Accumulation of actin microfilaments in adult rat hepatocytes cultured on collagen gel/nylon mesh.

Adult rat hepatocytes cultured on a collagen gel/nylon mesh support were shown previously to exhibit fetal properties as a function of time in culture and to accumulate numerous microfilament structures as seen with electron microscopy. The present study characterizes the accumulation and properties of such microfilaments. Electron microscopy revealed that microfilaments, 40 to 60 A in diameter, accumulated as a compact network almost exclusively beneath the plasma membrane at the interface with the culture medium. Treatment with heavy meromyosin resulted in the formation of the characteristic arrowhead complexes on these microfilaments, indicating that they contain actin. Extraction of glycerinated hepatocytes with low-ionic-strength solutions removed most of the microfilaments, which could also be disrupted by treatment of the cells with cytochalasin B. The disruption was reversible, and reappearance of the network of microfilaments, though less compact, was observed as early as 30 min after removal of cytochalasin B. At later times in culture, the microfilaments developed into a more compact and extensive network, and their reappearance was not dependent on protein synthesis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of proteins extracted from hepatocytes labeled with L-[35S]methionine or from acetone powders of the labeled hepatocytes showed a major band of a molecular weight of 42,000 that comigrated with skeletal muscle actin on the sodium dodecyl sulfate-polyacrylamide gels. The amount of radioactivity in this band increased with the age of the hepatocyte culture; this indicated an increase in the synthesis of the actin-like protein as well as a greater degree of polymerization into microfilaments. This finding was further confirmed by the demonstration of an increase in the proportion of the filamentous form of actin in the late cultures by means of a DNase I assay. The studies reported in this paper indicate that (a) the microfilaments in cultured hepatocytes contain actin, with properties similar to those found in other nonmuscle cells, and (b) the large accumulation of microfilaments is due in part to the synthesis of actin and the formation of new microfilaments.