Actin in wound-healing of rabbit corneal endothelium. II. Study by nitrobenzoxadiazole-phallacidin method

The distribution of F-actin in the rabbit corneal endothelial cells was studied in vivo and in culture using nitrobenzoxadiazole-conjugated phallacidin. In the normal cornea, the fluorescence showing the presence of F-actin was observed along the membrane of the endothelial cells, but little fluorescence was seen in the cytoplasm. During wound-healing processes after transcorneal freezing, the endothelial cells migrating to the wound area showed abundant fiber-like fluorescence in the cytoplasm. In about 28 days after the injury, the endothelial cells recovered normal shape and the pattern of actin localization became normal, ie, fiber-like fluorescence localization along the cell membrane. The endothelial cells were cultured for about one week and the explants were removed. After further culture for about two weeks the cultured cells became confluent forming a monolayer. At the center of this monolayer, a small wound was made, and changes in the cell shape and actin distribution were studied. The actin distribution in the undisturbed monolayer cells was similar to that seen in vivo, ie, fiber-like fluorescence along the cell membrane. After the wound production, many cells were seen to migrate toward the wound center, and abundant fluorescent fiber-like structures were observed throughout the cytoplasm. Addition of cytochalasin B to the culture medium suppressed cell migration in a dose-dependent manner. At a high cytochalasin B concentration the fiber-like fluorescence was not formed and scattered fluorescent speckles were observed. Further culture in cytochalasin B-free medium after exposure to this agent permitted a recovery of cell migration and formation of the fiber-like actin fluorescence. It was suggested that polymerization of actin filaments is activated in the migrating cells during wound-healing, and that cytochalasin B reversibly blocks this polymerization, thereby suppressing cell migration. Actin filament polymerization would constitute a significant part of the mechanism underlying cell migration and wound-healing.