Figure 4.

ITD inhibits constitutive retrograde trafficking of ManII as revealed by nocodazole-induced Golgi mini-stack formation at ER exit sites. Clone 9 cells were incubated with various concentrations of ITD for 10 min at 4°C, medium was replaced with 37°C MEM containing nocodazole (plus or minus ITD as appropriate), and cells were further incubated for 1 h at 37°C. Cells were then fixed and stained by immunofluorescence for ManII. (A-D) Immunofluorescence micrographs: (A) control, untreated cells with a central interconnected Golgi complex; (B) cells treated with nocodazole for 1 h reveal the complete loss of a central, intact Golgi complex, which was replaced by small mini-stacks at ER exit sites throughout the cytoplasm. (C) Addition of 10 μM ITD partially inhibited min-stack formation and the loss of the central Golgi complex; (D) addition of 25 μM ITD substantially inhibited mini-stack formation and loss of the Golgi complex. (E) Quantitation of immunofluorescence results in which cells staining for ManII were scored as having widely distributed mini-stacks (as in B), an intermediate amount of inhibition with some mini-stacks and partial retention of a central Golgi complex (as in C), or a more typical juxtanuclear Golgi complex and no mini-stacks (as in A and D). Results show the mean ± 1 SD (n ≥ 3 for all data points). n, nucleus.