Brefeldin A (BFA) disrupts the organization of the microtubule and the actin cytoskeletons

Previous inquiries into the effects of Brefeldin A (BFA) have largely conce ntrated on dynamics of ER-Golgi membrane traffic, predominantly after relat ively short treatments with the drug. We have now analyzed the effects of l ong BFA treatment on overall cell morphology, behavior of resident and cycl ing Golgi proteins, and microtubular and actin cytoskeletons organization. Prolonged (15 h or 40 h) treatment of normal rat kidney (NRK) cells with BF A caused dramatic swelling of the Endoplasmic Reticulum (ER) and shifted it s localization to the periphery of the cells, The Golgi complex was disasse mbled and Golgi proteins redistributed and persisted in partially distinct compartments. Prolonged BFA treatment resulted in marked disruption of the MT and actin cytoskeleton, Peripheral MT were absent and tubulin staining w as concentrated in short astral MT emanating from the microtubule organizin g center (MTOC). Actin stress fibers were largely absent and actin staining was concentrated within a perinuclear area. Within this region, actin loca lization overlapped that of the membran transport factor p115. EFA effects on Golgi structure and on MT and actin organization showed the same thresho ld - all could be partially reversed after 30 min and 15 h BFA treatment bu t were irreversible after 40 h incubation with the drug. The observed effec ts were not induced by signaling pathways involved in apoptotic phenomena o r in ER stress response pathways. These results suggest that BFA inhibits t he activity of key molecules that regulate MT and actin cytoskeleton dynami cs. The findings can be used as the basis for elucidating the molecular mec hanism of BFA action on the cytoskeleton.