 0.05). Means (indicated by horizontal lines) and SDs are given in Table I.
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fig5: Membrane structure at PBL activation sites. Laurdan-labeled PBLs were conjugated with beads coated with α-CD3 mAb (A), a combination of α-CD3 and CD28 mAb (B), or CD28 mAb alone (C) and were incubated for 0–60 min at 37°C. Imaging and GP quantification at the contact site were performed as described for Fig. 1. (A) All datasets are significantly different to each other (P < 0.001) with the exception of 23 and 60 min, for which P > 0.05. (B) The mean of 0-min incubation is significantly different from 7, 23, and 60 min (P < 0.001), and differences between 23 and 60 min were also significant (P < 0.05); all other sets of data were not significantly different. (C) Only the means of 0 min is significantly different from 7, 23, and 60 min (P < 0.05). Means (indicated by horizontal lines) and SDs are given in Table I.

Mentions: Full activation of primary resting T lymphocytes requires the engagement of costimulatory receptors in addition to the TCR and, most prominently, the engagement of CD28 with the B7 antigen receptor (Acuto and Michel, 2003). It was reported that TCR and CD28 engagement is required for GM1 polarization toward the site of T lymphocyte activation, which is interpreted as raft recruitment to the TCR activation site (Viola et al., 1999; Tavano et al., 2004). To test the role of CD28 costimulation in the formation of raftlike ordered membrane domains, we isolated resting peripheral blood lymphocytes (PBLs) from human donors and stimulated them with anti-CD3 (Fig. 5 A), anti-CD28 alone (Fig. 5 C), or anti-CD3/CD28-coated beads (Fig. 5 B). We followed the membrane structure at the contact site between 0 and 60 min after stimulation at 37°C. Conjugation with anti-CD3–coated beads showed a rapid condensation after 7 min (0.413 ± 0.06; Table I) followed by a significant decrease in lipid order (23 min; 0.313 ± 0.074). Stimulation with CD28 alone exhibited a lower initial membrane condensation than CD3 ligation, which remained stable for at least 60 min (0.248 ± 0.097). Costimulation with CD3 + CD28 also induced an initial increase of the mean GP value (7 min; 0.343 ± 0.060). Compared with CD3 stimulation alone, membrane condensation was sustained for longer (23 min; 0.354 ± 0.083). In conclusion, CD3 alone is sufficient for the initial formation of condensed membrane domains at the TCR activation site, yet costimulation with CD28 stabilizes condensed membrane structures for longer times.


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)

Membrane structure at PBL activation sites. Laurdan-labeled PBLs were conjugated with beads coated with α-CD3 mAb (A), a combination of α-CD3 and CD28 mAb (B), or CD28 mAb alone (C) and were incubated for 0–60 min at 37°C. Imaging and GP quantification at the contact site were performed as described for Fig. 1. (A) All datasets are significantly different to each other (P < 0.001) with the exception of 23 and 60 min, for which P > 0.05. (B) The mean of 0-min incubation is significantly different from 7, 23, and 60 min (P < 0.001), and differences between 23 and 60 min were also significant (P < 0.05); all other sets of data were not significantly different. (C) Only the means of 0 min is significantly different from 7, 23, and 60 min (P < 0.05). Means (indicated by horizontal lines) and SDs are given in Table I.
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Related In: Results  -  Collection

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fig5: Membrane structure at PBL activation sites. Laurdan-labeled PBLs were conjugated with beads coated with α-CD3 mAb (A), a combination of α-CD3 and CD28 mAb (B), or CD28 mAb alone (C) and were incubated for 0–60 min at 37°C. Imaging and GP quantification at the contact site were performed as described for Fig. 1. (A) All datasets are significantly different to each other (P < 0.001) with the exception of 23 and 60 min, for which P > 0.05. (B) The mean of 0-min incubation is significantly different from 7, 23, and 60 min (P < 0.001), and differences between 23 and 60 min were also significant (P < 0.05); all other sets of data were not significantly different. (C) Only the means of 0 min is significantly different from 7, 23, and 60 min (P < 0.05). Means (indicated by horizontal lines) and SDs are given in Table I.
Mentions: Full activation of primary resting T lymphocytes requires the engagement of costimulatory receptors in addition to the TCR and, most prominently, the engagement of CD28 with the B7 antigen receptor (Acuto and Michel, 2003). It was reported that TCR and CD28 engagement is required for GM1 polarization toward the site of T lymphocyte activation, which is interpreted as raft recruitment to the TCR activation site (Viola et al., 1999; Tavano et al., 2004). To test the role of CD28 costimulation in the formation of raftlike ordered membrane domains, we isolated resting peripheral blood lymphocytes (PBLs) from human donors and stimulated them with anti-CD3 (Fig. 5 A), anti-CD28 alone (Fig. 5 C), or anti-CD3/CD28-coated beads (Fig. 5 B). We followed the membrane structure at the contact site between 0 and 60 min after stimulation at 37°C. Conjugation with anti-CD3–coated beads showed a rapid condensation after 7 min (0.413 ± 0.06; Table I) followed by a significant decrease in lipid order (23 min; 0.313 ± 0.074). Stimulation with CD28 alone exhibited a lower initial membrane condensation than CD3 ligation, which remained stable for at least 60 min (0.248 ± 0.097). Costimulation with CD3 + CD28 also induced an initial increase of the mean GP value (7 min; 0.343 ± 0.060). Compared with CD3 stimulation alone, membrane condensation was sustained for longer (23 min; 0.354 ± 0.083). In conclusion, CD3 alone is sufficient for the initial formation of condensed membrane domains at the TCR activation site, yet costimulation with CD28 stabilizes condensed membrane structures for longer times.

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