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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 ISs. (A–H) Laurdan-labeled naive T cells were conjugated to CMRA-labeled DCs for 0 (A and E), 3 (B and F), 7 (C and G), or 23 (D and H) min. Before conjugation, APCs were incubated in the absence (A–D and I) or presence (E–H and J) of 2 μM antigen cytochrome c. Cell couples were fixed and imaged as described for JCaM2 cells in Image analysis. Insets show the corresponding transmission image with the orange fluorescence image of CMRA overlaid to identify APCs. Bars, 5 μm. Imaging and pseudocoloring were performed as described in Fig. 1. (I and J). GP values measured over the entire contact area between primary T cell–APC couples without (I) and with (J) conjugation of APCs with antigen. Means (indicated by horizontal lines) and SDs are shown in Table I. (I) Differences were found between the means of 0 and 3 min or 7 min (P < 0.05) and the means of 23 and 3 min or 7 min (P < 0.001); other comparisons did not reveal significant differences. (J) All means are significantly different to each other (P < 0.01 or P < 0.001).
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fig6: Membrane structure at ISs. (A–H) Laurdan-labeled naive T cells were conjugated to CMRA-labeled DCs for 0 (A and E), 3 (B and F), 7 (C and G), or 23 (D and H) min. Before conjugation, APCs were incubated in the absence (A–D and I) or presence (E–H and J) of 2 μM antigen cytochrome c. Cell couples were fixed and imaged as described for JCaM2 cells in Image analysis. Insets show the corresponding transmission image with the orange fluorescence image of CMRA overlaid to identify APCs. Bars, 5 μm. Imaging and pseudocoloring were performed as described in Fig. 1. (I and J). GP values measured over the entire contact area between primary T cell–APC couples without (I) and with (J) conjugation of APCs with antigen. Means (indicated by horizontal lines) and SDs are shown in Table I. (I) Differences were found between the means of 0 and 3 min or 7 min (P < 0.05) and the means of 23 and 3 min or 7 min (P < 0.001); other comparisons did not reveal significant differences. (J) All means are significantly different to each other (P < 0.01 or P < 0.001).

Mentions: We observed a significant increase of GP values at the contact zone after 3 min of incubation between T cells and APCs; both were in conjugates with (Table I; 0.370 ± 0.082) and without antigen (0.276 ± 0.109; n = 95; P < 0.001 ± antigen), but this was more intense and was sustained for longer in antigen-pulsed conjugates (Fig. 6). At 7 min, lipid order had decreased for cells that were conjugated without antigen (Fig. 6 C) but further increased (to 0.422 ± 0.071) for conjugates with antigen present (Fig. 6 G). At this time, in the presence of the antigen, the condensed membrane domains were predominately localized at the central region of the IS (Fig. 6 G), which is in contrast to the 23-min time point in which ordered domains had moved away from the central area and associated more with peripheral areas of the synapses (Fig. 6 H). The mean GP value of synapses at 23 min decreased significantly (0.300 ± 0.100) compared with that after a 7-min incubation but remained above the mean without antigen present (0.187 ± 0.083; P < 0.001 ± antigen). For more spatial information, we constructed three-dimensional (3D) images and z-sectioned them at the IS, using conjugates of Jurkat 8.2 cells because they yielded a better resolution in z-direction (Fig. 7). As observed in the IS of transgenic T lymphocytes, these cells exhibited an increase of GP value if stimulated by antigen-pulsed APCs (Fig. 7, A and C). Like in primary T cells, this membrane condensation occurred initially at the contact zone to the APC and later moved to more peripheral sites (Fig. 7 C). These images show a reorganization of membrane condensation at the IS to peripheral regions.


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 ISs. (A–H) Laurdan-labeled naive T cells were conjugated to CMRA-labeled DCs for 0 (A and E), 3 (B and F), 7 (C and G), or 23 (D and H) min. Before conjugation, APCs were incubated in the absence (A–D and I) or presence (E–H and J) of 2 μM antigen cytochrome c. Cell couples were fixed and imaged as described for JCaM2 cells in Image analysis. Insets show the corresponding transmission image with the orange fluorescence image of CMRA overlaid to identify APCs. Bars, 5 μm. Imaging and pseudocoloring were performed as described in Fig. 1. (I and J). GP values measured over the entire contact area between primary T cell–APC couples without (I) and with (J) conjugation of APCs with antigen. Means (indicated by horizontal lines) and SDs are shown in Table I. (I) Differences were found between the means of 0 and 3 min or 7 min (P < 0.05) and the means of 23 and 3 min or 7 min (P < 0.001); other comparisons did not reveal significant differences. (J) All means are significantly different to each other (P < 0.01 or P < 0.001).
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fig6: Membrane structure at ISs. (A–H) Laurdan-labeled naive T cells were conjugated to CMRA-labeled DCs for 0 (A and E), 3 (B and F), 7 (C and G), or 23 (D and H) min. Before conjugation, APCs were incubated in the absence (A–D and I) or presence (E–H and J) of 2 μM antigen cytochrome c. Cell couples were fixed and imaged as described for JCaM2 cells in Image analysis. Insets show the corresponding transmission image with the orange fluorescence image of CMRA overlaid to identify APCs. Bars, 5 μm. Imaging and pseudocoloring were performed as described in Fig. 1. (I and J). GP values measured over the entire contact area between primary T cell–APC couples without (I) and with (J) conjugation of APCs with antigen. Means (indicated by horizontal lines) and SDs are shown in Table I. (I) Differences were found between the means of 0 and 3 min or 7 min (P < 0.05) and the means of 23 and 3 min or 7 min (P < 0.001); other comparisons did not reveal significant differences. (J) All means are significantly different to each other (P < 0.01 or P < 0.001).
Mentions: We observed a significant increase of GP values at the contact zone after 3 min of incubation between T cells and APCs; both were in conjugates with (Table I; 0.370 ± 0.082) and without antigen (0.276 ± 0.109; n = 95; P < 0.001 ± antigen), but this was more intense and was sustained for longer in antigen-pulsed conjugates (Fig. 6). At 7 min, lipid order had decreased for cells that were conjugated without antigen (Fig. 6 C) but further increased (to 0.422 ± 0.071) for conjugates with antigen present (Fig. 6 G). At this time, in the presence of the antigen, the condensed membrane domains were predominately localized at the central region of the IS (Fig. 6 G), which is in contrast to the 23-min time point in which ordered domains had moved away from the central area and associated more with peripheral areas of the synapses (Fig. 6 H). The mean GP value of synapses at 23 min decreased significantly (0.300 ± 0.100) compared with that after a 7-min incubation but remained above the mean without antigen present (0.187 ± 0.083; P < 0.001 ± antigen). For more spatial information, we constructed three-dimensional (3D) images and z-sectioned them at the IS, using conjugates of Jurkat 8.2 cells because they yielded a better resolution in z-direction (Fig. 7). As observed in the IS of transgenic T lymphocytes, these cells exhibited an increase of GP value if stimulated by antigen-pulsed APCs (Fig. 7, A and C). Like in primary T cells, this membrane condensation occurred initially at the contact zone to the APC and later moved to more peripheral sites (Fig. 7 C). These images show a reorganization of membrane condensation at the IS to peripheral regions.

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