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First published online 30 September 2008
doi: 10.1242/jcs.032573

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The ENTH and C-terminal domains of Dictyostelium epsin cooperate to regulate the dynamic interaction with clathrin-coated pits

Department of Molecular Cell and Developmental Biology, Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, TX 78712, USA

View larger version (43K): [in this window] [in a new window]   Fig. 1. Dictyostelium epsin. (A) Schematic representation of the structural organization of epsin from Dictyostelium (Dd), S. cerevisae (Sc), C. elegans (Ce), Drosophila (Dm) and Homo sapiens (Hs). Each has a membrane-binding ENTH domain (yellow box) as well as an unstructured C-terminal region containing motifs for binding clathrin (blue boxes), a DPF/DPW motif that binds AP2 (red oval) and an NPF for binding EH-domain-containing proteins. (B) Dictyostelium epsin binds clathrin. Amylose resin coupled to maltose binding protein (MBP) or to MBP:epsin was incubated with Dictyostelium lysate. Whole cell lysates (WCL) and fractions that did not bind (Unbound; UB) or that did bind (Bound; B) to the resin were immunoblotted for clathrin (anti-CHC) or the -adaptin subunit of AP2 (-AP2). (C) Confocal images (surface focal plane) of a wild-type cell expressing epsin:GFP. (C') Confocal image (middle focal plane) from the same cell. (D,E) Epsin colocalizes extensively with clathrin and AP2. Confocal images (surface focal planes) from cells expressing epsin:GFP (green) and immunostained for clathrin (D) or AP2 (E, red). Scale bars: 5 µm.

View larger version (52K): [in this window] [in a new window]   Fig. 2. Epsin-null mutants display defects in cytokinesis and spore morphology. (A) An immunoblot of whole cell lysates of the wild type (WT) and epsin-null mutants (epsin-) stained with anti-epsin antibodies. (B) Wild type (WT) and epsin mutants (epsin-) develop into fruiting bodies. Scale bar: 0.2 mm. (C) DIC images of spores harvested from fruiting bodies of wild-type cells (WT), epsin-null mutants (epsin-), and epsin-null mutants expressing epsin:GFP (epsin- +epsin:GFP). Expression of epsin:GFP restores wild-type sore morphology. Scale bar: 5 µm. (D) Wild type (WT), epsin-null mutants (epsin-), clathrin-null mutants (clathrin-), and epsin-null mutants expressing epsin:GFP (epsin- +epsin:GFP) grown in suspension for 72 hours and stained with DAPI to visualize nuclei. Scale bar, 5 µm. (E) Ratio of spore width:length in wild type (WT), epsin-null mutants (epsin-) and epsin-null mutants expressing epsin:GFP (epsin- +epsinGFP), n=50 for each cell line. (F) Quantification of multinucleated cells in suspension cultures of wild type (WT), epsin mutants (epsin-), epsin mutants expressing epsin:GFP (epsin- +epsin:GFP) and CHC mutants (clathrin-); n=300 for each cell line. Error bars are s.e.m.

View larger version (47K): [in this window] [in a new window]   Fig. 3. Epsin requires CHC to form puncta on the membrane. (A,B) Clathrin and AP2 localization in epsin mutants is comparable to that in the wild type. Confocal images (surface focal planes) of (A) wild-type cells (WT) and (B) epsin-null mutants (epsin-) expressing clathrin:GFP (green) and immunostained for the -adaptin subunit of AP2 (-AP2) (red). (C) Subcellular fractionation of clathrin is similar in wild-type (WT) and epsin-null cells (epsin-). Immunoblot of samples probed with -CHC antibodies. Lys, lysate; LSP, low-speed (3000 g) pellet; LSS, low-speed supernatant; HSP, high-speed (100,000 g) pellet; HSS, high-speed supernatant. (D) Epsin localizes to the membrane but does not form puncta in CHC-null cells. Confocal image (middle focal plane) of CHC-null cells (CHC-) expressing epsin:GFP (E) Subcellular fractionation of epsin is altered in CHC mutants, but not in -adaptin-null mutants. Immunoblot probed with anti-epsin antibodies. (F) Epsin forms puncta that colocalize with AP2 on the membranes of CLC mutants. Confocal images (surface focal plane) of CLC-null mutants expressing epsin:GFP (green) and immunostained for the -adaptin subunit of AP2 (-AP2) (red). (G) Epsin forms reduced numbers of puncta at the plasma membrane in -adaptin-null mutants that colocalize with clathrin. Confocal images (surface focal plane) -adaptin mutants (AP2-) expressing epsin:GFP (green) and immunostained for clathrin (red). Scale bars, 5 µm.

View larger version (60K): [in this window] [in a new window]   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) Epsin253-677:GFP does not form puncta at the plasma membrane and does not colocalize with AP2. Epsin-null cells expressing epsin253-677:GFP (green) were fixed and immunostained for the -adaptin subunit of AP2 (-AP2) (red). (E) Epsin253-677:GFP cytoplasmic puncta overlap with clathrin puncta in the cytoplasm but not the plasma membrane. Epsin-null cells expressing epsin253-677:GFP (green) were fixed and immunostained with anti-clathrin antibody (red). (F,G) Epsin1-333:GFP uniformly decorates the plasma membrane. Epsin-null cells expressing epsin1-333:GFP (green) were fixed and immunostained with anti--adaptin antibody (F) or anti-clathrin antibody (G, red). Scale bar: 5 µm.

View larger version (74K): [in this window] [in a new window]   Fig. 5. The ENTH domain rescues phenotypic defects of epsin mutants. (A) DIC images of spores from the wild type (WT) and epsin mutants (epsin-) expressing epsin:GFP, epsin1-333:GFP, epsin253-677:GFP, and PH:epsin253-677:GFP. Scale bar: 5 µm. (B) Ratio of spore width:length in wild type (WT) and epsin-null mutants (epsin-) and epsin-null mutants expressing epsin:GFP, epsin1-333:GFP, epsin253-677:GFP, and PH:epsin253-677:GFP; n=50 for each cell line, error bars represent s.e.m. (C) Quantification of multinucleated cells in suspension cultures of wild-type (WT), epsin mutants (epsin-), clathrin mutants (clathrin-), and epsin mutants expressing epsin:GFP, epsin1-333:GFP, epsin253-677:GFP, and PH:epsin253-677:GFP; n=300 for each cell line. Error bars represent s.e.m.

View larger version (42K): [in this window] [in a new window]   Fig. 6. PH:epsin253-677:GFP forms large patches on the plasma membrane and cells expressing PH:epsin253-677:GFP mislocalize clathrin and AP2 to these patches (A,B). Wild-type cells expressing PH:epsin253-677: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:epsin253-677:GFP forms relatively static patches. Plasma membrane images from a time course of wild-type cells expressing either epsin:GFP (top row) or PH:epsin253-677:GFP (bottom row) were imaged under fluorescence microscopy. (D,E) Wild-type cells expressing PH:epsin253-677:GFP display clathrin-associated phenotypic defects, including enlarged contractile vacuoles and cytokinesis defects. (D) Wild-type cells expressing epsin:GFP or PH:epsin253-677: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:epsin253-677:GFP (green) were cultured in suspension for 3 days, then fixed and stained with DAPI to visualize nuclei (blue). Scale bar: 10 µm.

View larger version (88K): [in this window] [in a new window]   Fig. 7. EpsinT107A 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) ENTHR65A/K78A, but not ENTHT107A, 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. ENTHWT (left) and ENTHT107A (right) localize to the plasma membrane, whereas ENTHR65A/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). EpsinT107A (right) forms puncta similar to epsinWT (left). EpsinR65A/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. ENTHR65A/K78A (center) or ENTHT107A (right) cannot rescue spore morphology. Scale bars: 5 µm.

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