Dyn1–3 localize to lamellipodia and filopodia and colocalize with IRSp53.
A, lamellipodia (panels i, iii, and v) and filopodia (panels ii, iv, and vi) were imaged by confocal microscopy. F-actin (red) and endogenous Dyn1 (panels i and ii), Dyn2 (panels iii and iv), and Dyn3 (panels v and vi) (green) were localized as shown. Bar, 5 μm. Filopodia in boxed areas (panels ii, iv, and vi) are shown enlarged as inserts. Bar, 1 μm. The panels show merged and single channel fluorescence images of N1E-115 cells cultured on laminin-coated glass coverslips, immunostained for endogenous Dyn, and counter-stained with A568-phalloidin, as described under “Experimental Procedures.” B, fluorescence images of HeLa and COS-7 cells transfected with Dyn1-GFP (panel i) and Dyn3-GFP (panel ii), respectively, and imaged by confocal microscopy. Bar, 5 μm. Filopodia-like protrusions (arrowheads) induced by ectopic expression of Dyn1-GFP are shown enlarged as inserts. Bar, 1 μm. Membrane ruffles induced by Dyn3-GFP are indicated by arrows. C, representative single channel and merged fluorescence images of cells, transfected to express Dyn1-GFP (panel i), Dyn2-GFP (panel iv), or Dyn3-GFP (panel vii) (green) and mRFP-IRSp53 (panels ii, v, and viii) (red), under ring-TIRF microscopy. Filopodia in boxed areas (panels iii, vi, and ix) are shown enlarged as inserts. Areas of colocalization are indicated by arrowheads. Bar, 1 μm.