Spontaneous appearance of circular actin bands in cultured hepatocytes of adult rats

We demonstrated, using the phase-contrast microscope, fluorescence microscope, and scanning electron microscope, that circular actin bands appeared spontaneously on the dorsal surface of primary cultured hepatocytes of adult rats on days 2 and 3 of culture. These circular bands protruded from the dorsal surfaces of hepatocytes, the walls of which were very smooth and 2–3 μm in thickness. These structures were strongly stained with rhodamine-phalloidin (F-actin staining), and detected mainly in the areas in which cells were sparse. In number, these circular actin bands were very few on the first day of culture, more than 4000 per 35-mm dish on the second day, about 1500 on the third day, and very few on the fourth day. When insulin was omitted beginning 1 day after culture, the circular actin bands did not decrease in number. In serum-free medium, the number of circular actin bands decreased, although a considerable number of circular actin bands were observed. Wortmannin, a phosphatidylinositol 3-kinase (PI3K) inhibitor, completely inhibited the formation of spontaneously appearing circular actin bands within 10 min. These results indicate that PI3K plays a pivotal role in the formation of circular actin bands.