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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 condensation at the TCR activation site in Jurkat cells. Laurdan-labeled JCaM2 cells expressing WT LAT were conjugated with α-CD3–coated polystyrene beads and incubated for 0 (A), 3 (B), and 7 (C) min at 37°C. Cells were adhered, fixed, and simultaneously imaged for the Laurdan intensity in two channels (400–460 nm and 470–530 nm). Intensity images were converted to GP images as described in Image analysis. (A–C) GP images were pseudocolored (see scale in A of GP −0.5–1), and insets show differential interference contrast (DIC) images. Bars, 5 μm. (D) GP values at the bead–cell contact area were determined as outlined by the white dashed line in A. Means (indicated by horizontal lines) and SDs are given in Table I. All datasets are significantly different to each other (P < 0.001). (E) GP distribution of 0- (diamonds; n = 17), 3- (triangles; n = 18), and 7-min (squares; n = 20) incubations from one representative experiment. Distributions were fitted to two Gaussian populations (solid lines) with mean GP values at 0.215 and 0.402 (0 min), 0.228 and 0.406 (3 min), and 0.201 and 0.456 (7 min). The high GP population (shaded area) covered 6.6 (0 min), 17.7 (3 min), and 21.0% (7 min) of all Laurdan-stained pixels.
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fig1: Membrane condensation at the TCR activation site in Jurkat cells. Laurdan-labeled JCaM2 cells expressing WT LAT were conjugated with α-CD3–coated polystyrene beads and incubated for 0 (A), 3 (B), and 7 (C) min at 37°C. Cells were adhered, fixed, and simultaneously imaged for the Laurdan intensity in two channels (400–460 nm and 470–530 nm). Intensity images were converted to GP images as described in Image analysis. (A–C) GP images were pseudocolored (see scale in A of GP −0.5–1), and insets show differential interference contrast (DIC) images. Bars, 5 μm. (D) GP values at the bead–cell contact area were determined as outlined by the white dashed line in A. Means (indicated by horizontal lines) and SDs are given in Table I. All datasets are significantly different to each other (P < 0.001). (E) GP distribution of 0- (diamonds; n = 17), 3- (triangles; n = 18), and 7-min (squares; n = 20) incubations from one representative experiment. Distributions were fitted to two Gaussian populations (solid lines) with mean GP values at 0.215 and 0.402 (0 min), 0.228 and 0.406 (3 min), and 0.201 and 0.456 (7 min). The high GP population (shaded area) covered 6.6 (0 min), 17.7 (3 min), and 21.0% (7 min) of all Laurdan-stained pixels.

Mentions: To assess the degree of lipid condensation at TCR-signaling sites, T leukemic Jurkat–derived TCR signaling–competent JCaM2 cells expressing wild-type (WT) LAT were labeled with Laurdan (Gaus et al., 2003). The cells were conjugated to polystyrene beads that were coated with TR66 anti-CD3 mAb and were subsequently warmed to 37°C, leading to TCR activation at the bead–cell contact site (Harder and Kuhn, 2000). The bead–cell conjugates were imaged by two-photon laser scanning microscopy, and GP values were taken as described in Microscopy and in Cells and reagents (Gaus et al., 2003). After a 3- or 7-min incubation at 37°C, Laurdan fluorescence revealed a region of high GP at the area of the plasma membrane that was in contact with anti-CD3–coated beads in WT LAT JCaM2 cells (Fig. 1, A–C). This is indicative of membrane condensation at the bead–cell contact site. For quantification, we determined the mean GP at the region of membrane–bead contact (Fig. 1 A) for >50 images (Fig. 1 D). The contact zone was selected as the membrane area that was adjacent to the bead with a mean width across the plasma membrane of 0.48 ± 0.11 μm (∼2.5-fold the spatial resolution of the microscope). Mean GP values increased from 0.240 ± 0.095 at 0 min to 0.336 ± 0.091 at 3 min and 0.442 ± 0.085 at 7 min (Table I), indicating that stimulation of the TCR causes significant membrane condensation at the activation site. Fig. 1 E shows the GP distribution of all GP values (i.e., of total membranes) as a normalized histogram of >12 cell cross sections from one experiment. As described previously in macrophages (Gaus et al., 2003), a condensed membrane population with high mean GP can be discriminated in these histograms (Fig. 1 E, shaded area). Importantly, the proportion of these condensed membranes as a percentage of total membranes significantly increased from 6.6 to 21.0% upon stimulation (7 min) of the Jurkat cells with anti-CD3 beads. Such changes in GP distribution were not observed when cells were conjugated to beads that were coated with antitransferrin receptor (TfR) mAb (high GP membranes as percentage of total membranes: 9.5% at 0 min, 9.0% at 3 min, and 10.7% at 7 min), which do not stimulate TCR-mediated signaling events (Harder and Kuhn, 2000). As the TCR activation site (Fig. 1 A, highlighted area) represents 7–13% of the surface area of the cell cross sections, membrane condensation at the anti-CD3 bead contact site accounts for the majority of the TCR triggering–induced membrane condensation that is seen in Fig. 1 E, with non-TCR sites contributing ∼1–2% at 3 min and 4–5% at 7 min. The membrane condensation that is induced by TCR activation may be interpreted either as a de novo condensation of the lipid bilayer or as a coalescence of small preexisting rafts below the resolution of the microscope (<183 nm) at the bead–cell contact zone. Although we cannot distinguish between large continuous domains or clustered small ones, these experiments showed that condensed raftlike lipid domains, which are detectable by light microscopy, accumulate at the site of TCR activation by anti-CD3–coated beads.


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 condensation at the TCR activation site in Jurkat cells. Laurdan-labeled JCaM2 cells expressing WT LAT were conjugated with α-CD3–coated polystyrene beads and incubated for 0 (A), 3 (B), and 7 (C) min at 37°C. Cells were adhered, fixed, and simultaneously imaged for the Laurdan intensity in two channels (400–460 nm and 470–530 nm). Intensity images were converted to GP images as described in Image analysis. (A–C) GP images were pseudocolored (see scale in A of GP −0.5–1), and insets show differential interference contrast (DIC) images. Bars, 5 μm. (D) GP values at the bead–cell contact area were determined as outlined by the white dashed line in A. Means (indicated by horizontal lines) and SDs are given in Table I. All datasets are significantly different to each other (P < 0.001). (E) GP distribution of 0- (diamonds; n = 17), 3- (triangles; n = 18), and 7-min (squares; n = 20) incubations from one representative experiment. Distributions were fitted to two Gaussian populations (solid lines) with mean GP values at 0.215 and 0.402 (0 min), 0.228 and 0.406 (3 min), and 0.201 and 0.456 (7 min). The high GP population (shaded area) covered 6.6 (0 min), 17.7 (3 min), and 21.0% (7 min) of all Laurdan-stained pixels.
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Related In: Results  -  Collection

Show All Figures
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fig1: Membrane condensation at the TCR activation site in Jurkat cells. Laurdan-labeled JCaM2 cells expressing WT LAT were conjugated with α-CD3–coated polystyrene beads and incubated for 0 (A), 3 (B), and 7 (C) min at 37°C. Cells were adhered, fixed, and simultaneously imaged for the Laurdan intensity in two channels (400–460 nm and 470–530 nm). Intensity images were converted to GP images as described in Image analysis. (A–C) GP images were pseudocolored (see scale in A of GP −0.5–1), and insets show differential interference contrast (DIC) images. Bars, 5 μm. (D) GP values at the bead–cell contact area were determined as outlined by the white dashed line in A. Means (indicated by horizontal lines) and SDs are given in Table I. All datasets are significantly different to each other (P < 0.001). (E) GP distribution of 0- (diamonds; n = 17), 3- (triangles; n = 18), and 7-min (squares; n = 20) incubations from one representative experiment. Distributions were fitted to two Gaussian populations (solid lines) with mean GP values at 0.215 and 0.402 (0 min), 0.228 and 0.406 (3 min), and 0.201 and 0.456 (7 min). The high GP population (shaded area) covered 6.6 (0 min), 17.7 (3 min), and 21.0% (7 min) of all Laurdan-stained pixels.
Mentions: To assess the degree of lipid condensation at TCR-signaling sites, T leukemic Jurkat–derived TCR signaling–competent JCaM2 cells expressing wild-type (WT) LAT were labeled with Laurdan (Gaus et al., 2003). The cells were conjugated to polystyrene beads that were coated with TR66 anti-CD3 mAb and were subsequently warmed to 37°C, leading to TCR activation at the bead–cell contact site (Harder and Kuhn, 2000). The bead–cell conjugates were imaged by two-photon laser scanning microscopy, and GP values were taken as described in Microscopy and in Cells and reagents (Gaus et al., 2003). After a 3- or 7-min incubation at 37°C, Laurdan fluorescence revealed a region of high GP at the area of the plasma membrane that was in contact with anti-CD3–coated beads in WT LAT JCaM2 cells (Fig. 1, A–C). This is indicative of membrane condensation at the bead–cell contact site. For quantification, we determined the mean GP at the region of membrane–bead contact (Fig. 1 A) for >50 images (Fig. 1 D). The contact zone was selected as the membrane area that was adjacent to the bead with a mean width across the plasma membrane of 0.48 ± 0.11 μm (∼2.5-fold the spatial resolution of the microscope). Mean GP values increased from 0.240 ± 0.095 at 0 min to 0.336 ± 0.091 at 3 min and 0.442 ± 0.085 at 7 min (Table I), indicating that stimulation of the TCR causes significant membrane condensation at the activation site. Fig. 1 E shows the GP distribution of all GP values (i.e., of total membranes) as a normalized histogram of >12 cell cross sections from one experiment. As described previously in macrophages (Gaus et al., 2003), a condensed membrane population with high mean GP can be discriminated in these histograms (Fig. 1 E, shaded area). Importantly, the proportion of these condensed membranes as a percentage of total membranes significantly increased from 6.6 to 21.0% upon stimulation (7 min) of the Jurkat cells with anti-CD3 beads. Such changes in GP distribution were not observed when cells were conjugated to beads that were coated with antitransferrin receptor (TfR) mAb (high GP membranes as percentage of total membranes: 9.5% at 0 min, 9.0% at 3 min, and 10.7% at 7 min), which do not stimulate TCR-mediated signaling events (Harder and Kuhn, 2000). As the TCR activation site (Fig. 1 A, highlighted area) represents 7–13% of the surface area of the cell cross sections, membrane condensation at the anti-CD3 bead contact site accounts for the majority of the TCR triggering–induced membrane condensation that is seen in Fig. 1 E, with non-TCR sites contributing ∼1–2% at 3 min and 4–5% at 7 min. The membrane condensation that is induced by TCR activation may be interpreted either as a de novo condensation of the lipid bilayer or as a coalescence of small preexisting rafts below the resolution of the microscope (<183 nm) at the bead–cell contact zone. Although we cannot distinguish between large continuous domains or clustered small ones, these experiments showed that condensed raftlike lipid domains, which are detectable by light microscopy, accumulate at the site of TCR activation by anti-CD3–coated beads.

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