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 Ca2+ shift

Desmosome formation in MDCK cells was investigated using a Ca2+ shift. Following preliminary treatment with cycloheximide at 37degreesC, continued surface transport and subsequent endocytosis were minimized by incubating cells at 19degreesC to trap nascent glycoproteins within the Golgi body. Release into high Ca2+ medium (HCM) at 37degreesC 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 19degreesC.