Brock, Jane; (1997) The role of actin purse-strings in wound healing and in the natural movements of embryonic morphogenesis. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Skin wounds in limb-bud stage chick embryos re-epithelialise using a contracting filamentous actin cable which assembles in the basal layer of the wound edge epithelial bilayer within 2 minutes of wounding (Martin and Lewis, 1992a). Characterising the contractile apparatus by immunostaining reveals intracellular segments of the actin cable inserting into clustered adherens junctions at the cell-cell boundaries, as well as colocalisation of myosin II protein localising to the basal wound margin within 7 minutes of wounding. Epithelial cells at the wound edge are transiently permeabilised by the wounding process, allowing loading of small molecules into precisely the cells which assemble actin cable, thus providing a method of investigating the signals which regulate cable assembly. The Ras-related small GTPases regulate rapid reorganisations of the actin cytoskeleton in cultured fibroblasts (Ridley and Hall, 1992; Ridley et al, 1992). I show that co-loading of wound edge cells with fluorescent dextran and Rho inactivator, C3 transferase, blocks assembly of actin cable and prevents re-epithelialisation. By contrast, introducing dominant negative N17Rac into the wound edge epithelial cells has no affect on the healing process, suggesting that Rho, but not N17Rac, is the molecular switch regulating wound-induced assembly of an actin cable. The keratin intermediate filaments of embryonic epithelium are required for repair of wounds in Xenopus embryos (Torpey et al, 1992). Wounded limb-bud stage mouse embryos assemble a filamentous keratin-8 cable in wound edge epithelial cells in addition to the actin cable described above. In contrast to the Xenopus depletion experiments, I show that transgenic knock-out mouse embryos null for K8 (Baribault et al, 1993; 1994) completely re-epithelialise their wounds within 24 hours just like their wild-type litter mates demonstrating that despite an inability to assemble keratin filaments, wound repair proceeds normally. Finally I report preliminary experiments characterising an in vitro wound healing model of a gut epithelial cell line, Caco-2BBE, which may enable more detailed studies of the mechanisms of purse-string versus crawling motility.

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