Inhibition of cytoskeletal reorganization stimulates actin and tubulin syntheses during injury-induced cell migration in the corneal endothelium

Abstract

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.

Colchicine inhibits pressure-induced tumor cell implantation within surgical wounds and enhances tumor-free survival in mice

“…Further studies would be required to determine how colchicine pharmacokinetics in humans compare with those in mice, the effect of treating recipient mice with colchicine, whether a lower colchicine dose similarly reduces tumor cell adhesion, and whether humans can tolerate a single preoperative dose of the magnitude used in the present study. Although chronic colchicine therapy can inhibit the healing of fractures, corneal epithelium, gastric mucosa, and contracting skin wounds (49)(50)(51)(52), such effects from a single preoperative dose of colchicine are less likely, because most postsurgical wound healing would occur after the single dose of colchicine had been metabolized. Alternatively, other agents might target this adhesion-stimulating pathway.…”

Soybean agglutinin binding to corneal endothelial cell surfaces disrupts in situ monolayer integrity and actin organization and interferes with wound repair

“…During injuryinduced cell migration, endothelial cells reorganize their actin CMBs into stress fibers (Gordon et al 1982;Gordon and Staley 1990;Ichijima et al 1993;Petroll et al 1995). This reorganization depends on a repatterning of actin that is independent of new protein synthesis (Gordon and Buxar 1997). In contrast, in SBA-treated tissues, these blunted pyramidal-shaped cells migrate into the injury site but fail to demonstrate stress fiber organization by fluorescence microscopy.…”

“…Transferring the tissues back into standard medium, without the inhibitor, results in a higher level of SBA binding (c), indicating that some cellular toxicity occurs at this concentration. Bar 50 µm zation of circumferential actin bundles (the CMBs) into distinct stress fibers within migrating cells (Gordon et al 1982;Gordon and Staley 1990;Ichijima et al 1993;Petroll et al 1995;Gordon and Buxar 1997;Gordon et al 2005) with fibronectin deposition occurring along the cell/matrix interface (Sabet and Gordon 1989). Our studies indicate that injury to the endothelium also causes a puromycinsensitive upregulation of the SBA-binding protein on the cell surface.…”

“…Nevertheless, although we demonstrate that puromycin is effective in abolishing SBA binding, both actinomycin D and α-amanitin with the time frames and at the concentrations used in this study fail to prevent SBA binding to the cell surface. In contrast, we have previously shown that actinomycin D at the concentration used (0.05 µg/ml) effectively inhibits actin synthesis in endothelial cells (Gordon and Buxar 1997). Our present results suggest that post-transcriptional regulation of the SBA-binding protein (s) acts as an inherent means of facilitating a response to adverse conditions in order to maintain or assist in the reformation of an intact tissue monolayer.…”

Scraped-Wounding Causes Activation and Association of C-Src Tyrosine Kinase with Microtubules in Cultured Keratinocytes.

“…These processes are associated with dynamic regulation of contacts with the extracellular matrix, i.e., hemidesmosomes and focal contacts, and adherens junctions. Dynamic rearrangements of the cytoskeletons of microfilaments and microtubules are thought to be also profoundly involved in cell migration in vitro at the earliest phase of wound healing (Gordon and Staley, 1990;Gordon and Buxar, 1997;Nakamura et al, 1991, Ettenson andGotlieb, 1992;Baschong et al, 1997).…”