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Functional implications of plasma membrane condensation for T cell activation.

Rentero C, Zech T, Quinn CM, Engelhardt K, Williamson D, Grewal T, Jessup W, Harder T, Gaus K - PLoS ONE (2008)

Bottom Line: Upon 7KC treatment, T cell antigen receptor (TCR) triggered calcium fluxes and early tyrosine phosphorylation events appear unaltered.However, signaling complexes form less efficiently on the cell surface, fewer phosphorylated signaling proteins are retained in the plasma membrane and actin restructuring at activation sites is impaired in 7KC-enriched cells resulting in compromised downstream activation responses.Our data emphasizes lipids as an important medium for the organization at T cell activation sites and strongly indicates that membrane condensation is an important element of the T cell activation process.

View Article: PubMed Central - PubMed

Affiliation: Centre for Vascular Research, University of New South Wales and the Department of Haematology, Prince of Wales Hospital, Sydney, Australia.

ABSTRACT
The T lymphocyte plasma membrane condenses at the site of activation but the functional significance of this receptor-mediated membrane reorganization is not yet known. Here we demonstrate that membrane condensation at the T cell activation sites can be inhibited by incorporation of the oxysterol 7-ketocholesterol (7KC), which is known to prevent the formation of raft-like liquid-ordered domains in model membranes. We enriched T cells with 7KC, or cholesterol as control, to assess the importance of membrane condensation for T cell activation. Upon 7KC treatment, T cell antigen receptor (TCR) triggered calcium fluxes and early tyrosine phosphorylation events appear unaltered. However, signaling complexes form less efficiently on the cell surface, fewer phosphorylated signaling proteins are retained in the plasma membrane and actin restructuring at activation sites is impaired in 7KC-enriched cells resulting in compromised downstream activation responses. Our data emphasizes lipids as an important medium for the organization at T cell activation sites and strongly indicates that membrane condensation is an important element of the T cell activation process.

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TIRF microscopy images of TCR activation sites of sterol-enriched T cells.Sterol-treated Jurkat cells were activated for 10 min on anti-CD3 mAb-coated glass coverslips, fixed and stained for phosphotyrosine (pY, A), phalloidin (B), ZAP70 phosphorylated at tyrosine 319 (D), LAT phosphorylated at tyrosine 191 (E) or PLCγ1 phosphorylation at tyrosine 783 (F). T cell activation sites were imaged by TIRF microscopy with a penetration depth of ∼100 nm. Panel C show the merged images with pY in green and F-actin in red. Bar 5 μm.
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pone-0002262-g007: TIRF microscopy images of TCR activation sites of sterol-enriched T cells.Sterol-treated Jurkat cells were activated for 10 min on anti-CD3 mAb-coated glass coverslips, fixed and stained for phosphotyrosine (pY, A), phalloidin (B), ZAP70 phosphorylated at tyrosine 319 (D), LAT phosphorylated at tyrosine 191 (E) or PLCγ1 phosphorylation at tyrosine 783 (F). T cell activation sites were imaged by TIRF microscopy with a penetration depth of ∼100 nm. Panel C show the merged images with pY in green and F-actin in red. Bar 5 μm.

Mentions: We independently monitored the formation of TCR activation clusters using total internal reflection fluorescence (TIRF) microscopy in sterol-treated Jurkat T cells (Fig. 7). The cells were allowed to settle on anti-CD3 mAb coated coverslips for 10 min at 37°C, fixed and probed for total tyrosine phosphorylation (pY, Fig. 7A and colored green in Fig. 7C), stained with phalloidin for F-actin (Fig. 7B and colored red in Fig. 7C), probed for ZAP70 phosphorylated at tyrosine 319 (Fig. 7D), LAT phosphorylated at tyrosine 191 (Fig. 7E) or PLCγ1 phosphorylated at tyrosine 783 (Fig. 7F). In control and cholesterol-loaded cells, a large number of tyrosine phosphorylation spots were visible at the activation site surrounded by a ring of F-actin as described previously [31], [32]. With increasing levels of 7KC, activation sites were smaller, tyrosine phosphorylation positive spots reduced in number and brightness (quantification in Fig. S4A) and the F-actin ring became less pronounced (Fig. S4B) with some small and bright F-actin spots visible in 7KC-enriched cells. Jurkat cells plated on poly-L-lysine- or transferrin receptor (TfR)-coated coverslips, under all sterol treatments spread and formed similarly bright phalloidin-stained lamellipodia (data not shown) indicating that 7KC-enriched cells are capable of actin polymerisation but are specifically deficient in producing TCR signals to mediate formation of actin rings. The intensity of phospho-ZAP70 staining was similar in control cells and cells enriched with cholesterol or 2:1 CH:7KC but reduced significantly at higher doses of 7KC (Fig. S4C). In contrast, phospho-LAT was significantly reduced in all sterol conditions (Fig. S4D) correlating with the reduced TCR surface expression (Fig. 3B). Importantly, as in immuno-isolated TCR activation sites, 7KC enrichment reduced phospho-LAT at the cell surface in a dose-dependent manner and to a greater extent than cholesterol enrichment. Similarly, PLCγ1 was significantly lower in 7KC-enriched cells compared to cholesterol-enriched or control cells so that the degree of pLAT correlate well with pPLCγ1. In summary, the microscopy data of reduced tyrosine phosphorylated proteins at the cell surface agrees extremely well with our biochemical observations of diminished signaling complexes in the plasma membrane of 7KC-enriched cells. Taken together, our data suggests that 7KC modulates the location of TCR signaling proteins with less signaling proteins recruited or retain at the cell surface and this impacts more severely on LAT or actin restructuring than on upstream components such as ZAP70 or elements of the CD3 complex.


Functional implications of plasma membrane condensation for T cell activation.

Rentero C, Zech T, Quinn CM, Engelhardt K, Williamson D, Grewal T, Jessup W, Harder T, Gaus K - PLoS ONE (2008)

TIRF microscopy images of TCR activation sites of sterol-enriched T cells.Sterol-treated Jurkat cells were activated for 10 min on anti-CD3 mAb-coated glass coverslips, fixed and stained for phosphotyrosine (pY, A), phalloidin (B), ZAP70 phosphorylated at tyrosine 319 (D), LAT phosphorylated at tyrosine 191 (E) or PLCγ1 phosphorylation at tyrosine 783 (F). T cell activation sites were imaged by TIRF microscopy with a penetration depth of ∼100 nm. Panel C show the merged images with pY in green and F-actin in red. Bar 5 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2384009&req=5

pone-0002262-g007: TIRF microscopy images of TCR activation sites of sterol-enriched T cells.Sterol-treated Jurkat cells were activated for 10 min on anti-CD3 mAb-coated glass coverslips, fixed and stained for phosphotyrosine (pY, A), phalloidin (B), ZAP70 phosphorylated at tyrosine 319 (D), LAT phosphorylated at tyrosine 191 (E) or PLCγ1 phosphorylation at tyrosine 783 (F). T cell activation sites were imaged by TIRF microscopy with a penetration depth of ∼100 nm. Panel C show the merged images with pY in green and F-actin in red. Bar 5 μm.
Mentions: We independently monitored the formation of TCR activation clusters using total internal reflection fluorescence (TIRF) microscopy in sterol-treated Jurkat T cells (Fig. 7). The cells were allowed to settle on anti-CD3 mAb coated coverslips for 10 min at 37°C, fixed and probed for total tyrosine phosphorylation (pY, Fig. 7A and colored green in Fig. 7C), stained with phalloidin for F-actin (Fig. 7B and colored red in Fig. 7C), probed for ZAP70 phosphorylated at tyrosine 319 (Fig. 7D), LAT phosphorylated at tyrosine 191 (Fig. 7E) or PLCγ1 phosphorylated at tyrosine 783 (Fig. 7F). In control and cholesterol-loaded cells, a large number of tyrosine phosphorylation spots were visible at the activation site surrounded by a ring of F-actin as described previously [31], [32]. With increasing levels of 7KC, activation sites were smaller, tyrosine phosphorylation positive spots reduced in number and brightness (quantification in Fig. S4A) and the F-actin ring became less pronounced (Fig. S4B) with some small and bright F-actin spots visible in 7KC-enriched cells. Jurkat cells plated on poly-L-lysine- or transferrin receptor (TfR)-coated coverslips, under all sterol treatments spread and formed similarly bright phalloidin-stained lamellipodia (data not shown) indicating that 7KC-enriched cells are capable of actin polymerisation but are specifically deficient in producing TCR signals to mediate formation of actin rings. The intensity of phospho-ZAP70 staining was similar in control cells and cells enriched with cholesterol or 2:1 CH:7KC but reduced significantly at higher doses of 7KC (Fig. S4C). In contrast, phospho-LAT was significantly reduced in all sterol conditions (Fig. S4D) correlating with the reduced TCR surface expression (Fig. 3B). Importantly, as in immuno-isolated TCR activation sites, 7KC enrichment reduced phospho-LAT at the cell surface in a dose-dependent manner and to a greater extent than cholesterol enrichment. Similarly, PLCγ1 was significantly lower in 7KC-enriched cells compared to cholesterol-enriched or control cells so that the degree of pLAT correlate well with pPLCγ1. In summary, the microscopy data of reduced tyrosine phosphorylated proteins at the cell surface agrees extremely well with our biochemical observations of diminished signaling complexes in the plasma membrane of 7KC-enriched cells. Taken together, our data suggests that 7KC modulates the location of TCR signaling proteins with less signaling proteins recruited or retain at the cell surface and this impacts more severely on LAT or actin restructuring than on upstream components such as ZAP70 or elements of the CD3 complex.

Bottom Line: Upon 7KC treatment, T cell antigen receptor (TCR) triggered calcium fluxes and early tyrosine phosphorylation events appear unaltered.However, signaling complexes form less efficiently on the cell surface, fewer phosphorylated signaling proteins are retained in the plasma membrane and actin restructuring at activation sites is impaired in 7KC-enriched cells resulting in compromised downstream activation responses.Our data emphasizes lipids as an important medium for the organization at T cell activation sites and strongly indicates that membrane condensation is an important element of the T cell activation process.

View Article: PubMed Central - PubMed

Affiliation: Centre for Vascular Research, University of New South Wales and the Department of Haematology, Prince of Wales Hospital, Sydney, Australia.

ABSTRACT
The T lymphocyte plasma membrane condenses at the site of activation but the functional significance of this receptor-mediated membrane reorganization is not yet known. Here we demonstrate that membrane condensation at the T cell activation sites can be inhibited by incorporation of the oxysterol 7-ketocholesterol (7KC), which is known to prevent the formation of raft-like liquid-ordered domains in model membranes. We enriched T cells with 7KC, or cholesterol as control, to assess the importance of membrane condensation for T cell activation. Upon 7KC treatment, T cell antigen receptor (TCR) triggered calcium fluxes and early tyrosine phosphorylation events appear unaltered. However, signaling complexes form less efficiently on the cell surface, fewer phosphorylated signaling proteins are retained in the plasma membrane and actin restructuring at activation sites is impaired in 7KC-enriched cells resulting in compromised downstream activation responses. Our data emphasizes lipids as an important medium for the organization at T cell activation sites and strongly indicates that membrane condensation is an important element of the T cell activation process.

Show MeSH
Related in: MedlinePlus