Healing of incisional wounds in the embryonic chick wing bud:characterization of the actin purse-string and demonstration of arequirement for Rho activation

摘要

Small skin wounds in the chick embryo do not heal by lamellipodial crawling of cells at the wound edge as a skin wound does in the adult, but rather by contraction of an actin purse-string that rapidly assembles in the front row of epidermal cells (Martin, P., and J. Lewis. 1992. Nature (Lond.). 360:179-183). To observe the early time course of actin purse-string assembly and to characterize other cytoskeletal components of the contractile machinery, we have followed the healing of incisional or slash wounds on the dorsum of the chick wing; these wounds take only seconds to create and heal within approximately 6 h. Healing of the epithelium depends on a combination of purse-string contraction and zipper-like closure of the gap between the cut edges of the epithelium. Confocal laser scanning microscope studies show that actin initially aligns into a cable at the wound margin in the basal layer of the epidermis within approximately 2 min of wounding. Coincident with actin cable assembly, we see localization of cadherins into clusters at the wound margin, presumably marking the sites where segments of the cable in adjacent cells are linked via adherens junctions. A few minutes later we also see localization ofmyosin II at the wound margin, as expected if myosin is being recruitedinto the cable to generate a contractile force for wound healing. At thetime of wounding, cells at the wound edge become transiently leaky,allowing us to load them with reagents that block the function of two smallGTPases, Rho and Rac, which recently have been shown to play key roles inreorganiztion of the actin cytoskeleton in tissue-culture cells (Hall, A.1994. Annu. Rev. Cell Biol. 10:31-54). Loading wound edge epidermal cellswith C3 transferase, a bacterial exoenzyme that inactivates endogenous Rho,prevents assembly of an actin cable and causes a failure of healing. Nosuch effects are seen with N17rac, a dominant inhibitory mutant Racprotein. These findings support the view that in this system the actincable is required for healing-both the purse-string contraction and thezipping up-and that Rho is required for formation of the actin cable.