Fig. 6. PH:epsin_253-677:GFP forms large patches on the plasma membrane and cells expressing PH:epsin_253-677:GFP mislocalize clathrin and AP2 to these patches (A,B). Wild-type cells expressing PH:epsin_253-677:GFP (green) were fixed and immunostained with anti--adaptin antibody (A) or anti-clathrin antibody (B) (red). Images were acquired under wide-field fluorescence microscopy (focal plane in the middle of the cell). Scale bar: 5 µm. (C) Epsin forms dynamic membrane puncta, but PH:epsin_253-677:GFP forms relatively static patches. Plasma membrane images from a time course of wild-type cells expressing either epsin:GFP (top row) or PH:epsin_253-677:GFP (bottom row) were imaged under fluorescence microscopy. (D,E) Wild-type cells expressing PH:epsin_253-677:GFP display clathrin-associated phenotypic defects, including enlarged contractile vacuoles and cytokinesis defects. (D) Wild-type cells expressing epsin:GFP or PH:epsin_253-677:GFP were shifted from medium to water and imaged under DIC optics. Arrows indicate contractile vacuoles. Scale bar: 5 µm. (E) Wild-type cells expressing epsin:GFP or PH:epsin_253-677:GFP (green) were cultured in suspension for 3 days, then fixed and stained with DAPI to visualize nuclei (blue). Scale bar: 10 µm.