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Desmosomes: interconnected calcium-dependent structures of remarkable stability with significant integral membrane protein turnover

Reinhard Windoffer, Monika Borchert-Stuhlträger and Rudolf E. Leube*

Department of Anatomy, Johannes Gutenberg-University Mainz, Becherweg 13, 55128 Mainz, Germany



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  		Fig. 1. Epifluorescence microscopy of cell lines stably expressing fluorescent human Dsc2a chimeras. Clone PDc-13 (A-C') was obtained after transfection of human hepatocellular carcinoma-derived PLC cells with construct C-Dsc2a.GFP-1 coding for fusion protein Dsc2a.GFP and selection with neomycin. Clone MDc-2 (D-H') was generated from canine kidney-derived MDCK cells by transfection with construct C-Dsc2a. YFP-2 coding for chimera Dsc2a. YFP and selection with hygromycin. The fluorescence elicited by the transgenic fusion proteins in methanol/acetone-fixed cells is shown in the micrographs on the left (A-H) and compared with the indirect immunofluorescence obtained after reaction with primary antibodies against GFP (anti-GFP), desmoplakin (anti-Dp), desmoglein (anti-Dsg), plakoglobin (anti-Pg), plakophilin 2 (anti-Pp2) and plakophilin 3 (anti-Pp3). Note the similar punctate fluorescence pattern in each picture pair, except for plakoglobin, which is detected in additional plasma membrane domains. Bars, 10 µm.

 


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  		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 (Mr) 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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  		Fig. 3. Time-lapse recording of epifluorescence in PDc-13 cells expressing fluorescent Dsc2a chimera Dsc2a. GFP. Pictures were taken every 2 minutes for 3 hours and are shown in Movie 2 (jcs.biologist.org/supplemental ). Large arrows demarcate desmosomes that remained in place for the entire observation period with little changes in shape and fluorescence intensity. A slowly rotating vacuolar structure in the cytoplasm is denoted by a small arrow; a rare fusion event of desmosomes is demarcated by an arrowhead. Bar, 5 µm.

 


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  		Fig. 4. Time-space diagrams depicting coordinated desmosome-motility in PDc-13 cells (A) and MDc-2 cells (B) stably expressing fluorescent Dsc2a chimeras. The diagram on the left was derived from a time-lapse fluorescence recording of a cell contact region that was imaged for 3 hours at 2 minute intervals (Movie 3; jcs.biologists.org/supplemental ); the diagram at right was derived from a time-lapse fluorescence recording of a desmosomal array that was monitored for 10 hours at 5 minute intervals (Movie 4). Time is plotted along the y-axis in hours (h), whereas movement in the 2D space dimensions is plotted along the x- and z-axis in µm. The rough and sometimes interrupted surface of the desmosomal trajectories in MDc-2 cells is the result of the comparatively long recording intervals. Note the coordinated movement, constant arrangement, size and shape of the depicted desmosomes, whereas interdesmosomal distance varies considerably, albeit in a coordinated fashion (compare, for example, time points marked by arrows).

 


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  		Fig. 5. Double-epifluorescence microscopy of living MDc-2 cells stably expressing Dsc2a chimera Dsc2a. YFP, including a transiently transfected cell synthesizing, in addition, fluorescent CK 18 chimera HK 18-CFP. The picture shown is taken from a time-lapse recording (Movie 5; jcs.biologists.org/supplemental ). Fluorescence is depicted in false colors in the movie for better visualization, denoting Dsc2a. YFP-containing desmosomes in red and HK 18-CFP-positive CKFs in green. All micrographs of the movie and the picture shown in the figure consist of five superimposed focal recordings that were taken at each time point after excitation at 498 nm (YFP) and 436 nm (CFP). Bar, 5 µm.

 


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  		Fig. 6. Epifluorescence microscopy of live MDc-2 monolayer monitoring the distribution of fluorescent chimera Dsc2a.YFP for 23.3 hours. The pictures are taken from a time-lapse recording (Movie 6; jcs.biologists.org/supplemental ), for which seven focal planes were imaged every 5 minutes. The projection images are shown. Individual cells are labeled by letters, and daughter cells that are generated during the observation period are denoted by ' and ''. Note the maintenance of desmosomes for very long periods (arrows). Bar, 10 µm.

 


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  		Fig. 7. Detail taken from Fig. 6 to demonstrate the characteristic stages of desmosome distribution during mitosis at higher magnification. The entire image series is provided as Movie 7 (recording intervals 5 minutes). Each picture consists of seven focal planes that were superimposed. Note the transient increase of diffuse fluorescence (265 minutes, 285 minutes), ongoing fusion of desmosomes (large arows), the enrichment of fluorescent puncta around the cleavage furrow (thin arrows in 285 min) and the continued presence of desmosomes at all time points. Bar, 5 µm.

 


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  		Fig. 8. Double epifluorescence microscopy of live MDc-2 cells showing the distribution of Dsc2a.YFP and HK18-CFP during mitosis. Images at each time point represent the projection of six pictures (z-distance of 0.5 µm) recording the fluorescence emitted after excitation at 498 nm (YFP chimera) and 436 nm (CFP chimera). The complete series of images is provided as Movie 8 (jcs.biologists.org/supplemental ) (recording intervals 5 minutes; same color code as in Movie 5). Note the continued presence of Dsc2a. YFP-positive desmosomes throughout cell division, and the considerable alterations in the HK18-CFP-containing CKF system with some residual desmosome-associated material. Bar 5 µm.

 


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  		Fig. 9. Time-lapse fluorescence microscopy of cells expressing fluorescent Dsc2a chimeras depicting alterations in desmosomal cadherin distribution upon reduction of Ca++. Cells were transferred from SCM (+ Ca++) to LCM (-Ca++) as indicated. (A) Confocal laser scan microscopy of MDc-2 cells using a large pinhole. The pictures are taken from Movie 9 (1 minute recording intervals) depicting the internalization of Dsc2a. YFP-containing desmosomal particles and the continued presence of diffuse non-desmosomal Dsc2a. YFP fluorescence at the cell surface. Bar, 10 µm. (B) 3D reconstruction of z-stacks each consisting of five epifluorescence micrographs showing the disintegration of large Dsc2a. GFP-labeled desmosomal structures into smaller particles in PDc-13 cells after the reduction of the Ca++ concentration (arrows). The complete sequence is provided as Movie 10 (1 minute recording intervals; jcs.biologists.org/supplemental ). Bar, 5 µm. (C) Projection images of z-stacks, each consisting of five focal planes recording epifluorescence of Dsc2a.GFP in PDc-13 cells. Note the fusion of small fluorescent desmosomal particles after the reduction of Ca2+ (arrows). The entire image series is presented in Movie 11 (2 minute recording intervals). Bar, 5 µm. (D) Overlay of projected fluorescence pictures (5 focal planes) and corresponding phase contrast micrographs obtained from Movie 12 (jcs.biologists.org/supplemental ), which was recorded in PDc-13 cells. Note the overall reduction of desmosomes together with the fusion of desmosomes (black arrows) and fission of desmosomes (white arrows) after removal of calcium. Bar, 10 µm.

 


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  		Fig. 10. Time-lapse epifluorescence microscopy of MDc-2 cells monitoring the distribution of Dsc2a. YFP in response to a transient 5 minute reduction of Ca2+. The entire sequence is shown as Movie 13 (jcs.biologists.org/supplemental ; 2 minute recording intervals). Note the uptake of fluorescent puncta into the cytoplasm shortly after the pulse of LCM and their continued presence in this location (small arrows), whereas small dots re-appear at the cell surface (large arrows). Bar, 10 µm.

 


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  		Fig. 11. FRAP analyses in MDc-2 and PDc-13 cells. A representative series of photomicrographs of single confocal sections that were recorded with a large pinhole is depicted for MDc-2 (A) and PDc-13 cells (B). The bleached areas are demarcated by boxes. Note the strong desmosomal fluorescence before bleaching (prebleach); this fluorescence is completely destroyed by bleaching (bleached) but recovers to a considerable degree within 30 minutes (5 min, 30 min). Bars, 5 µm. (C) The graphs show digital representations of pooled results from MDc-2 (n=6) and PDc-13 cells (n=10). The means of the relative fluorescence intensities are blotted as functions of time. (D) Confocal fluorescence recording of bleaching in PDc-13 cells; microscope settings were adjusted for high spatial resolution (small pinhole). E,E' show a high magnification of the bleached area prior to bleaching and after a 30 minute recovery period. Note the re-emergence of fluorescence in desmosomal structures (arrows in D,E,E'). Bars, 10 µm in D, 1 µm in E'.

 

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© The Company of Biologists Ltd 2002