Microcystin-LR and kinetics of cytoskeletal reorganization in hepatocytes, kidney cells, and fibroblasts.

Abstract

Microcystin-LR (MCLR) is a cyanobacterial hepatotoxin that inhibits protein phosphatases 1 and 2A. To characterize cytoskeletal changes over time, hepatocytes were incubated with the toxin at 13.3 microM for 0, 2, 4, 6, 8, 16, 32, or 64 minutes. Changes in the hepatocytes were compared to those in cultured kidney cells and skin fibroblasts incubated with the toxin at 133 microM for 0, 2, 4, 8, 12, 16, or 24 hours. Cells were fixed and incubated with rhodamine-conjugated phalloidin, or primary antibodies against beta-tubulin and either vimentin or cytokeratin intermediate filaments (IFs), followed by fluorescein-conjugated secondary antibodies. The number of affected cells per 400 counted (NAC) with alterations in a specific cytoskeletal element were determined at each time point. In fibroblasts as well as kidney cells, changes occurred first in IFs, followed by microtubules (MTs), and later microfilaments (MFs). In some hepatocytes, IFs were affected first, but after 16 minutes, the NAC with altered MTs exceeded the NAC with alterations in other cytoskeletal elements. In both hepatocytes and non-hepatocytes, IFs and MTs condensed and collapsed around the nucleus. MFs similarly collapsed, but some of the actin radiated outward, producing a star-like appearance. The similarity of the cytoskeletal changes induced by MCLR in hepatocytes and non-hepatocytes suggests a common mechanism of action. Differences among cell types in sequential cytoskeletal alterations may be due to differences in phosphorylation of intracellular proteins.