A single layer of squamous epithelial cells termed the "endothelium" resides upon its natural basement membrane (Descemet's membrane) along the posterior surface of the vertebrate cornea. A well-defined circular freeze injury to the center of the tissue exposes the underlying basement membrane and results in the directed migration of surrounding cells into the wound center. This cellular translocation is characterized by the reorganization of the actin and tubulin cytoskeletons. During migration, circumferential microfilament bundles are replaced by prominent stress fibers while microtubules, observed as delicate lattices in non-injured cells, become organized into distinct web-like patterns. To determine whether this cytoskeletal reorganization requires actin or tubulin synthesis, injured rabbit endothelia were organ cultured for various times and metabolically labeled with 35S-methionine/cystine (250 microCi/ml) for the final 6 h of each experiment. Analysis of actin and tubulin immunoprecipitates indicated no significant increases in 35S incorporation occurred during the course of wound repair when compared to isotope incorporation in noninjured tissues. However, when cytoskeletal reorganization was hampered, either by pre-treating tissues with 7 microM phalloidin to stabilize their circumferential microfilament bundles, or culturing in the presence of 10(-8)M colchicine to dissociate microtubules, 35S incorporation increased significantly into both actin and tubulin immunoprecipitates at 48 h post-injury. Furthermore, in both cases, exposure to actinomycin D substantially suppressed isotope incorporation. These results indicate that cytoskeletal rearrangement of microfilaments and microtubules during wound repair, in corneal endothelial cells migrating along their natural basement membrane, utilizes existing actin and tubulin subunits for filament reorganization. Disrupting this disassembly/reassembly process prevents cytoskeletal restructuring and leads to the subsequent initiation of actin and tubulin syntheses, as a result of increased transcriptional activity.

A single layer of squamous epithelial cells termed the "endothelium" resides upon its natural basement membrane (Descemet's membrane) along the posterior surface of the vertebrate cornea. A well-defined circular freeze injury to the center of the tissue exposes the underlying basement membrane and results in the directed migration of surrounding cells into the wound center. This cellular translocation is characterized by the reorganization of the actin and tubulin cytoskeletons. During migration, circumferential microfilament bundles are replaced by prominent stress fibers while microtubules, observed as delicate lattices in non-injured cells, become organized into distinct web-like patterns. To determine whether this cytoskeletal reorganization requires actin or tubulin synthesis, injured rabbit endothelia were organ cultured for various times and metabolically labeled with 35S-methionine/cystine (250 microCi/ml) for the final 6 h of each experiment. Analysis of actin and tubulin immunoprecipitates indicated no significant increases in 35S incorporation occurred during the course of wound repair when compared to isotope incorporation in noninjured tissues. However, when cytoskeletal reorganization was hampered, either by pre-treating tissues with 7 microM phalloidin to stabilize their circumferential microfilament bundles, or culturing in the presence of 10(-8)M colchicine to dissociate microtubules, 35S incorporation increased significantly into both actin and tubulin immunoprecipitates at 48 h post-injury. Furthermore, in both cases, exposure to actinomycin D substantially suppressed isotope incorporation. These results indicate that cytoskeletal rearrangement of microfilaments and microtubules during wound repair, in corneal endothelial cells migrating along their natural basement membrane, utilizes existing actin and tubulin subunits for filament reorganization. Disrupting this disassembly/reassembly process prevents cytoskeletal restructuring and leads to the subsequent initiation of actin and tubulin syntheses, as a result of increased transcriptional activity.