7-nitrobenz-2-oxa-1-3-diazole-phallacidin and Corneal-Injuries

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.

Topics: Actins; Amanitins; Animals; Cell Division; Cornea; Corneal Injuries; Endothelium; Female; Histocytochemistry; Male; Microscopy, Fluorescence; Rats; Rats, Inbred Strains

The fluorescent derivative of the actin-binding toxin phallacidin, 7-nitrobenz-2-oxa-1,3 diazole phallacidin, has been used to cytologically demonstrate the presence of actin in lens epithelium, corneal endothelium, and retinal pigment epithelium. In these noninjured tissues, no stress fibers are observed and fluorescence is confined mainly to an area at or near the cell membrane, although some diffuse cytoplasmic staining can also be seen. However, following injury to either the lens epithelium or corneal endothelium of rats and frogs, stress fibers are detected, but only in those cells that migrate into the wound area. Cells on the periphery of each tissue do not partake in would repair and thus maintain their normal appearance. After the tissue has regenerated, stress fibers disappear, and those cells involved in the injury response return to their normal morphology. When rabbit corneal endothelium is placed in tissue culture, stress fibers are observed as the cells migrate away from the initial explant. Upon reaching confluency, these cells spread out and each is surrounded by thick actin-containing bands. Furthermore, they exhibit some stress cables within their cytoplasm. This is in contrast to their appearance in vivo where stress fibers are absent and fluorescence is limited to a region near the cell membrane.

Topics: Actins; Amanitins; Animals; Cell Movement; Cornea; Corneal Injuries; Cytoplasm; Cytoskeleton; Endothelium; Epithelium; Lens, Crystalline; Microscopy, Fluorescence; Pigment Epithelium of Eye; Rana pipiens; Rats; Rats, Inbred Strains