Biochemical requirements for BFA-induced Golgi
disassembly in microtubule-disrupted semi-intact cells. (A) CHO-GT
cells were treated with nocodazole to depolymerize microtubules and
then permeabilized by SLO. The drug-treated semi-intact cells were
incubated with BFA/ATP (ATP), BFA/ATP/cytosol (cytosol), or
BFA/ATP/NEM-treated cytosol (NEM-cyto) at 32°C for 80 min, fixed, and
then subjected to morphometric analysis (Golgi disassembly assay). The
drug-treated semi-intact cells incubated with BFA/ATP/NEM-treated
cytosol at 32°C for 80 min were washed and then further incubated
with BFA/ATP/NEM-treated cytosol/His-NSF at 32°C for 60 min. Again,
morphometric analysis was performed (NEM-cyto+His-NSF). The protein
concentration of the cytosol used was 2.5 mg/ml. At least 300 cells
were counted for each sample. Cells were counted in three randomly
selected fields. Three independent experiments were performed, and
means and SDs were calculated. (B) Golgi tubules in the presence or
absence of nocodazole treatment. Nocodazole-treated (+Noc) or
-untreated (−Noc) semi-intact cells were incubated with
BFA/ATP/NEM-treated cytosol at 32°C for 80 min. Then, microtubules
(MT) were visualized by the immunofluorescence method with the use of
anti-α-tubulin antibody. Bar, 10 μm.