ALTERATIONS IN MICROTUBULES, INTERMEDIATE FILAMENTS, AND MICROFILAMENTS INDUCED BY MICROCYSTIN-LR IN CULTURED-CELLS

Microcystin-LR (MCLR) is a cyanobacterial hepatotoxin that inhibits in tracellular serine/threonine protein phosphatases causing disruption o f actin microfilaments (MFs) and intermediate filaments (Ifs) in hepat ocytes. This study compared the effects of MCLR on the organization of MFs, Ifs, and microtubules (MTs) in hepatocytes and nonhepatocyte cel l hues and determined the sequence of toxin-induced changes in these c ytoskeletal components. Rat renal epithelial cells and fibroblasts wer e incubated with MCLR at 100 or 200 mu M for 6-18 hr. Rat hepatocytes in primary culture were exposed to the toxin at 1 or 10 mu M for 2-64 min. Cells were fixed and incubated with primary antibodies against be ta-tubulin, actin, and vimentin or cytokeratin Ifs, followed by gold-l abeled secondary antibodies with silver enhancement of the gold probe. The fraction of fibroblasts and hepatocytes with altered cytoskeletal morphology was evaluated as a function of MCLR dose and exposure time to assess the sequence of changes in cytoskeletal components. Changes in fibroblasts and some hepatocytes were characterized initially by d isorganization of IFs, followed rapidly by disorganization of MTs, wit h the progressive collapse of both cytoskeletal components around cell nuclei. Many hepatocytes exhibited MT changes prior to effects on IF structure. Alterations in MFs occurred later and included initial aggr egation of actin under the plasma membrane, followed by condensation i nto rosette-like structures and eventual complete collapse into a dens e perinuclear bundle. The similarity of effects among different cell t ypes suggests a common mechanism of action, but the independent kineti cs of IF and MT disruption in hepatocytes suggests that there may be a t least 2 sites of phosphorylation that lead to cytoskeletal alteratio ns.