The localization of actin in dividing corneal endothelial cells demonstrated with nitrobenzoxadiazole phallacidin

Abstract

The distribution of actin in dividing endothelial cells of the rat cornea was studied by fluorescence microscopy by means of the nitrobenzoxadiazole conjugated derivative of the actin-binding toxin phallacidin (NBD-Ph). In normal noninjured tissue, fluorescence is limited to an area at or near the plasma membrane. Twenty-four hours after a corneal freeze injury, stress fibers are detected but only in those cells that are migrating into the wound area. By 48 h post-injury, cells in various stages of mitosis can be identified. During metaphase, anaphase, and telophase, diffuse cytoplasmic staining is observed, although the spindle region remains free of fluorescence. At various sites along the plasma membrane, fluorescence appears stronger compared to other regions. During the latter two stages of proliferation, NBD-Ph positive material can be seen within cell processes. In addition, a band of this material is observed within the region that corresponds to the cleavage furrow. As the daughter cells separate, actin can be detected within the cytoplasmic bridge. The results indicate that NBD-Ph can be used to study the distribution of actin in cells that were proliferating in vivo, and these patterns appear similar to those obtained with immunological methods on cultured cells.