Fig. 2. Characterization of cell lines MDc-2 and PDc-13 stably expressing fluorescent Dsc2a chimeras. (A) Electron microscopy of MDc-2 cells demonstrating the normal ultrastructural appearance of desmosomes with a desmoglea-filled intercellular gap (small arrows) and symmetrical cytoplasmic plaque regions (large arrows) together with inserting IF bundles (arrowheads). Bar, 100 nm. (B-E) Immunoelectron microscopy showing abundant immunogold labelling (silver amplification) for GFP epitopes throughout the desmosomal plaque regions in MDc-2 cells (B) and PDc-13 cells (C). Arrowheads, IF bundles; arrows, desmosomal plaques. Controls are shown in D (GFP-negative cells) and E (no primary antibodies). Arrows demarcate cytoplasmic background label. Bars, 150 nm in B, 100 nm in C-E. (F) 3D reconstruction from epifluorescence micrographs that were recorded as a z-stack of eight consecutive focal planes: demonstration of the spatial dimensions of desmosomes and their arrangement in methanol/acetone-fixed MDc-2 cells. The reconstruction is also provided as movie (Movie 1; jcs.biologists.org/supplemental ). Bar, 5 µm. (G) Immunoblot of 50 µg polypeptides that were derived from total cell lysates, postnuclear supernatants and 100,000 g pellets and separated by 8% SDS-PAGE. Detection of both the green and yellow fluorescent Dsc2a fusion proteins with polyclonal GFP antibodies (anti-GFP) in cDNA-transfected PDc-13 and MDc-2 cells but not in wild-type PLC and MDCK cells. The positions of coelectrophoresed molecular weight markers are shown on the left, and the relative molecular mass (M_r) is given in units of 1,000. ^*Position of endogenous protein crossreacting with the GFP antibodies. (H) Immunoblots of immunoprecipitates obtained from MDc-2 cells that had been lysed either in standard immunoprecipitation buffer (RIPA) or in immunoprecipitation buffer lacking SDS (—SDS) or in immunoprecipitation buffer without SDS and deoxycholate (—SDS, —DOC). For precipitation, antibodies against GFP were used that were omitted in the negative controls. The precipitates were probed either with antibodies against GFP to detect chimera Dsc2a.YFP or with antibodies against the desmosomal plaque protein plakoglobin. Note that plakoglobin is specifically coprecipitated with the chimera when the immunoprecipitation buffers lack SDS. The same molecular weight standards were used as in (G). ^*Position of Dsc2a.YFP degradation product.

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