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First published online 13 December 2005
doi: 10.1242/jcs.02722

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ß2-chimaerin provides a diacylglycerol-dependent mechanism for regulation of adhesion and chemotaxis of T cells

¹ Department of Immunology and Oncology, Centro Nacional de Biotecnología/CSIC, Cantoblanco, E-28049 Madrid, Spain
² Department of Immunology, Centro de Investigaciones Biológicas/CSIC, Ramiro de Maeztu 9, E-28040 Madrid, Spain
³ Department of Molecular Pharmacology, University of Pennsylvania Medical School, 3620 Hamilton Walk, Philadelphia, PA 19104, USA

View larger version (40K): [in a new window]   Fig. 1. ß2-chimaerin is expressed in lymphoid cells. (A) Expression of ß2-chimaerin mRNA was determined in Jurkat T cells (JK) by RT-PCR using specific oligonucleotides; 1, 2 and ß2-expressing plasmids were used as controls. (B) ß2-chimaerin protein expression was analyzed in total lysates of the indicated lymphoid cell lines by western blotting with anti-ß2-chimaerin antibody. Membranes were re-probed with anti-tubulin antibody as a protein loading control. H, HT; HD, HDLM2; J, Jurkat; K, Karpas; M, Molt-4; N, Namalwa; SU, SU-DHL1; T, Toledo. (C) Subcellular localization of GFP-ß2-chimaerin in Jurkat T cells was examined by confocal microscopy (green, left), the Golgi was stained with the BODIPY TR fluorescent marker ceramide (red, center); merged fluorescence is shown in the right panel. Bar, 3 µm.

View larger version (41K): [in a new window]   Fig. 2. PMA-dependent regulation of GFP-ß2-chimaerin in intact Jurkat cells. (A) Jurkat T cells were transfected with vectors encoding GFP-ß2-chimaerin C1 domain (GFP-C1), GFP-ß2-chimaerin, GFP-ß2-chimaerin Q32A and GFP-ß2-chimaerin I130A. At 24 hours post-transfection, cells were suspended in HBSS and plated on poly-D,L-lysine-coated chamber slides. Slides were mounted on a 37°C plate and analyzed by confocal microscopy. PMA was added at 100 ng/ml after the first frame, and images captured every 10 seconds. Time-lapse confocal recording is shown in Movies 1-3 in the supplementary material. Images are shown at the indicated times to illustrate subcellular protein localization. (B) Jurkat T cells were transfected with GFP-ß2-chimaerin or GFP-ß2-chimaerin C1 domain mutant (GFP-F215G). At 24 hours post-transfection, cells were treated as in A. PMA (800 ng/ml) was added after the first frame, and images captured every 10 seconds. Time-lapse confocal recording is shown in Movie 4 in supplementary material. Images are shown at the indicated times to illustrate subcellular protein localization. (C) Jurkat T cells were co-transfected with a EE-tagged, constitutive active (RacV12) Rac mutant and either GFP-ß2-chimaerin or GFP-F215G mutant. At 24 hours post-transfection, cells were stimulated with PMA (100 ng/ml) for 3 minutes, fixed, stained with anti-EE antibodies (red) and images acquired by confocal microscopy. GFP-ß2-chimaerin localization (green) is shown in the left column, RacV12 expression (red) is shown in the middle column, merge images are shown on the right. Time-lapse confocal recording of a similar experiment performed in living cells is shown. Bars, 5 µm.

View larger version (37K): [in a new window]   Fig. 3. GFP-ß2-chimaerin-expressing Jurkat T cells show reduced PMA-induced F-actin polymerization. Jurkat T cells transfected with (A) GFP-ß2-chimaerin or (B) a GFP-coupled dominant-negative (RacN17) Rac mutant were stimulated with PMA (800 ng/ml, 5 minutes) or unstimulated (-), fixed and processed to determine GFP-ß2-chimaerin (A) or GFP-RacN17 (B) localization (green) and actin polymerization by Rhodamine-phalloidin staining (red). Control cells not expressing GFP constructs are indicated with an asterisk. Phase-contrast micrographs show the cells analyzed (left). Images are representative of the field observed for each condition. (C) Quantification of F-actin polymerization of experiments described in A and B was determined by analyzing intensity of fluorescence of Rhodamine-phalloidin staining. Values are the mean ± s.d. fluorescence intensity (arbitrary units) corresponding to 20 cells for each condition. Bars, 5 µm.

View larger version (60K): [in a new window]   Fig. 4. GFP-ß2-chimaerin-expressing Jurkat T cells show reduced CXCL12-dependent cell spreading to immobilized VCAM-1. Jurkat T cells were transiently transfected with vectors encoding GFP, GFP-ß2-chimaerin or GFP-ß2-chimaerin plus RacV12. After 24 hours, cells were suspended in HBSS and plated onto VCAM-1-coated slides. Slides were mounted on a 37°C plate and analyzed by confocal microscopy. CXCL12 (200 ng/ml) was added after the first frame and images captured every 10 seconds. Time-lapse confocal recording is shown in Movie 5 in supplementary material. Images are shown at the indicated times to illustrate cell spreading. Bar, 10 µm.

View larger version (30K): [in a new window]   Fig. 5. GFP-ß2-chimaerin-expressing Jurkat T cells show reduced CXCL12/PMA-induced Rac activation and adhesion to immobilized VCAM-1. (A) Control Jurkat cells (JK) or Jurkat cells stably transfected with GFP-ß2-chimaerin (JKß2) were plated onto fibronectin and stimulated with CXCL12 (200 ng/ml, 60 minutes) or PMA (100 ng/ml, 30 minutes). Cells were lysed and Rac-GTP determined by pull-down assays with PAK-RBD-GST (see Materials and Methods). Samples were separated by SDS-PAGE and Rac-GTP levels determined by western blotting with anti-Rac antibody. One-tenth of each sample was analyzed for total Rac level and ß2-chimaerin expression. (B) Jurkat cells were transfected with GFP or GFP-ß2-chimaerin-expressing vectors; GFP-positive cells were sorted after 24 hours. Sorted cells were added to wells coated with BSA or sVCAM-1, either alone (medium) or co-immobilized with CXCL12. Where indicated, cells were stimulated with PMA (100 ng/ml, 5 minutes) before addition to sVCAM-1-coated wells. Cells were allowed to adhere at 37°C for 2 minutes. Unbound cells were washed and the extent of adhesion determined as described (see Materials and Methods). Data represent the mean ± s.d. of at least three independent experiments performed in triplicate.

View larger version (27K): [in a new window]   Fig. 6. Effect of a GFP-ß2-chimaerin GAP-defective mutant on CXCL12/PMA-induced adhesion. (A) Vectors encoding GFP, GFP-ß2-chimaerin or a ß2-chimaerin GAP-defective mutant bearing a deletion of the EIE sequence (GFP-EIE) were transfected into HEK293 cells. At 24 hours post-transfection, cells were lysed and Rac-GTP levels determined by pull-down assays with PAK-RBD-GST. One-tenth of each sample was analyzed for total Rac level and ß2-chimaerin expression. (B) Jurkat T cells were transfected with the indicated plasmids; cells were sorted 24 hours post-transfection and GFP-positive cells tested for adhesion as in Fig. 5. Data represent the mean ± s.d. of at least three independent experiments performed in triplicate.

View larger version (26K): [in a new window]   Fig. 7. Effect of GFP-ß2-chimaerin on CXCL12/PMA-induced adhesion of primary blood T lymphocytes. Human peripheral blood mononuclear cells were prepared, and T lymphocytes were isolated and nucleofected as described in the Materials and Methods. After 6 hours, cells were added to wells coated with sVCAM-1 alone (medium), or co-immobilized with CXCL12 and allowed to adhere (37°C, 2 minutes). Where indicated, cells were stimulated with PMA (100 ng/ml, 5 minutes) before addition to the wells. The inset shows endogenous ß2-chimaerin expression in cell lysates of Jurkat and hPBLs, determined by western blotting. Tubulin expression was measured as a control of total protein levels. Data represent the mean ± s.d. of two independent experiments performed in triplicate.

View larger version (19K): [in a new window]   Fig. 8. CXCL12- and PMA-dependent regulation of ß2-chimaerin requires a functional C1 domain. (A) Jurkat T cells were transfected with a GFP-ß2-chimaerin C1 domain mutant (GFP-F215G). At 24 hours post-transfection, cells were sorted and GFP-positive cells tested for PMA-induced adhesion to sVCAM-1 as in Fig. 5. Data represent the mean ± s.d. of two independent experiments performed in triplicate. (B) Jurkat cells were transfected with GFP-ß2-chimaerin F215G and plated onto VCAM-1-coated slides, mounted on a 37°C plate and analyzed by confocal microscopy. CXCL12 (200 ng/ml) was added after the first frame and images are shown at the indicated times to illustrate cell spreading. Time-lapse confocal recording is shown in Movie 6 in supplementary material. (C) Jurkat T cells transfected with the indicated plasmids were sorted and GFP-positive cells were tested for CXCL12-induced adhesion to sVCAM-1 as in Fig. 5. Data represent the mean ± s.d. of two independent experiments performed in triplicate. Bar, 5 µm.

View larger version (26K): [in a new window]   Fig. 9. GFP-ß2-chimaerin expression enhances CXCL12-dependent migration of Jurkat T cells. (A) The chemotactic response of parental (control) and GFP-ß2-chimaerin cells towards CXCL12 (200 ng/ml) was determined in a 5-µm-pore chemotaxis chamber (see Materials and Methods). Data are expressed as a percentage of input cells, and are shown as the mean ± s.d. of at least three independent experiments. (B) Chemotactic responses of parental (control) and GFP-ß2-chimaerin-expressing cells towards CXCL12 at the concentrations indicated were determined at 1 hour post-stimulation. Data are expressed as in A, showing the mean ± s.d. of at least three independent experiments. (C) The chemotactic response of parental, GFP- and GFP-ß2-chimaerin-expressing cells towards CXCL12 (200 ng/ml) was determined 1 hour post-stimulation. Before seeding into the chamber, cells were incubated with the indicated concentrations of U73122 or EGTA (30 minutes) or with PTx (100 ng/ml overnight) at 37°C. Data are expressed as in (A), and are shown as the mean ± s.d. of at least three independent experiments.

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