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.