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

High-pressure freezing and freeze-substitution were used to study Golgi ult rastructure and its brefeldin A-induced transformations in HepG2 human hepa toma cells. Cryoimmobilization arrested subcellular dynamics within millise conds, thus considerably improving the temporal resolution in monitoring th e very early effects of high brefeldin concentrations at the ultrastructura l level (i.e., 20 mu g/ml brefeldin applied for 35 s to 8 min), Moreover, t his approach ruled out possible cumulative and/or synergistic effects of th e drug and fixatives, Several findings differed from studies based on chemi cal 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 a nd 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 sta cks, starting tubulation and fragmentation. After 3-5 min the Golgi apparat us was completely replaced by loose meshworks of straight tubules of differ ent sizes and staining properties; also frequent mere bent tubules and vesi cles forming glomerule-like structures. After 8 min all kinds of Golgi-deri ved structures had aggregated within huge clusters. The morphologically hig hly distinct structures found after brefeldin treatment could in part be co rrelated with particular Gels domains in the control cells. (C) 2000 Academ ic Press.