Effects of cytochalasin B on actin and myosin association with particle binding sites in mouse macrophages: implications with regard to the mechanism of action of the cytochalasins.

Abstract

The intracellular distribution of F-actin and myosin has been examined in mouse peritoneal macrophages by immunofluorescence microscopy. In resting, adherent cells, F-actin was distributed in a fine networklike pattern throughout the cytoplasm. Myosin, in contrast, was distributed in a punctate pattern. After treatment with cytochalasin B (CB), both proteins showed a coarse punctate pattern consistent with a condensation of protein around specific foci. After CB-pretreated cells were exposed to opsonized zymosan particles, immunofluorescent staining for F-actin and myosin showed an increased staining under particle binding sites. Transmission electron microscope (TEM) examination of whole-cell mounts of such preparations revealed a dense zone of filaments beneath the relatively electron-translucent zymosan particles. At sites where particles had detached during processing, these filament-rich areas were more clearly delineated. At such sites dense arrays of filaments that appeared more or less randomly oriented were apparent. The filaments could be decorated with heavy meromyosin, suggesting that they were composed, in part, of F-actin and were therefore identical to the structures giving rise to the immunofluorescence patterns. After viewing CB-treated preparations by whole-mount TEM, we examined the cells by scanning electron microscopy (SEM). Direct SEM comparison of the filament-rich zones seen by TEM showed that these structures resulted from the formation of short lamellipodial protrusions below the site of particle binding. Electron micrographs of thin-sectioned material established that these lamellipodial protrusions were densely packed with microfilaments that were in part associated with the cytoplasmic surface of the plasma membrane. The formation of particle-associated lamellipodia did not appear to represent merely a slower rate of ingestion in the presence of CB, because they formed within minutes of particle contact with the cell membrane and were not followed by particle ingestion even after a 1-h or longer incubation. Furthermore, their formation required cellular energy. These results suggest that cytochalasin B blocks phagocytosis of large particles by affecting the distances over which any putative actomyosin-mediated forces are generated.