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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: 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.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.

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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Signaling activities upon TCR triggering in whole cell lysates.A. Control and sterol-enriched Jurkat cells were activated with 5 μg of UCHT1 (anti-CD3 mAb) for the indicated periods of time. Whole cell lysates were probed for ZAP70 phosphorylated at tyrosine 319. B. Quantification of tyrosine 319 phosphorylation of ZAP70. The data show the mean and range of two independent experiments. C. Multiplex analysis of T cell signaling. 2×106 sterol-enriched Jurkat wt cells were activated with 5 μg/ml of anti-CD3 UCHT1 antibody for 0-15 min at 37°C. Non-site specific tyrosine phosphorylation of CD3ε, Lck, ZAP70, LAT and ERK1/2 (Tyr185/Tyr187) as well as serine phosphorylation of CREB (Ser133) in whole cell lysates was assessed by multiplex microbead suspension assay. The data is one representative experiment; error bars represent standard deviations. Legend shown in B applies to data in C.
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pone-0002262-g005: Signaling activities upon TCR triggering in whole cell lysates.A. Control and sterol-enriched Jurkat cells were activated with 5 μg of UCHT1 (anti-CD3 mAb) for the indicated periods of time. Whole cell lysates were probed for ZAP70 phosphorylated at tyrosine 319. B. Quantification of tyrosine 319 phosphorylation of ZAP70. The data show the mean and range of two independent experiments. C. Multiplex analysis of T cell signaling. 2×106 sterol-enriched Jurkat wt cells were activated with 5 μg/ml of anti-CD3 UCHT1 antibody for 0-15 min at 37°C. Non-site specific tyrosine phosphorylation of CD3ε, Lck, ZAP70, LAT and ERK1/2 (Tyr185/Tyr187) as well as serine phosphorylation of CREB (Ser133) in whole cell lysates was assessed by multiplex microbead suspension assay. The data is one representative experiment; error bars represent standard deviations. Legend shown in B applies to data in C.

Mentions: We further tested early signaling events upon TCR triggering in whole cell lysates by probing for tyrosine phosphorylation with immunoblotting (Fig. 5A and B) or multiplex analysis using a microbead suspension assay (Fig. 5C). Surprisingly, we found no differences in the degree or rate of specific (Fig. 5B) or total tyrosine phosphorylation of CD3ζ, Lck, ZAP70, LAT, ERK and CREB (Fig. 5C). We also found no differences between the five cell conditions in PLCγ1 phosphorylation upon TCR stimulation. Differences within an assay such as the lower phosphorylation of Lck in cholesterol-enriched T cells were not reproduced in independent experiments. It is possible that the assays employed here, particularly immunoblotting, are not sensitive enough to detect small changes in overall phosphorylation rate but it appears that signaling activities in whole cell lysates via tyrosine phosphorylation are unaltered in sterol-enriched T cells.


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)

Signaling activities upon TCR triggering in whole cell lysates.A. Control and sterol-enriched Jurkat cells were activated with 5 μg of UCHT1 (anti-CD3 mAb) for the indicated periods of time. Whole cell lysates were probed for ZAP70 phosphorylated at tyrosine 319. B. Quantification of tyrosine 319 phosphorylation of ZAP70. The data show the mean and range of two independent experiments. C. Multiplex analysis of T cell signaling. 2×106 sterol-enriched Jurkat wt cells were activated with 5 μg/ml of anti-CD3 UCHT1 antibody for 0-15 min at 37°C. Non-site specific tyrosine phosphorylation of CD3ε, Lck, ZAP70, LAT and ERK1/2 (Tyr185/Tyr187) as well as serine phosphorylation of CREB (Ser133) in whole cell lysates was assessed by multiplex microbead suspension assay. The data is one representative experiment; error bars represent standard deviations. Legend shown in B applies to data in C.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2384009&req=5

pone-0002262-g005: Signaling activities upon TCR triggering in whole cell lysates.A. Control and sterol-enriched Jurkat cells were activated with 5 μg of UCHT1 (anti-CD3 mAb) for the indicated periods of time. Whole cell lysates were probed for ZAP70 phosphorylated at tyrosine 319. B. Quantification of tyrosine 319 phosphorylation of ZAP70. The data show the mean and range of two independent experiments. C. Multiplex analysis of T cell signaling. 2×106 sterol-enriched Jurkat wt cells were activated with 5 μg/ml of anti-CD3 UCHT1 antibody for 0-15 min at 37°C. Non-site specific tyrosine phosphorylation of CD3ε, Lck, ZAP70, LAT and ERK1/2 (Tyr185/Tyr187) as well as serine phosphorylation of CREB (Ser133) in whole cell lysates was assessed by multiplex microbead suspension assay. The data is one representative experiment; error bars represent standard deviations. Legend shown in B applies to data in C.
Mentions: We further tested early signaling events upon TCR triggering in whole cell lysates by probing for tyrosine phosphorylation with immunoblotting (Fig. 5A and B) or multiplex analysis using a microbead suspension assay (Fig. 5C). Surprisingly, we found no differences in the degree or rate of specific (Fig. 5B) or total tyrosine phosphorylation of CD3ζ, Lck, ZAP70, LAT, ERK and CREB (Fig. 5C). We also found no differences between the five cell conditions in PLCγ1 phosphorylation upon TCR stimulation. Differences within an assay such as the lower phosphorylation of Lck in cholesterol-enriched T cells were not reproduced in independent experiments. It is possible that the assays employed here, particularly immunoblotting, are not sensitive enough to detect small changes in overall phosphorylation rate but it appears that signaling activities in whole cell lysates via tyrosine phosphorylation are unaltered in sterol-enriched T cells.

Bottom Line: 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.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.

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