Desmosome assembly in MDCK cells: transport of precursors to the cell surface occurs by two phases of vesicular traffic and involves major changes in centrosome and Golgi location during a Ca(2+) shift.
Desmosome formation in MDCK cells was investigated using a Ca(2+) shift. Following preliminary treatment with cycloheximide at 37 degrees C, continued surface transport and subsequent endocytosis were minimized by incubating cells at 19 degrees C to trap nascent glycoproteins within the Golgi body. Release into high Ca(2+) medium (HCM) at 37 degrees C resulted in junction formation as well as relocation of the Golgi body and centrosomes to a subapical location. Desmosome formation occurred in two stages over 2 h, the first occurring within 30 min of the shift to HCM, in 60-nm vesicles containing chiefly Dsc2 and lower concentrations of Dsg and E-cadherin distributed to the entire cell surface. Much of this material was subsequently endocytosed. The second stage involved transport of Dsg, E-cadherin, plakoglobin, and beta-catenin, in more complex vesicles some 200 nm in size, directed to possible nucleation sites on the developing basolateral surface. Plaque proteins such as desmoplakin I/II were added subsequently. Stage-two vesicles, but possibly not those of stage one, were accessible to endocytic markers via retrograde transport from multivesicular bodies prelabeled at 19 degrees C.
Burdett
Ian D J
ID
Division of Membrane Biology, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, United Kingdom. iburdet@nimr.mrc.ac.uk
Sullivan
Kate H
KH
D000818:Animals; D015820:Cadherins / Q000096:biosynthesis / Q000648:ultrastructure; D020013:Calcium Signaling / Q000502:physiology; D002448:Cell Adhesion / Q000502:physiology; D002462:Cell Membrane / Q000378:metabolism* / Q000648:ultrastructure; D002478:Cells, Cultured; D018385:Centrosome / Q000378:metabolism* / Q000648:ultrastructure; D003598:Cytoskeletal Proteins / Q000096:biosynthesis / Q000648:ultrastructure; D051180:Desmoplakins; D003896:Desmosomes / Q000378:metabolism* / Q000648:ultrastructure; D004285:Dogs; D004705:Endocytosis / Q000502:physiology; D004847:Epithelial Cells / Q000378:metabolism* / Q000648:ultrastructure; D006056:Golgi Apparatus / Q000378:metabolism* / Q000648:ultrastructure; D007150:Immunohistochemistry; D008854:Microscopy, Electron; D015335:Molecular Probes; D011498:Protein Precursors / Q000096:biosynthesis* / Q000648:ultrastructure; D021381:Protein Transport / Q000502:physiology*; D015534:Trans-Activators; D022161:Transport Vesicles / Q000378:metabolism* / Q000648:ultrastructure; D051176:beta Catenin; D051185:gamma Catenin; D021601:trans-Golgi Network / Q000378:metabolism / Q000648:ultrastructure
D015820:Cadherins; D003598:Cytoskeletal Proteins; D051180:Desmoplakins; D015335:Molecular Probes; D011498:Protein Precursors; D015534:Trans-Activators; D051176:beta Catenin; D051185:gamma Catenin