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Fig. 7. Epsin^T107A localizes to plasma membrane puncta but does not rescue spore morphology. (Top of figure) Schematic representation of epsin, showing the location of point mutations. (A) ENTH^R65A/K78A, but not ENTH^T107A, exhibits impaired binding to PtdIns(4,5)P₂. PtdIns and PtdIns(4,5)P₂ (PI and PI(4,5)P₂, respectively) were spotted onto nitrocellulose membrane and then incubated with lysate from Dictyostelium cells expressing either wild-type or mutant ENTH:GFP. Membranes were probed with anti-GFP antibody. (B) Wild-type or mutant ENTH:GFP was expressed in epsin-null cells and imaged by epifluorescence wide-field microscopy (epi); focal planes from the middle of cells. ENTH^WT (left) and ENTH^T107A (right) localize to the plasma membrane, whereas ENTH^R65A/K78A (center) does not. (C) Wild-type or mutant epsin:GFP was expressed in epsin-null cells and imaged by total internal reflection microscopy (TIRF). Epsin^T107A (right) forms puncta similar to epsin^WT (left). Epsin^R65A/K78A (center) does not form puncta on the plasma membrane. (D) Epsin-null cells expressing wild-type or mutant ENTH:GFP were allowed to develop on starvation plates. Spores were harvested and imaged under DIC optics. ENTH^R65A/K78A (center) or ENTH^T107A (right) cannot rescue spore morphology. Scale bars: 5 µm.

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