Figure 9.

Time-lapse analysis of living COS-7 cells during recovery from BFA directly demonstrates dynamitin-induced inhibition of ER-to-Golgi transport. COS-7 cultures were transiently transfected with a GFP–NAGT-I fusion protein either alone (A–E) or with myc-tagged dynamitin (F–J). More than 90% of overexpressing cells in fixed, cotransfected cultures were found to express both transfected constructs. After a 20-min exposure to BFA, the drug was washed out and the GFP–NAGT-I fluorescence was recorded by time-lapse microscopy at 3- to 7-s intervals. (A–E) Cytoplasmic region beneath the nucleus with reforming Golgi complex at upper left corner. (F–J) Cytoplasmic region between nucleus at upper left corner and cell periphery at lower right corner. Control cells typically exhibited rapid centripetal movements of newly formed GFP–NAGT-I–positive vesicles (A–E, arrows; arrowhead indicates static vesicle for reference), leading to the reformation of a perinuclear Golgi complex (A–E, upper left corner). Similar labeled vesicles appeared throughout the cytoplasm of dynamitin-transfected cells, but no sustained movements were observed in any direction, as seen in F–J. Note partial alignments of vesicles in F–J, forming short linear arrays suggestive of interactions with microtubules. Bars, 1 μm.