Kinetics of each process of BFA-induced Golgi
disassembly in nocodazole-untreated (A–D) and -treated (E–H)
semi-intact CHO-GT cells. Intact CHO-GT cells were treated with
nocodazole (2 μg/ml) as described in the text to depolymerize
microtubules and then permeabilized by SLO. (A and E) Drug-untreated or
-treated semi-intact cells were incubated with BFA/ATP/cytosol at
32°C for various periods, fixed, and then subjected to morphometric
analysis (Golgi disassembly assay). Dotted lines indicate the
simulation curves, which were simultaneously fitted to the data plots
according to the successive reaction model shown in Figure 10. (B and
F) The cells were incubated with BFA/ATP/NEM-treated cytosol at 32°C
for various periods and then analyzed as described above (Golgi tubule
formation assay). (C and G) The cells were incubated with
BFA/ATP/NEM-treated cytosol at 32°C for 60 min to generate the Golgi
tubules. After being washed, the cells were further incubated with
BFA/ATP/cytosol at 32°C for various periods, fixed, and subjected to
morphometric analysis (Golgi tubule fusion assay). The protein
concentration of the cytosol used was 2.3 mg/ml. Three hundred cells
were counted in three randomly selected fields for each time point.
Three independent experiments were performed, and means and SDs were
calculated. (D and H) Simulated curves for the Golgi disassembly
process. With the use of the rate constants obtained from the Golgi
tubule formation assay (B and F) and the Golgi tubule fusion assay (C
and G), simulated curves for the Golgi disassembly process according to
the successive reaction model were drawn. ○, intact Golgi; ▵,
tubular Golgi; ●, fused Golgi.