Figure 5.

Acute degradation of GRASP55 and/or 65 during interphase does not affect the cis to trans polarization of Golgi stacks. (A and B) Cells were treated with doxycycline (dox) for 6 h, IAA was added for an additional 2 h, and cells were prepared for immunofluorescence analysis. IAA treatment did not change the localization of the cis-Golgi marker Golgin-84 (green) and the trans-Golgi protein Golgin-97 (red) to the perinuclear Golgi ribbon. Scale bar, 10 μm. (C and D) Cells incubated with doxycycline were treated with IAA or IAA with nocodazole (noc) for an additional 2 h to depolymerize microtubules and to disperse the Golgi ribbon into individual stacks. Cells were then immunostained for Golgin-84 (green), Golgin-97 (red), and DNA (blue). Insets show a magnified Golgi stack with the white line marking the line scan of the fluorescence intensities shown in the graphs. Scale bar, 10 µm. Inset scale bar, 1 µm. (E and F) Polarized Golgi stacks reassemble in cells depleted of GRASPs. Cells treated with doxycycline were incubated with IAA and nocodazole for 2 h, followed by BFA to fragment the Golgi stacks. BFA was then removed, and nocodazole (plus dox and IAA) was maintained to allow reformation of individual Golgi stacks. The cells were fixed and immunostained for Golgin-84 (green) and Golgin-97 (red) and labeled for DNA (blue). Insets show a magnified Golgi stack with the white line marking the line scan of the fluorescence intensities shown in the graphs. Scale bar, 10 µm. Inset scale bar, 1 µm.