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First published online July 12, 2005
doi: 10.1242/10.1242/jcs.02442

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Cold-induced coalescence of T-cell plasma membrane microdomains activates signalling pathways

Anthony I. Magee^1,*, Jeremy Adler^2,* and Ingela Parmryd^1,3,

¹ Division of Biomedical Sciences, Imperial College Faculty of Medicine, Exhibition Road, South Kensington, London, SW7 2AZ, UK
² Department of Developmental Biology, The Wenner-Gren Institute, Stockholm University, 106 91 Stockholm, Sweden
³ Department of Cell Biology, The Wenner-Gren Institute, Stockholm University, 106 91 Stockholm, Sweden

View larger version (71K): [in a new window]   Fig. 1. Patching of GM1-CT-B in Jurkat cells fixed at different temperatures. (A) Jurkat T cells were PFA-fixed at the indicated temperatures and treated with either CT-B-rhodamine or CT-B-Alexa Fluor 594 followed anti-CT-B to induce patching. Single confocal equatorial sections show fluorescence in the rhodamine channel. (B) Cells treated with CT-B-Alexa Fluor 594 and patched with anti-CT-B at 37°C prior to fixation. Scale bars: 10 µm.

View larger version (34K): [in a new window]   Fig. 2. Temperature dependence of Lck, CD3 and TfR distribution. Jurkat T cells were fixed at the indicated temperatures and stained with anti-Lck, anti-CD3 or anti-TfR followed by FITC-conjugated secondary antibodies. (A) Single confocal equatorial sections show fluorescence in the fluorescein channel. Arrowheads indicate plasma membrane regions with very low fluorescence intensities in cells with a patchy distribution of fluorophore. Images are typical for the respective populations. Scale bars: 5 µm. (B) Perimeter traces of the cells shown in A. The plasma membrane was manually marked with sequential points and adjacent points were then joined by line segments to delineate the membrane. The fluorescence intensities around the membrane were copied into a one dimensional image. The x and y axes show the intensity range and total intensity, respectively. (C) Accumulative intensity distribution histograms for populations of cells; n=10/11.

View larger version (65K): [in a new window]   Fig. 3. Localisation of Lck in relation to the TfR and CD3 in Jurkat cells at 0°C. Jurkat T cells were fixed at 0°C and stained with mouse anti-Lck and rabbit anti-TfR or rabbit anti-Lck and mouse anti-CD3, followed by anti-mouse-Alexa Fluor 594 and anti-rabbit-FITC or anti-rabbit-Alexa Fluor 488 antibodies. Scale bars: 5 µm.

View larger version (53K): [in a new window]   Fig. 4. Temperature dependence of GM1 distribution and the effect of Triton X-100 extraction. Jurkat T cells were fixed at the specified temperatures and, where indicated, extracted with 1% Triton X-100 on ice for 5 minutes. The cells were then stained with CT-B-rhodamine or CT-B-Alexa Fluor 594. Single equatorial confocal sections show fluorescence in the rhodamine channel. Arrowheads indicate plasma membrane regions where GM1 has been extracted. Scale bars: 5 µm.

View larger version (82K): [in a new window]   Fig. 5. Temperature-dependence of tyrosine phosphorylation and ERK activation in Jurkat cells. Cells were incubated at the indicated temperatures for 10 minutes, of which, anti-CD3 (OKT3) at 1 µg/ml was present for the last 5 minutes in the stimulated aliquots (lanes +). Lysates were analysed by western blotting for (A) tyrosine phosphorylation (4G10) or (B) ERK activation (anti-phospho-p44/42 MAP kinase). Gels were loaded on an equal protein basis. (C) Cells incubated at 10°C for 5 minutes were stimulated with 1 µg/ml OKT3 for 5 minutes at 10°C and lysed in 1% NP40-containing buffer. Precleared lysates were used for immunoprecipitation with either anti-ZAP-70, anti-LAT or anti-Lck rabbit antisera coupled to protein A Sepharose beads with dimethylpimelidate. Precipitated proteins were recovered by glycine elution and analysed by western blotting for tyrosine phosphorylation. The arrow indicates the band in the Lck lane. Molecular mass markers are indicated (kDa). Fluorographs shown are representative of 11, nine and three experiments, respectively.

View larger version (81K): [in a new window]   Fig. 6. Correlation of tyrosine phosphorylation and GM1 distribution. (A) Jurkat T cells were fixed at the indicated temperatures, permeabilised and stained with CT-B-Alexa Fluor 594 and 4G10 (anti-pTyr) followed by anti-mouse FITC-conjugated secondary antibody. Single equatorial confocal sections show fluorescence in the rhodamine and FITC channels. Scale bars: 5 µm. For cells fixed at 0°C, the Pearson correlation coefficient between GM1-CT-B and tyrosine phosphorylation at the plasma membrane was 0.488±0.032 (mean±s.e.m.; n=10).

View larger version (125K): [in a new window]   Fig. 7. Distribution of tyrosine phosphorylated proteins. A, TX-DRMs were prepared from Jurkat cells incubated and stimulated with 1 µg/ml OKT3 (+) or left unstimulated (–) at either 10 or 37°C. TX-DRMs from 107 cells were loaded on the gel. (B) TX-DRMs (DRM), Triton-soluble proteins (SP) and pellets (P) from cells incubated at 37°C were loaded on an equal cell basis. Molecular mass markers are indicated (kDa). Fluorographs shown are representative of two experiments.

View larger version (87K): [in a new window]   Fig. 8. Genetic requirement for cold-induced tyrosine phosphorylation. Jurkat-derived cell lines J.RT3-T3.5 (TCR/CD3 deficient) and JCam-1.6 (Lck deficient) were examined for cold-induced tyrosine phosphorylation. Cells were incubated at the indicated temperatures for 10 minutes. Lysates were analysed by western blotting for tyrosine phosphorylation (4G10). Gels were loaded on an equal protein basis, confirmed by probing for ERK (anti-p44/42 MAP kinase). For the J.RT3-T3.5 cells, the two panels correspond to different exposure times of the same membrane. Molecular mass markers are indicated (kDa). Fluorographs shown are representative of four experiments.

View larger version (111K): [in a new window]   Fig. 9. Temperature dependence of tyrosine phosphorylation in CD4+ human primary T cells. Cells were incubated at the indicated temperatures for 10 minutes, of which, anti-CD3 (OKT3) at 1 µg/ml was present for the last 5 minutes in the stimulated aliquots (lanes +). Lysates were analysed by western blotting for (A) tyrosine phosphorylation (4G10) or (B) ERK activation (anti-phospho-p44/42 MAP kinase). Gels were loaded on an equal protein basis. Molecular mass markers are indicated (kDa). Fluorographs shown are representative of three (A) and two (B) experiments.

View larger version (57K): [in a new window]   Fig. 10. Model of signalling from the T-cell receptor. In the resting T cell, the TCR and Lck, as well as other signalling molecules, exist in small plasma membrane domains with a lipid environment excluding deactivating phosphatases. The circumference to area ratio of the domains is high so any tyrosine phosphorylated proteins within the domains quickly get dephosphorylated upon contact with the surrounding phosphatases. Upon ligation of the TCR, the domains coalesce to larger entities decreasing their circumference to area ratio, thus reducing chance encounters with surrounding phosphatases and thereby increasing the lifetime of any tyrosine phosphorylated proteins and related signalling. Upon cooling, the small domains aggregate as a result of phase separation creating large platforms for sustained signalling.

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