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First published online 28 September 2004
doi: 10.1242/jcs.01380

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Tetraspanin CD82 controls the association of cholesterol-dependent microdomains with the actin cytoskeleton in T lymphocytes: relevance to co-stimulation

¹ INSERM U396, Hôpital Saint Louis, 1 avenue Claude Vellefaux 75010 Paris, France
² Unité d'Immunologie Virale, Institut Pasteur, 25, 28 rue du Docteur Roux, 75724 Paris CEDEX 15, France
³ Département de Biologie Cellulaire, Institut Cochin, INSERM U567, CNRS UMR 8104, Université Paris V. 22, rue Méchain, 75014 Paris, France
⁴ INSERM U268, Institut Andre-Lwoff, Hopital Paul-Brousse, 7 rue Guy Moquet, BP 8, 94801 Villejuif CEDEX, France

View larger version (60K): [in a new window]   Fig. 1. Tetraspanin molecules co-cap with GM1 gangliosides together with F-actin. (Aa-d,Ba-d) Capping and labeling of GM1 gangliosides at the Jurkat T-cell surface induced by FITC-labeled avidin cross-linking (at 37°C) of biotin-labeled CTx. Surface proteins (CD9, CD81, CD82 or CD71) and actin were visualized on capped and fixed cells by labeling with specific mAbs, followed by Cy3-conjugated secondary reagent and rhodamine-conjugated phalloidin. (Be-h) Reciprocal experiments in which surface proteins were first labeled with specific antibodies and induced to cap with Cy3-coupled secondary antibodies. GM1 and actin were stained on capped and fixed cells as previously. (Ae-h) Control experiments in which cells were stained without any capping with indicated antibody and Cy3 secondary reagent, GM1 or rhodamine-phalloidin at 4°C. In all pictures, red represents actin, GM1 is shown in green and surface proteins (tetraspanins or CD71) are in dark blue. (A) Cross-linking of GM1. (Aa-c,e-g) Single color analysis. (Ad,h) Merge of three images. (B) Only merged images are shown. (Ba-d) Cross-linking of GM1. (Be-h) Cross-linking of CD82 (e), CD81 (f), CD9 (g) or CD71 (h). Dark blue indicates CD82 (a,e), CD81(b,f), CD9 (c,g) or CD71 (d,h). White colors and arrows indicate clusters of tetraspanin, actin and GM1.

View larger version (39K): [in a new window]   Fig. 2. Distribution of different tetraspanins (CD9, CD81 and CD82), raft markers (LAT) and non-raft molecules (CD71) in sucrose gradients. 2x107 Jurkat cells (A,B) or PHA blasts from healthy donors (C) were lysed at 4°C in MNE buffer supplemented with either 1% BRIJ-58 (A, left) or 0.5% Triton X-100 (A, right, B, C). Lysates were centrifuged overnight at 4°C in a three-step (42.5%, 30%, 5%) sucrose gradient and 12 fractions of 280 µl were collected from the top. An equal amount of each fraction (B,C) or a pool of four sequential fractions (1-4, 5-8, 9-12) (A) was subjected to non-reducing SDS-PAGE and immunoblotting was carried out using antibodies against molecules indicated in the figures.

View larger version (50K): [in a new window]   Fig. 3. Tetraspanin density distribution on a sucrose gradient depends on the presence of cholesterol but not of tetraspanin post-translational modifications. 2x107 Jurkat cells were lysed at 4°C in MNE buffer supplemented with 0.5% Triton X-100. Lysates were centrifuged overnight at 4°C in a three-step (42.5%, 30%, 5%) sucrose gradient and 12 fractions of 280 µl were collected from the top. After non-reducing SDS-PAGE, immunoblotting was carried out with anti-CD82 (A, bottom, B) and anti-CD9 (A, top, C) antibodies. (A) Jurkat-cell lysis was performed in the presence or absence of 0.1% saponin, a specific cholesterol detergent, as indicated. (B) Before Jurkat-cell lysis, cells were treated overnight with the glycosylation inhibitor tunicamycin. (C) Daudi cells expressing wild-type CD9, or unpalmitoylated CD9 were analysed. Lanes were loaded with 30 µl (A, top, all lanes; B,C, lanes 1-6) or 6 µl (B,C, lanes 7-12) of each fraction.

View larger version (88K): [in a new window]   Fig. 4. Low- and intermediate-density fractions originate from poorly solubilized microdomains of the plasma membrane. (A) 2x107 Jurkat cells were biotin-labeled and lysed at 4°C in MNE supplemented with 0.5% Triton X-100. After overnight centrifugation in a three-step (42.5 %, 30%, 5%) sucrose gradient, 12 fractions of 280 ml were collected from the top and four sequential fractions were pooled (1-4, 5-8, 9-12). An equal amount of each pool was either left untreated (cell lysate) or was immunoprecipitated with avidin coated beads (IPP: Avidin) before SDS-PAGE analysis and blotting with avidin (left) or anti-CD9 antibody (right). (B) 2x107 cells were lysed and fractionated by sucrose gradient as above. A pool of fractions 2-3 (F2-3, left) or 6-7 (F6-7, right) were washed, concentrated and analysed by electron microscopy. Bar, 1 µm.

View larger version (42K): [in a new window]   Fig. 5. The specific tetraspanin density distribution on a sucrose gradient is dependent on actin polymerization state. 2x107 Jurkat cells were lysed at 4°C in MNE supplemented with 0.5% Triton X-100, and centrifuged overnight at 4°C in a three-step (42.5%, 30%, 5%) sucrose gradient, and 12 fractions of 280 ml were collected from the top. Proteins resolved by SDS-PAGE were analyzed by immunoblots with indicated mAbs. (A) Before lysis, cells were treated for 2 hours with 2 µM latrunculin (a depolymerizing agent), and an equal amount of the pool of fractions (1-4) or (5-8) is shown. CD71* represent an overexpressed immunoblot of CD71. Numbers under each plots represent densitometric values normalized to the untreated pool of fractions 5-8, except the LAT blot, which is normalized to the untreated pool of fractions 1-4. (B) Fifteen minutes before lysis, cells were electroporated with or without phalloidin, lanes 1-6 were loaded with 30 µl of each fraction, lanes 7-12 with only 6 µl.

View larger version (38K): [in a new window]   Fig. 6. Tetraspanin engagement modifies the distribution of CD82 in sucrose density gradient. 2.107 Jurkat cells were cultured for 15 minutes at 37°C on plates coated, with unspecific antibodies or antibodies against CD9, CD53, CD63, CD81, or CD82. The cells were lysed in MNE supplemented with 0.5% Triton X-100 and centrifuged overnight at 4°C in a three-step (42.5%, 30%, 5%) sucrose gradient, 12 fractions of 280 µl were collected from the top, and the insoluble pellet was recovered by addition of 50 µl SDS sample buffer. (A) CD82 immunoblot of the 12 fractions recovered from unstimulated (top) or CD82-stimulated (bottom) Jurkat cells. (B) Four sequential fractions were pooled (1-4 and 5-8), resolved together with the insoluble pellet by SDS-PAGE and immunoblotted with mAbs against CD82, CD81 or CD9. Stimulation conditions are indicated at the top.

View larger version (59K): [in a new window]   Fig. 7. MßCD treatment inhibits all CD82 signaling events. Jurkat cells, treated or not with 5 mM MßCD for 30 minutes, were stimulated for various times at 37°C on antibody-coated plates. 0, nonspecific mAbs; CD3s, suboptimal doses of OKT3 (0.5 µg ml–1); CD3o, optimal doses of OKT3 (20 µg ml–1); CD82, 50 µg ml–1 C11. (A,B) 2x105 Jurkat cells were cultured for 1 hour on antibody-coated plates. (A) The number of adherent cells was evaluated by crystal violet staining, followed by densitometric analysis at 520 nm. (B) Adherent cells were fixed, permeabilized, stained with rhodamine-phalloidin and visualized by confocal microscopy. Bar, 10 µm. (C) 2x105 Jurkat cells were cultured for 10 minutes on antibody-coated plates and lysed in SDS sample buffer. Proteins were resolved by SDS-PAGE and tyrosine phosphorylations were evaluated by immunoblotting with the anti-phosphotyrosine antibody 4G10. Cell treatments and stimulations are indicated at the top. Arrows indicate proteins for which phosphorylation was reduced by MßCD treatment.

View larger version (88K): [in a new window]   Fig. 8. CD82 concentrates in the IS upon contact with APCs. T8-1 cells expressing YFP-tagged CD82 (YFP-CD82) and labeled with the calcium indicator Fura 2 were added to antigen-pulsed L625.7 fibroblasts. (A) Merged phase-contrast images and YFP fluorescence at the beginning of the experiment. Cell 1, uniformly stained, did not move; cell 2 moved rapidly as indicated by the arrow. (B) Merged images of phase-contrast microscopy and either Fura-2-fluorescence ratio (left; low calcium concentration is indicated in blue and high calcium concentration in red) or YFP fluorescence (right) after 20 minutes of contact between T cells and APCs. In T cells with a high intracellular calcium level (1-4), CD82 is relocalized to the T-cell/APC contact area. (C) After 20 minutes of contact with APCs, cells were fixed, permeabilized and labeled with rhodamine-phalloidin. Cells were analysed simultaneously for YFP and rhodamine fluorescence and phase-contrast microscopy. The phase-contrast image is merged with YFP fluorescence (left), rhodamine fluorescence (middle) or both (right).