Cryopreparation provides new insight into the effects of brefeldin A on the structure of the HepG2 Gel apparatus

High-pressure freezing and freeze-substitution were used to study Gel ultrastructure and its brefeldin A-induced transformations in HepG2 human hepatoma cells. Cryoimmobilization arrested subcellular dynamics within milliseconds, thus considerably improving the temporal resolution in monitoring the very early effects of high brefeldin concentrations at the ultrastructural level (i.e., 20 mu g/ml brefeldin applied for 35 s to 8 min). Moreover, this approach ruled out possible cumulative and/or synergistic effects of the drug and fixatives. Several findings differed from studies based on chemical fixation. In particular, Golgi breakdown did not proceed gradually but occurred in distinct steps. We found a conspicuous lag between the absence of nonclathrin coats on Golgi membranes after 30 s of brefeldin treatment and the disassembly of the stacks, which did not start until after 90 to 120 s. At this time, domains at the trans and cis faces separated from the stacks, starting tubulation and fragmentation. After 3-5 min the Golgi apparatus was completely replaced by loose meshworks of straight tubules of different sizes and staining properties; also frequent mere bent tubules and vesicles forming glomerule-like structures. After 8 min all kinds of Golgi-derived structures had aggregated within huge clusters. The morphologically highly distinct structures found after brefeldin treatment could in part be correlated with particular Golgis domains in the control cells. (C) 2000 Academic Press.