Desmosomes: interconnected calciumdependent structures of remarkable stability with significant integral membrane protein turnover
Windoffer R, Borchert-Stuhlträger M, Leube RE, 2002
Desmosomes are prominent cell adhesion structures that are major stabilizing elements, together with the attached cytoskeletal intermediate filament network, of the cytokeratin type in epithelial tissues. To examine desmosome dynamics in tightly coupled cells and in situations of decreased adhesion, fluorescent desmosomal cadherin desmocollin 2a (Dsc2a) chimeras were stably expressed in human hepatocellular carcinoma-derived PLC cells (clone PDc-13) and in Madin-Darby canine kidney cells (clone MDc-2) for the continuous monitoring of desmosomes in living cells.
The hybrid polypeptides integrated specifically and without disturbance into normal-appearing desmosomes that occurred in association with typical cytokeratin filament bundles. Tracking of labeled adhesion sites throughout the cell cycle by time-lapse fluorescence microscopy revealed that they were immobile and that they maintained their structural integrity for long periods of time. Time-space diagrams further showed that desmosomal positioning was tightly controlled, even during pronounced cell shape changes, although the desmosomal arrays extended and contracted, suggesting that they were interconnected by a flexible system with intrinsic elasticity. Double-fluorescence microscopy detecting Dsc2a chimeras together with fluorescent cytokeratin 18 chimeras revealed the association and synchronous movement of labeled desmosomes and fluorescent cytokeratin filaments. Only a minor destabilization of desmosomes was observed during mitosis, demonstrated by increased diffuse plasma membrane fluorescence and the fusion of desmosomes into larger structures.
Desmosomes did not disappear completely at any time in any cell, and residual cytokeratin filaments remained in association with adhesion sites throughout cell division. On the other hand, a rapid loss of desmosomes was observed upon calcium depletion, with irreversible uptake of some desmosomal particles. Simultaneously, diffusely distributed desmosomal cadherins were detected in the plasma membrane that retained the competence to nucleate the reformation of desmosomes after the cells were returned to a standard calcium-containing medium. To examine the molecular stability of desmosomes, exchange rates of fluorescent chimeras were determined by fluorescence recovery after photobleaching, thereby identifying considerable Dsc2a turnover with different rates of fluorescence recovery for PDc-13 cells (36+/-17% recovery after 30 minutes) and MDc-2 cells (60+/-20% recovery after 30 minutes). Taken together, our observations suggest that desmosomes are pliable structures capable of fine adjustment to functional demands despite their overall structural stability and relative immobility.
Double epifluorescence microscopy of living MDc-2 cells co-expressing Dsc2a chimera Dsc2a.YFP and cytokeratin 18 chimera HK18-CFP. Fluorescence is depicted in false color in the movie for better visualization, showing Dsc2a.YFP-containing desmosomes in red and HK18-CFP-positive CKFs in green. All frames of the movie consist of five superimposed focal recordings that were taken at each time point after excitation at 498 nm (YFP) and 436 nm (CFP).
Detail taken from movie 6 to demonstrate the characteristic stages of desmosome distribution during mitosis at higher magnification. Each frame consists of 7 projected focal planes. Note the transient increase of diffuse fluorescence, the increased fusion of desmosomes, the enrichment of fluorescent puncta around the cleavage furrow, and the continued presence of desmosomes at all time points.
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 6 pictures (z-distance of 0.5 µm) recording the fluorescence emitted after excitation at 498 nm (YFP chimera) and 436 nm (CFP chimera). Note the continued presence of Dsc2a.YFP-positive desmosomes throughout cell division and the considerable alterations in the HK18-CFP-containing cytokeratin filament system with some residual desmosome-associated material.
Time-lapse fluorescence microscopy of MDc-2 cells expressing fluorescent Dsc2a chimeras depicting alterations in desmosomal cadherin distribution upon reduction of Ca2+. Cells were transferred from standard calcium medium (+ Ca2+) to low calcium medium (- Ca2+) as indicated. A large pinhole was used for confocal laser scan microscopy. The movie shows the internalization of Dsc2a.YFP-containing desmosomal particles and the continued presence of diffuse non-desmosomal Dsc2a.YFP fluorescence at the cell surface.
me-lapse fluorescence microscopy of cells expressing fluorescent Dsc2a chimeras depicting alterations in desmosomal cadherin distribution upon reduction of Ca2+. Cells were transferred from standard calcium medium (+ Ca2+) to low calcium medium (- Ca2+) as indicated. 3D-reconstructions of
z-stacks each consisting of 5 epifluorescence micrographs show the disintegration of large Dsc2a.GFP-labeled desmosomal structures into smaller particles in PDc-13 cells after the reduction of the Ca2+ concentration (arrows).
Time-lapse fluorescence microscopy of cells expressing fluorescent Dsc2a chimeras depicting alterations in desmosomal cadherin distribution upon reduction of Ca2+. Cells were transferred from standard calcium medium (+ Ca2+) to low calcium medium (- Ca2+) as indicated. Projection images of
z-stacks are shown, each consisting of 5 focal planes recording the epifluorescence of Dsc2a.GFP in PDc-13 cells. Note the fusion of small fluorescent desmosomal particles after the reduction of Ca2+.
Time-lapse fluorescence microscopy of cells expressing fluorescent Dsc2a chimeras depicting alterations in desmosomal cadherin distribution upon reduction of Ca2+. Cells were transferred from standard calcium medium (+ Ca2+) to low calcium medium (- Ca2+) as indicated. Overlay of projected fluorescence pictures (5 focal planes) and corresponding phase contrast micrographs were obtained from 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.
Time-lapse epifluorescence microscopy of MDc-2 cells monitoring the distribution of Dsc2a.YFP in response to a transient 5 minute reduction of Ca2+. Note the uptake of fluorescent puncta into the cytoplasm shortly after the pulse of low calcium medium and their continued presence in this location, while small dots re-appear at the cell surface.
