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The heavy accumulation of beta 1 and beta 4 at the wound bed interface in migrating cells suggests that these subunits may be involved in attachments of migrating cells to extracellular matrix proteins in the wound. The accumulation of ptyr in the same region further suggests that integrin-ligand interaction in keratinocytes modulates cell behavior through phosphorylated proteins. The fact that freshly isolated newt keratinocytes could adhere and spread on fibronectin or collagen shows that these cells are constitutively activated. This view is supported by the absence of any evidence that the beta 1 in migrating keratinocytes is larger and therefore more mature than beta 1 in normal keratinocytes. By comparison, beta 1 integrins on human keratinocytes are not constitutively activated (Takashima and Grinnell, 1985; Toda et al., 1987; Guo et al., 1990, 1991), a difference that may explain why epidermal wound healing is faster in newts than in humans.

The interaction of migrating newt epidermal cells with the extracellular matrix protein, fibronectin, was studied. Pieces of nitrocellulose coated with intact human plasma fibronectin or proteolytically derived fragments were implanted into wounded limbs so that the coated nitrocellulose served as wound bed for migrating epidermal cells as they attempted to form a wound epithelium. Epidermal cells migrated very poorly on nitrocellulose pieces coated with (a) a 27-kD amino-terminal heparin-binding fragment, (b) a 46-kD gelatin-binding fragment, (c) a combined 33-and 66-kD carboxy-terminal heparin-binding preparation representing peptide sequences in the A and B chains, respectively, or (d) a 31-kD carboxyterminal fragment from the A chain, containing a free sulfhydryl group. In contrast, epidermal cells readily migrated onto nitrocellulose coated with a mixture of fragments from the middle of the molecule (80-125kD) that bind neither heparin nor gelatin. Attempts to block migration on fibronectin-coated nitrocellulose using 1810, a monoclonal antibody that blocks Chinese hamster ovary cell attachment to fibronectin, were unsuccessful despite saturation of the epitope against which I810 is directed. In contrast, a polyclonal anti-fibronectin antibody did inhibit migration. These results show that the ability of fibronectin to support newt epidermal cell migration is not shared equally by all regions of the molecule, but is restricted to a domain in the middle third. They also suggest that the site supporting migration is separate and distinct from the site mediating Chinese hamster ovary cell attachment.

Pieces of coverslip glass, polycarbonate filters, or coverslip plastic, coated with fibrinogen or type I collagen, were implanted under one edge of a fresh skin wound on adult newt hind limbs so that the implant served as wound bed for migrating epidermal cells as they attempted to form a wound epithelium. Migratory events were then analyzed by phase contrast and electron microscopy. Phase-contrast microscopy revealed two types of lamellipodia on leading edge cells: one which was attached broadly to the cell body and one attached by a long, thin stalk. Stalkless forms were by far the most common type and we believe they provide the motive force for cell movement. Stalked-forms often moved at distinct angles to the direction of sheet movement, suggesting that they may be sensory appendages. Phase photographs of the leading edge of migrating sheet 4 hours and 8 hours after implantation showed that all cells that were on the leading edge at 4 hours continued to advance for the next 4 hours, demonstrating clearly that under these circumstances the distalmost cells do not become immobile upon contact with the substrate as others have suggested. TEM revealed that migrating sheets were modified monolayers and that regardless of proximodistal location in the sheet, and even in the intact skin adjoining a wound, each epidermal cell adjacent to the substrate puts forth a lamellipodium which underlaps the cell in front. This and the behavior of sheets as they were teased or pulled from the implant suggest strongly that all basal cells contribute to movement of the sheet by interacting with the substrate.(ABSTRACT TRUNCATED AT 250 WORDS)