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

Abstract Desmosome formation in MDCK cells was investigated using a Ca 2+ shift. Following preliminary treatment with cycloheximide at 37°C, continued surface transport and subsequent endocytosis were minimized by incubating cells at 19°C to trap nascent glycoproteins within the Golgi body. Release into high Ca 2+ medium (HCM) at 37°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 β-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°C.