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First published online 27 February 2007
doi: 10.1242/jcs.03415

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Myosin IIA is involved in the endocytosis of CXCR4 induced by SDF-1

¹ Servicio de Inmunología, Hospital Universitario de la Princesa, Diego de León, 62, 28006 Madrid, Spain
² Departamento de Biología Molecular y Centro de Biología Molecular Severo Ochoa (CSIC-UAM), Universidad Autónoma de Madrid, 28049 Madrid, Spain

View larger version (39K): [in this window] [in a new window]   Fig. 1. Confocal microscopy analysis of SDF-1-induced endocytosis of MIIA and CXCR4. (A) Confocal analysis of MIIA- and CXCR4-staining in PBLs untreated or treated with 300 nM SDF-1 for the indicated time periods. Quantification of the fluorescence signal of CXCR4 and MIIA in the cytosolic compartment versus the plasma membrane was performed on the complete 3D confocal stack for n=10 cells from two independent experiments, and represented as the mean percentage of internalization ± s.d.; *P<0.05. (B) Confocal analysis of MIIA, CXCR4 and CD45 in J77 T cells untreated or treated with 300 nM SDF-1 for the indicated periods of time. CD45 is depicted as a control of plasma membrane molecule that is not internalized in response to SDF-1. Bars, 5 µm.

View larger version (20K): [in this window] [in a new window]   Fig. 2. Overexpression of MIIA tail-GFP prevents the ligand-induced endocytosis of CXCR4 in T cells. (A) Flow cytometry analysis of CXCR4 surface expression in untransfected J77 cells and J77 cells transfected with GFP, MIIA tail-GFP and MIIA head-GFP after 30-minute treatment with 300 nM SDF-1, or 1-hour treatment PMA 100 ng/ml. CXCR4 plasma membrane expression levels in untreated samples were not altered by the transfection of the different GFP constructs and were in the range of 100-130 MFI units in different experiments. Data are represented as the mean ± s.d. of the percentages of CXCR4 surface expression of three independent experiments *P<0.05 or a representative experiment for MIIA head-GFP. (B) Flow cytometry analysis of cell surface expression of transferrin receptor after treatment with 500 µg/ml transferrin for 1 hour. Data represented are the mean ± s.d. of three independent experiments. (C) Flow cytometry analysis showing the kinetics of CXCR4 membrane expression in J77 cells untransfected or transfected with MIIA tail-GFP. Data represent the mean percentage of CXCR4 expression ± s.d. with respect to the expression at 0 minutes of three independent experiments.

View larger version (26K): [in this window] [in a new window]   Fig. 3. Interference with MIIA expression or function inhibits the ligand-induced endocytosis of CXCR4. (A) Western blot analysis of J77 cells transfected with siRNA oligonucleotides and stained for MIIA, MIIB and tubulin. Numbers depict the quantification of MIIA and MIIB expression, corrected with tubulin loading and related to cells transfected with the negative control oligonucleotide. (B) Flow cytometry analysis of cell surface expression of CXCR4 in cells transfected with siRNA targeting MIIA after 30-minute treatment with 300 nM SDF-1. CXCR4 plasma membrane expression levels in untreated samples were not altered by the transfection of the different siRNA oligonucleotides and were in the range of 90-120 MFI units in the different experiments. Data represent the percentage of CXCR4 with respect to untreated cells (mean± s.d.) of six independent experiments *P<0.05. (C) Flow cytometry analysis showing cell surface expression of CXCR4 in cells pretreated for 30 minutes with 50 or 100 µM blebbistatin and then exposed to 300 nM SDF-1 at 37°C for 60 minutes. CXCR4 plasma membrane expression levels before SDF-1 addition were not altered by blebbistatin treatment and ranged 80-100 MFI units in the different experiments. Data represent the percentage of CXCR4 expression with respect to that at 0 minutes in a representative experiment (n=6 experiments). (D) Confocal microscopy analysis of J77 cells transfected with siRNA targeting MIIA (MIIA1) and negative control siRNA. Cells were treated with 300 nM SDF-1 at 37°C for 30 minutes when indicated, and stained for MIIA and clathrin. Bar, 5 µm.

View larger version (36K): [in this window] [in a new window]   Fig. 4. Functional effects of MIIA targeting. (A) Increase in the migratory response of J77 cells transfected with MIIA tail-GFP to 100 nM SDF-1. Data represent the percentages of migrated cells in response to SDF-1, normalized with respect to untransfected (Ut) cells treated with SDF-1 (mean ± s.e.m.) of three independent experiments *P<0.05. (B) Increase in the migratory response of J77 cells transfected with siRNA oligonucleotides for MIIA (MIIA1 and MIIA2) to 100 nM SDF-1. Data represented are the percentages of migrated cells in response to SDF-1, normalized with respect to cells with no SDF-1-treatment (mean ± s.d.) of three independent experiments *P<0.05. (C) Loss of the anti-HIV-1 fusogenic capacity of SDF-1 in cells transfected with MIIA tail-GFP, in Env-HIV-mediated cell fusion experiments, by using MIIA tail-GFP-transfected HeLa cells, at non-saturating concentrations of the chemokine. The level of cell fusion was evaluated by quantitative measurement of chemiluminescence, using a kit that couples -galactosidase and luciferase activity (-gal reporter gene assay); results are shown as the mean ± s.e.m. of three independent experiments *P<0.05. (D) Series of images of a representative experiment of those quantified in C, showing the block of Env-mediated cell-to-cell fusion by SDF-1 at different concentrations by using a fusion model, based on HeLa cells transfected with MIIA tail-GFP. The number and size of syncytia formed were monitored by X-Gal staining. Images were acquired with a 10x objective.

View larger version (18K): [in this window] [in a new window]   Fig. 5. MIIA interacts with CXCR4 and -arrestin. (A) Direct interaction between CXCR4 and MIIA in protein-protein binding assays. [35S]Met-MIIA pulldown assays were attempted with GST (lanes 1 and 3) or GST-CXCR4 (lanes 2 and 4) and sepharose-bound proteins were resolved by SDS-PAGE. After Coomassie-Blue staining – lane 3 (GST) and lane 4 (GST-CXCR4) – gels were developed by autoradiography (lanes 1 and 2). (B) Direct interaction between -arrestin and MIIA. [35S]Met-MIIA was mixed with recombinant -arrestin 1 and -arrestin 2, precipitated with an anti-pan--arrestin pAb (384-397) and the sepharose-bound proteins were analyzed by autoradiography. (C) Interaction of endogenous MIIA with -arrestin. J77 cells were treated with 300 nM SDF-1 for the indicated periods of time, lysed and precipitated with anti-CD45 (D3/9 mAb) and polyclonal anti-pan -arrestin (172-268). Gels were then analyzed by western blot, revealing both MIIA and -arrestin 1 (mAb 38-44); Lys, lysate. The lane labeled Ab shows unspecific bands from the immunoprecipitating antibody in the absence of cellular lysates. Numbers indicate the quantification of the MIIA band corrected with the amount of -arrestin immunoprecipitated in each lane relative to no SDF treatment. Arrows indicate major myosin bands. Additional upper and lower minor bands correspond to a fraction of the protein retained at the interface with the stacking gel and to a degradation product, respectively.

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