title: Desmosomes: interconnected calcium-dependent structures of remarkable stability with significant integral membrane protein turnover.
abstract: 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.
authors.affiliation: Department of Anatomy, Johannes Gutenberg-University Mainz, Becherweg 13, 55128 Mainz, Germany.
authors.forename: Reinhard
authors.initials: R
authors.lastname: Windoffer
authors.forename: Monika
authors.initials: M
authors.lastname: Borchert-Stuhlträger
authors.forename: Rudolf E
authors.initials: RE
authors.lastname: Leube
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002277:Carcinoma; D002448:Cell Adhesion / Q000502:physiology*; D002453:Cell Cycle / Q000502:physiology; D002460:Cell Line; D051187:Desmocollins; D003896:Desmosomes / Q000737:chemistry / Q000378:metabolism*; D004285:Dogs; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism*; D017930:Genes, Reporter; D006801:Humans; D007633:Keratins / Q000378:metabolism; D008113:Liver Neoplasms; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D008854:Microscopy, Electron; D008856:Microscopy, Fluorescence; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D013997:Time Factors
chemical_list: C081561:DSC2 protein, human; D051187:Desmocollins; D008562:Membrane Glycoproteins; D011993:Recombinant Fusion Proteins; D007633:Keratins; D002118:Calcium