Figure 4

Mena11a homotetramers alter F-actin organization at the leading edge, target to tips of filopodia and decrease filopodia formation.

(A) Western blot of lysates from MV^D7 cells expressing GFP, Mena or Mena11a and MCF7 cells. Membranes were probed with anti pan-Mena and GFP antibodies. α-Tubulin is used as the loading control. (B) 3D-SIM images of EGFP-Mena in MV^D7 EGFP-Mena cells (top) and EGFP-Mena11a in MV^D7 EGFP-Mena11a cells (bottom). Phalloidin staining shows F-actin. Scale bar, 10 μm. Red arrowheads: Mena and Mena11a localize properly to the leading edge of lamellipodia. (C) Platinum replica EM of actin cytoskeleton in MV^D7 cells expressing GFP, Mena and Mena11a, stimulated with 100 ng/ml PDGF-BB for 5 minutes. Scale bar, 250 nm. Images representative of seven independent experiments. (D) Immunofluorescence of the spreading assay (MV^D7 cells plated on 20 μg/ml laminin). The three spreading phenotypes depicted are: smooth-edge (left panel), ruffled-edge (middle panel) and filopodial (right panel). F-actin visualized by phalloidin labeling. Scale bar, 10 μm. (E) Immunofluorescence of spreading MV^D7 cells expressing EGFP-Mena (left panel) and EGFP-Mena11a (right panel). Fascin antibodies are used as a filopodial marker. Scale bar, 10 μm. For EGFP-Mena MV^D7 cells, inset at 10X magnification shows Mena localization at the tips of filopodia. For EGFP-Mena11a MV^D7 cells, inset at 11X magnification shows Mena11a localization at the tips of filopodia. (F) Percent of spreading MV^D7 cells expressing GFP, Mena, and Mena11a with the filopodial phenotype. Results represent triplicates, >930 cells analyzed. Error bars represent SEM. One-way ANOVA *p < 0.05, **p < 0.01, n.s.: not significant.