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Local changes in lipid environment of TCR microclusters regulate membrane binding by the CD3ε cytoplasmic domain.

Gagnon E, Schubert DA, Gordo S, Chu HH, Wucherpfennig KW - J. Exp. Med. (2012)

Bottom Line: Release of the CD3ε cytoplasmic domain from the membrane is accompanied by a substantial focal reduction in negative charge and available PS in TCR microclusters.These changes in the lipid composition of TCR microclusters even occur when TCR signaling is blocked with a Src kinase inhibitor.Local changes in the lipid composition of TCR microclusters thus render the CD3ε cytoplasmic domain accessible during early stages of T cell activation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02115, USA. etienne.gagnon@umontreal.ca

ABSTRACT
The CD3ε and ζ cytoplasmic domains of the T cell receptor bind to the inner leaflet of the plasma membrane (PM), and a previous nuclear magnetic resonance structure showed that both tyrosines of the CD3ε immunoreceptor tyrosine-based activation motif partition into the bilayer. Electrostatic interactions between acidic phospholipids and clusters of basic CD3ε residues were previously shown to be essential for CD3ε and ζ membrane binding. Phosphatidylserine (PS) is the most abundant negatively charged lipid on the inner leaflet of the PM and makes a major contribution to membrane binding by the CD3ε cytoplasmic domain. Here, we show that TCR triggering by peptide--MHC complexes induces dissociation of the CD3ε cytoplasmic domain from the plasma membrane. Release of the CD3ε cytoplasmic domain from the membrane is accompanied by a substantial focal reduction in negative charge and available PS in TCR microclusters. These changes in the lipid composition of TCR microclusters even occur when TCR signaling is blocked with a Src kinase inhibitor. Local changes in the lipid composition of TCR microclusters thus render the CD3ε cytoplasmic domain accessible during early stages of T cell activation.

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Reduction of negative charge at the inner leaflet of the T cell synapse during early stages of TCR engagement by peptide-MHC. (A and B) B-A8 T cells expressing the anionic lipid probe R-pre-mRFP were stimulated with HA-DR4/ICAM-1 lipid beads and imaged by confocal microscopy at 10-s intervals. Top row: typical cell showing bead (blue) and R-pre binding to inner leaflet (red) as synapse is formed. Bottom row shows intensity of R-pre fluorescence using pseudo-color gradient; quantification within or outside of synapse (B). Images representative of n > 3 experiments. Bar, 5 µm. (C and E) B-A8 T cells expressing both R-pre-mRFP and ZAP-70-eGFP probes (C) or MyrPalm-eGFP probe (E) were stimulated with HA-DR4/ICAM-1 lipid beads and imaged by confocal microscopy. Bottom rows show higher magnification (white box) and pseudo-color gradient of fluorescence intensity. Dotted line represents cross section for cells shown in D and F. Images representative of n > 3 experiments. Bar, 10 µm. (D and F) Fluorescence signals of bead (blue), R-pre (red), and ZAP-70 (green; D) or MyrPalm (green; F) across cells in C and E (dotted lines). (G) Population analysis of ratio between PM fluorescence within or outside bead–cell interface for at least 30 cells per group, error bars show SEM. P-value was calculated using an unpaired two-tailed Student’s t test with a 99% CI. n > 10 cells per experiment.
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fig4: Reduction of negative charge at the inner leaflet of the T cell synapse during early stages of TCR engagement by peptide-MHC. (A and B) B-A8 T cells expressing the anionic lipid probe R-pre-mRFP were stimulated with HA-DR4/ICAM-1 lipid beads and imaged by confocal microscopy at 10-s intervals. Top row: typical cell showing bead (blue) and R-pre binding to inner leaflet (red) as synapse is formed. Bottom row shows intensity of R-pre fluorescence using pseudo-color gradient; quantification within or outside of synapse (B). Images representative of n > 3 experiments. Bar, 5 µm. (C and E) B-A8 T cells expressing both R-pre-mRFP and ZAP-70-eGFP probes (C) or MyrPalm-eGFP probe (E) were stimulated with HA-DR4/ICAM-1 lipid beads and imaged by confocal microscopy. Bottom rows show higher magnification (white box) and pseudo-color gradient of fluorescence intensity. Dotted line represents cross section for cells shown in D and F. Images representative of n > 3 experiments. Bar, 10 µm. (D and F) Fluorescence signals of bead (blue), R-pre (red), and ZAP-70 (green; D) or MyrPalm (green; F) across cells in C and E (dotted lines). (G) Population analysis of ratio between PM fluorescence within or outside bead–cell interface for at least 30 cells per group, error bars show SEM. P-value was calculated using an unpaired two-tailed Student’s t test with a 99% CI. n > 10 cells per experiment.

Mentions: We introduced a monomeric RFP (mRFP)–tagged version of R-pre (R-pre-mRFP) into B-A8 T cells to examine possible changes in PM lipid composition during early stages of T cell activation (Fig. 4 A). As previously described, the probe primarily localized to the inner leaflet of the PM (Fig. 4, A and C; Yeung et al., 2006). When T cells were activated through the TCR using HA-DR4/ICAM-1 lipid beads, a rapid reduction in R-pre-mRFP fluorescence at the PM was observed within the synapse (Fig. 4, A and B). This decrease was observed soon after T cell–APB contact, and occurred concomitant with an increase in ZAP-70-eGFP fluorescence at the interface (a peak of ZAP-70 fluorescence was visualized at the interface, despite substantial cytosolic ZAP-eGFP fluorescence; Fig. 4, C and D). It was important to assess the possibility of steric hindrance by recruited signaling molecules. We constructed a control protein that was also anchored to the membrane through a lipid tail: eGFP with an N-terminal myristoylation and dual palmitoylation signals (MyrPalm-eGFP). This probe also associated primarily with the inner leaflet of the PM (Fig. 4 E; Hashimoto-Tane et al., 2010). Only small changes in MyrPalm-eGFP fluorescence were observed at the T cell–APB interface when T cells expressing this control protein were stimulated with APB (Fig. 4, E-G).


Local changes in lipid environment of TCR microclusters regulate membrane binding by the CD3ε cytoplasmic domain.

Gagnon E, Schubert DA, Gordo S, Chu HH, Wucherpfennig KW - J. Exp. Med. (2012)

Reduction of negative charge at the inner leaflet of the T cell synapse during early stages of TCR engagement by peptide-MHC. (A and B) B-A8 T cells expressing the anionic lipid probe R-pre-mRFP were stimulated with HA-DR4/ICAM-1 lipid beads and imaged by confocal microscopy at 10-s intervals. Top row: typical cell showing bead (blue) and R-pre binding to inner leaflet (red) as synapse is formed. Bottom row shows intensity of R-pre fluorescence using pseudo-color gradient; quantification within or outside of synapse (B). Images representative of n > 3 experiments. Bar, 5 µm. (C and E) B-A8 T cells expressing both R-pre-mRFP and ZAP-70-eGFP probes (C) or MyrPalm-eGFP probe (E) were stimulated with HA-DR4/ICAM-1 lipid beads and imaged by confocal microscopy. Bottom rows show higher magnification (white box) and pseudo-color gradient of fluorescence intensity. Dotted line represents cross section for cells shown in D and F. Images representative of n > 3 experiments. Bar, 10 µm. (D and F) Fluorescence signals of bead (blue), R-pre (red), and ZAP-70 (green; D) or MyrPalm (green; F) across cells in C and E (dotted lines). (G) Population analysis of ratio between PM fluorescence within or outside bead–cell interface for at least 30 cells per group, error bars show SEM. P-value was calculated using an unpaired two-tailed Student’s t test with a 99% CI. n > 10 cells per experiment.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3526357&req=5

fig4: Reduction of negative charge at the inner leaflet of the T cell synapse during early stages of TCR engagement by peptide-MHC. (A and B) B-A8 T cells expressing the anionic lipid probe R-pre-mRFP were stimulated with HA-DR4/ICAM-1 lipid beads and imaged by confocal microscopy at 10-s intervals. Top row: typical cell showing bead (blue) and R-pre binding to inner leaflet (red) as synapse is formed. Bottom row shows intensity of R-pre fluorescence using pseudo-color gradient; quantification within or outside of synapse (B). Images representative of n > 3 experiments. Bar, 5 µm. (C and E) B-A8 T cells expressing both R-pre-mRFP and ZAP-70-eGFP probes (C) or MyrPalm-eGFP probe (E) were stimulated with HA-DR4/ICAM-1 lipid beads and imaged by confocal microscopy. Bottom rows show higher magnification (white box) and pseudo-color gradient of fluorescence intensity. Dotted line represents cross section for cells shown in D and F. Images representative of n > 3 experiments. Bar, 10 µm. (D and F) Fluorescence signals of bead (blue), R-pre (red), and ZAP-70 (green; D) or MyrPalm (green; F) across cells in C and E (dotted lines). (G) Population analysis of ratio between PM fluorescence within or outside bead–cell interface for at least 30 cells per group, error bars show SEM. P-value was calculated using an unpaired two-tailed Student’s t test with a 99% CI. n > 10 cells per experiment.
Mentions: We introduced a monomeric RFP (mRFP)–tagged version of R-pre (R-pre-mRFP) into B-A8 T cells to examine possible changes in PM lipid composition during early stages of T cell activation (Fig. 4 A). As previously described, the probe primarily localized to the inner leaflet of the PM (Fig. 4, A and C; Yeung et al., 2006). When T cells were activated through the TCR using HA-DR4/ICAM-1 lipid beads, a rapid reduction in R-pre-mRFP fluorescence at the PM was observed within the synapse (Fig. 4, A and B). This decrease was observed soon after T cell–APB contact, and occurred concomitant with an increase in ZAP-70-eGFP fluorescence at the interface (a peak of ZAP-70 fluorescence was visualized at the interface, despite substantial cytosolic ZAP-eGFP fluorescence; Fig. 4, C and D). It was important to assess the possibility of steric hindrance by recruited signaling molecules. We constructed a control protein that was also anchored to the membrane through a lipid tail: eGFP with an N-terminal myristoylation and dual palmitoylation signals (MyrPalm-eGFP). This probe also associated primarily with the inner leaflet of the PM (Fig. 4 E; Hashimoto-Tane et al., 2010). Only small changes in MyrPalm-eGFP fluorescence were observed at the T cell–APB interface when T cells expressing this control protein were stimulated with APB (Fig. 4, E-G).

Bottom Line: Release of the CD3ε cytoplasmic domain from the membrane is accompanied by a substantial focal reduction in negative charge and available PS in TCR microclusters.These changes in the lipid composition of TCR microclusters even occur when TCR signaling is blocked with a Src kinase inhibitor.Local changes in the lipid composition of TCR microclusters thus render the CD3ε cytoplasmic domain accessible during early stages of T cell activation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02115, USA. etienne.gagnon@umontreal.ca

ABSTRACT
The CD3ε and ζ cytoplasmic domains of the T cell receptor bind to the inner leaflet of the plasma membrane (PM), and a previous nuclear magnetic resonance structure showed that both tyrosines of the CD3ε immunoreceptor tyrosine-based activation motif partition into the bilayer. Electrostatic interactions between acidic phospholipids and clusters of basic CD3ε residues were previously shown to be essential for CD3ε and ζ membrane binding. Phosphatidylserine (PS) is the most abundant negatively charged lipid on the inner leaflet of the PM and makes a major contribution to membrane binding by the CD3ε cytoplasmic domain. Here, we show that TCR triggering by peptide--MHC complexes induces dissociation of the CD3ε cytoplasmic domain from the plasma membrane. Release of the CD3ε cytoplasmic domain from the membrane is accompanied by a substantial focal reduction in negative charge and available PS in TCR microclusters. These changes in the lipid composition of TCR microclusters even occur when TCR signaling is blocked with a Src kinase inhibitor. Local changes in the lipid composition of TCR microclusters thus render the CD3ε cytoplasmic domain accessible during early stages of T cell activation.

Show MeSH
Related in: MedlinePlus