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Rho GTPases link cytoskeletal rearrangements and activation processes induced via the tetraspanin CD82 in T lymphocytes

Alix Delaguillaumie¹, Cécile Lagaudrière-Gesbert^*, Michel R. Popoff² and Hélène Conjeaud^1,

¹ U332 – Institut National de la Santé et la Recherche Médicale. Institut Cochin de Génétique Moléculaire, 22 rue Méchain, 75014 Paris, France
² Toxines Microbiennes, Institut Pasteur, 28 rue du Dr Roux 75724 Paris Cedex15, France
^* Present address: Harvard Medical School, Dept of Pathology, D2-143, 200 Longwood Avenue, Boston MA-02115, USA

View larger version (24K): [in a new window]   Fig. 1. CD82 engagement triggers tyrosine phosphorylation of Vav and SLP76. Jurkat cells were cultured for 10 minutes at 37°C on uncoated plates or plates coated with anti-CD82 (C11, 50 µg/ml). (A) upper panel, after lysis (5x106 cells) in SDS sample buffer and SDS-PAGE analysis, tyrosine-phosphorylated proteins were analyzed by immunoblotting with 4G10 antibody. Stimulation, lane 1; no mAb, lane 2; anti-CD3o (optimal OKT3, 20 µg/ml); anti-CD82 (C11, 50 µg/ml). (A) Middle and lower panels, after dehybridization, the same membrane was re-probed with anti-Vav1 rabbit (middle panel) or anti-SLP76 sheep antisera (lower panel). (B) After solubilization (20x106 cells) in lysis buffer, 500 µg of each samples were immunoprecipitated with anti-Vav1 (rabbit polyclonal IgG), anti-SLP76 (sheep polyclonal IgG.) or control antibodies (C1, rabbit IgG; C2, sheep IgG). Immunoprecipitates were resolved by 7.5% SDS-PAGE and blotted with the anti-phosphotyrosine mAb 4G10 (upper panel), monoclonal mouse anti-Vav1 (middle panel) or polyclonal sheep anti-SLP76 (lower panel). The data shown are representative of three independent experiments.

View larger version (96K): [in a new window]   Fig. 2. Rho-GTPase-inactivation inhibits CD82-induced morphological changes. (a,b) Untreated Jurkat cells (a) and Jurkat cells treated (b) for two hours with 0.1 µg/ml of Toxin B from C. difficile were cultured for one hour at 37°C, on anti-CD82-coated plates (C11, 50 µg/ml). (c,d) Jurkat cells were cultured at 37°C on anti-CD82-coated plates (C11, 50 µg/ml). After one hour, 0.1 µg/ml of Toxin B was added (d), or not (c), to the culture medium and the cells were cultured for another two hours on anti-CD82 coated plates. After removing non-adherent cells, Jurkat cells were fixed, permeabilized and stained with rhodamine-phalloidin. The data shown are representative of five independent experiments. Bar represents 10 µm.

View larger version (59K): [in a new window]   Fig. 3. Inactivation of RhoA inhibits CD82-induced morphological changes. Jurkat cells were electroporated with Lucifer Yellow (1 mg/ml) with or without 20 µg/ml C3 exoenzyme from C. botulinum. After two hours in culture, cells were tested for their response to CD82 stimulation, as described in the legend of Figure 1. (a) Jurkat cells electroporated with Lucifer Yellow alone. (b) Cells electroporated with both Lucifer Yellow and C3 exoenzyme. The data shown are representative of three independent experiments. Bar represents 10 µm.

View larger version (77K): [in a new window]   Fig. 4. Expression of N17Rac1 or N17Cdc42 inhibits most of the CD82-induced morphological changes but different cytoskeletal rearrangements still occur. Jurkat cells were transfected with 3 µg GFP and 15 µg of empty vector (a), c-myc-tagged N17Rac1 (b,d) or N17Cdc42 (c,e) as described in Materials and Methods and sorted by flow cytometry by their GFP expression after 48 hours in culture. Cells were stimulated for two hours by culture on anti-CD82-coated plates and assayed for morphological changes as described in the legend of Fig. 1. The data shown are representative of three independent experiments. Bar represents 10 µm.

View larger version (39K): [in a new window]   Fig. 5. CD82-specific translocation to the detergent insoluble fraction is independent of Rho GTPases. Jurkat cells, treated with 0.1 µg/ml Toxin B for two hours or untreated, were cultured for 15 minutes at 37°C on antibody-coated plates (anti-CD82: c11, 50 µg/ml; anti-CD9:Syb1, 50 µg/ml; anti-CD81: Z81, 50 µg/ml). After SDS-PAGE analysis, proteins of the soluble (equivalent to 105 cells per lane) and insoluble fractions (equivalent to 2x106 cells per lane) were probed by immunoblotting with anti-CD82 or anti-CD81 mAbs. (A) Stimulation: lane 1, 0; lane 2, anti-CD82; lane 3, anti-CD9; lane 4, anti-CD81. The left and right panels represent the soluble and insoluble fractions respectively. The lower and upper panels represent western blots with anti-CD82 and anti-CD81 antibodies, respectively. (B) Stimulation: lanes 1-2, no mAb; lanes 3-4, anti-CD82 mAbs (C11, 50 µg/ml). Treatment: lanes 1, 3, no addition; lanes 2, 4, 0.1 µg/ml Toxin B. Left panel and right panels represent soluble and insoluble fractions, respectively. The data are representative of three independent experiments.

View larger version (34K): [in a new window]   Fig. 6. Rho GTPases and cytoskeleton participate in Vav and SLP76 phosphorylation. Jurkat cells, treated with 0.1 µg/ml of Toxin B for two hours or with cytochalasin for 30 minutes or left untreated were cultured for 15 minutes at 37°C on plates coated with anti-CD82 (C11, 50 µg/ml) or left uncoated. After lysis (5x106 cells) in SDS sample buffer, SDS-PAGE analysis was performed. Upper panel, immunoblot with anti-phosphotyrosine antibodies (4G10). Stimulation: lanes 1, no mAb; lane 2-4, anti-CD82. Cell treatment: lanes 1-2, untreated cells; lanes 3, Toxin B; lane 4, cytochalasin. Lower panel: after dehybridization, the same membrane was re-probed with anti-ZAP70 rabbit antisera.

View larger version (54K): [in a new window]   Fig. 7. Inactivation of all the Rho GTPases inhibits CD82 cosignaling activity. (A,B) Jurkat cells (5x106 cells) were cultured for 15 minutes at 37°C on antibody-coated or non-coated plates. After lysis in SDS sample buffer, SDS-PAGE analysis was performed. (A) An immunoblot with anti-phosphotyrosine antibodies 4G10. Left panel: cells treated with (lanes 2, 5 and 7) or without (lanes 1, 3, 4 and 6), 0.1 µg/ml Toxin B for two hours. Stimulation: lanes 1-2, no mAb; lane 3, suboptimal doses of anti-CD3 (OKT3: 0.5 µg/ml); lanes 4-5, optimal doses of anti-CD3 (OKT3: 20 µg/ml); lanes 6-7, anti-CD82 and suboptimal doses of anti-CD3 (C11: 50 µg/ml, OKT3: 0.5 µg/ml). Right panel: before stimulation Jurkat cells were transfected with 3 µg/ml GFP vector and 15 µg/ml of N17Rho GTPases or empty vector and sorted by their levels of GFP expression after 48 hours in culture. (B) After dehybridization, the same membrane was reprobed with anti-ZAP70 rabbit antiserum. (C) Jurkat cells (20x106 cells), treated or not with 0.1 µg/ml toxin B for two hours, were cultured for 15 minutes at 37°C on antibody-coated plates. After solubilization in lysis buffer, 500 µg proteins of each sample were immuno-precipitated with anti-ZAP70 or anti-LAT rabbit antisera. Immunoprecipitates were resolved by 7.5% (for ZAP) or 12% (for LAT) SDS-PAGE and blotted with the anti-phosphotyrosine mAb 4G10. The data shown are representative of four independent experiments.

View larger version (194K): [in a new window]   Fig. 8. Surface-expressed YFP-CD82 localized in contact areas with anti-CD3 coated beads. Jurkat cells (2x105) were transfected by electroporation with 15 µg of YFP-CD82. After 48 hours in culture, the cells were stimulated, or not (A), by culturing at 37°C with 2x105 control IgG2a (B) or anti-CD3-coated (C) beads. After 15 minutes of contact, cells were plated on microscope slides, fixed and analyzed by fluorescent microscopy.

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