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Toxicologic Pathology
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Journal Article

Alterations in Microtubules, Intermediate Filaments, and Microfilaments Induced by Microcystin-LR in Cultured Cells

Mark L. Wickstrom

Department of Veterinary Biosciences, University of Illinois, US EPA, 8 HWM-SM, 999 18th Street, Suite 500, Denver, Colorado 80202-2466

Safdar A. Khan

Department of Veterinary Biosciences, University of Illinois

Wanda M. Haschek

Department of Veterinary Pathobiology, University of Illinois, Urbana, Illinois 6801

John F. Wyman

Naval Medical Research Institute/Toxicology Detachment, Wright Patterson AFB, Ohio

John E. Eriksson

Turku Center for Biotechnology, Biocity, Turku, Finland

David J. Schaeffer

Department of Veterinary Biosciences, University of Illinois

Val R. Beasley

Department of Veterinary Biosciences, University of Illinois

Microcystin-LR (MCLR) is a cyanobacterial hepatotoxin that inhibits intracellular serine/threonine protein phosphatases causing disruption of actin microfilaments (MFs) and intermediate filaments (IFs) in hepatocytes. This study compared the effects of MCLR on the organization of MFs, IFs, and microtubules (MTs) in hepatocytes and nonhepatocyte cell lines and determined the sequence of toxin-induced changes in these cytoskeletal components. Rat renal epithelial cells and fibroblasts were incubated with MCLR at 100 or 200 µM for 6-18 hr. Rat hepatocytes in primary culture were exposed to the toxin at 1 or 10 µm for 2-64 min. Cells were fixed and incubated with primary antibodies against β-tubulin, actin, and vimentin or cytokeratin IFs, followed by gold-labeled 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 disorganization of IFs, followed rapidly by disorganization of MTs, with 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 aggregation of actin under the plasma membrane, followed by condensation into rosette-like structures and eventual complete collapse into a dense perinuclear bundle. The similarity of effects among different cell types suggests a common mechanism of action, but the independent kinetics of IF and MT disruption in hepatocytes suggests that there may be at least 2 sites of phosphorylation that lead to cytoskeletal alterations.

Key Words: Cyanobacteria • microcystin • protein phosphatase inhibitors • cytoskeleton • hepatocytes

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Toxicologic Pathology, Vol. 23, No. 3, 326-337 (1995)
DOI: 10.1177/019262339502300309
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