Fig. 4. Both domains of epsin are required for targeting to clathrin-coated pits. (A) Schematic representation of GFP-labeled epsin truncation and chimera constructs. Black bars indicate clathrin-binding motifs, ovals indicate DPF/W AP2 binding motifs and rectangles indicate NPF motifs for binding EH-domain-containing proteins. (B-G) Wide-field fluorescence microscopy (focal planes from the middle of cells). (B,C) Epsin:GFP colocalizes with AP2 in epsin-null cells. Epsin-null cells expressing epsin:GFP (green) were fixed and immunostained with anti--adaptin antibody (B) or anti-clathrin antibody (C, red). (D) Epsin_253-677:GFP does not form puncta at the plasma membrane and does not colocalize with AP2. Epsin-null cells expressing epsin_253-677:GFP (green) were fixed and immunostained for the -adaptin subunit of AP2 (-AP2) (red). (E) Epsin_253-677:GFP cytoplasmic puncta overlap with clathrin puncta in the cytoplasm but not the plasma membrane. Epsin-null cells expressing epsin_253-677:GFP (green) were fixed and immunostained with anti-clathrin antibody (red). (F,G) Epsin_1-333:GFP uniformly decorates the plasma membrane. Epsin-null cells expressing epsin_1-333:GFP (green) were fixed and immunostained with anti--adaptin antibody (F) or anti-clathrin antibody (G, red). Scale bar: 5 µm.