Synthesis of Mallory body, intermediate filament, and microfilament proteins in liver cell primary cultures. An electron microscopic autoradiography assay

Abstract

Background: The synthesis of Mallory body (MB) and cytoskeletal proteins in hepatocytes have not been studied in vitro. To visualize the incorporation of radiolabeled amino acid into MBs and cytoskeletal filaments separately in the same cell, we developed a new technique using high resolution electron microscopic autoradiography.

Experimental design: Primary cultures of hepatocytes isolated from griseofulvin (GF)-fed mice and control mice were used. The hepatocytes labeled with [35S]methionine were extracted with detergent. The detergent-resistant residual cytoskeleton was characterized by polyacrylamide gel electrophoresis and autoradiography. For light microscopic and electron microscopic autoradiography, the hepatocytes were labeled with [3H]methionine or [3H]leucine, then extracted with detergent and then embedded in plastic. To obtain high resolution of the labeled cytoskeleton, exposed ultrathin sections were treated by the gold latensification and physical developing methods and then examined by electron microscopy.

Results: Autoradiography of polyacrylamide gel electrophoresis showed [35S]methionine was incorporated into three major bands: cytokeratin 55, cytokeratin 49, and actin in control and GF hepatocytes. In GF hepatocytes, high molecular weight proteins that remained near the top of the gel (MB-derived proteins) were also labeled. The electron microscopic autoradiography revealed that the intermediate filaments, microfilaments, MBs, and nuclear matrix were labeled and the resolution of the labeling was high enough to localize the labeled amino acids incorporated into the filaments. The background was very low. The labeling using [3H]methionine was greater than [3H]leucine, but the pattern of labeling was the same. In control mice, hepatocytes intermediate filaments and microfilaments were heavily labeled at the cell border. In GF-fed mice, the MBs were heavily labeled compared with the intermediate filaments at the cell borders within the same cells. The intensity of labeling varied from cell to cell.

Conclusions: These results suggested that the newly synthesized cytokeratin monomers were incorporated into the already polymerized filamentous network as well as the MB filaments in cell culture.