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First published online 30 April 2003
doi: 10.1242/jcs.00433

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WIP participates in actin reorganization and ruffle formation induced by PDGF

Inés M. Antón^1,2,3,*, Stephen P. Saville^,1,2, Michael J. Byrne^1,2, Claudia Curcio³, Narayanaswamy Ramesh^1,2, John H. Hartwig^4,5 and Raif S. Geha^1,2

¹ Division of Immunology, Children's Hospital, Harvard Medical School, Boston, MA 02115, USA
² Department of Pediatrics, Harvard Medical School, Boston, MA 02115, USA
³ Dipartimento di Scienze Cliniche e Biologiche, Università degli Studi di Torino, Ospedale San Luigi Gonzaga, Orbassano 10043 TO, Italy
⁴ Division of Experimental Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115, USA
⁵ Department of Medicine, Harvard Medical School, Boston, MA 02115, USA

View larger version (112K): [in a new window]   Fig. 1. WIP overexpression increases PDGF-induced dorsal ruffle formation. (A) Fluorescence micrographs of TRITC-phalloidin stained NIH 3T3 fibroblasts transfected with control plasmid (3T3pcDNA) or with WIP coding sequence (3T3pcDNA-WIP) before and after PDGF challenge. Cells were grown on glass coverslips, serum starved for 4 days in the presence of 0.5% FBS, stimulated or not with PDGF-bb (50 ng ml-1) for 8 minutes and then fixed and labeled with TRITC-phalloidin to visualize actin filaments. Indicated cells (Wort+PDGF) were pretreated with 12 nM wortmannin for 30 minutes at 37°C and then stimulated with PDGF and stained as described. Magnification 400x. (B) Electron micrographs of cortical actin network of NIH 3T3pcDNA-WIP (left) and NIH 3T3pcDNA (right) fibroblasts stimulated as described above. (C) Frames of phase-contrast microscopy capture of NIH 3T3pcDNA and NIH 3T3pcDNA-WIP fibroblasts grown on glass coverslips and stimulated at 37°C with PDGF for the indicated times. Frames were processed using Microsoft PowerPoint software. Arrows point to membrane ruffles. Magnification 400x.

View larger version (102K): [in a new window]   Fig. 2. Subcellular distribution of WIP in the presence or absence of PDGF. Fluorescence micrographs of NIH 3T3 fibroblasts transfected with control plasmid (3T3pcDNA) (A) or with plasmid containing WIP coding sequence (3T3pcDNA-WIP) (B). Cells were grown and fixed as described in Fig. 1A and then labeled with rabbit serum specific for WIP followed by anti-rabbit Alexa488 (anti-WIP) plus TRITC-phalloidin to visualize actin filaments (Actin). Arrows point to dorsal/circular ruffles areas. Magnification 600x.

View larger version (83K): [in a new window]   Fig. 3. Distribution of WIP along the membrane ruffle structure by confocal microscopy. NIH 3T3pcDNA-WIP fibroblasts were grown, stimulated with PDGF, and stained as described in Fig. 2. Immunocytochemical analysis was carried out using a polyclonal antibody against a WIP peptide (anti-WIP) and F-actin was stained with TRITC-phalloidin (Actin). 11 equidistant stacks were recorded with a confocal microscope system. The bottom, middle and top stacks of a representative field of cells containing ruffles (arrows) are shown. Magnification 1000x.

View larger version (15K): [in a new window]   Fig. 4. Microinjection of anti-WIP IgG inhibits PDGF-induced ruffle formation. The proportion of NIH 3T3pcDNA cells developing ruffles after PDGF stimulation is plotted against antibody injection. Columns represent the proportion of cells with ruffles among noninjected cells (–, 78%), cells injected with control rabbit IgG (IgG, 72%) and cells injected with anti-WIP rabbit IgG (-WIP, 54%). Results are representative of three independent experiments. Similar results were obtained after injection of anti-WIP IgG in WIP-transfected fibroblasts (data not shown).

View larger version (105K): [in a new window]   Fig. 5. PDGF-induced actin reorganization is altered in WIP-/- primary fibroblasts. (A) Lysates of lung-derived fibroblasts (Fib) from WIP+/+ and WIP-/- mice were subjected to western blot analysis using anti-WIP rabbit serum as probe. (B) WIP+/+ and WIP-/- primary murine fibroblasts were serum starved (Medium) or serum starved and stimulated with PDGF for different times (PDGF). TRITC-phalloidin-stained cells were visualized on a Nikon Eclipse E800 microscope using 40x or 20x objectives. Lower magnification fields are depicted at 15 minutes to include more cells, which represent a more accurate proportion of ruffling cells. Arrows indicate ruffle formation.

View larger version (80K): [in a new window]   Fig. 6. WIP binding to actin is essential for PDGF-induced ruffle formation. (A) GST pull-down assay. The indicated GST-WIP constructs (5 µg per 10 µl beads) were incubated with 0.05 µM G-actin. After SDS-PAGE, samples were blotted with anti-actin monoclonal antibody. (B) Fluorescence micrographs of TRITC-phalloidin-stained Swiss 3T3 fibroblasts transfected with control plasmid (3T3pcDNA), WIP coding sequence (3T3pcDNA-WIP) or the deletion mutant WIP43-54 (3T3pcDNA-WIP43-54), before and after PDGF challenge. Cells were grown on glass coverslips, serum starved for 3 days in the presence of 0.5% FBS, stimulated or not with PDGF (50 ng ml-1) for 8 minutes and then fixed and labeled with TRITC-phalloidin to visualize actin filaments. Magnification 400x.