Location of a fibronectin domain involved in newt epidermal cell migration.

Abstract

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.

Fibronectin Is More Active than Fibrin or Fibrinogen in Promoting Staphylococcus aureus Adherence to Inserted Intravascular Catheters

“…Previous reports confirmed that the amino-terminal 27-kDa tryptic fragment bound specifically to S. aureus in suspension [15] or after adsorption on surfaces [16,41]. We also found a significant adherence-promoting activity in a mixture of 190-and 200-kDa tryptic fragments lacking both the amino-and carboxy-terminal ends of fibronectin [27,28]. The mixture of 190-and 200-kDa fragments adsorbed on gelatin-precoated cannula segments in parallel with intact fibronectin promoted S. aureus Cowan I adherence at equivalent amounts of adsorbed protein, although to a slightly lower extent (figure 4B).…”

“…aureus-binding domain [15,26] and previously referred to as the 180-kDa carboxy-terminal fragment [15], was prepared by trypsin digestion as previously described in detail [15]. These fragments, appearing as two closely spaced bands on SDS-PAGE (190 and 200 kDa), have subsequently been shown to be produced from the A and B chains, respectively [27], and to contain the collagen, heparin, and cell-binding regions, but not the carboxy-terminal end including the disulfide bonds connecting both subunits of fibronectin [27,28]. The mixture of 190-and 200-kDa fragments was purified from the 27-kDa fragment by chromatography on DEAE-Sepharose.…”

Role of fibronectin in normal wound healing

“…A later study by Donaldson et al . attempted to localise the domain of fibronectin that mediates this migration. They demonstrated that new epidermal cell migration is restricted to a domain in the middle third of the gene.…”

Limb regeneration in higher vertebrates: Developing a roadmap

“…In urodeles, the initial response to the injury is the formation of a fibrin clot that covers the wound surface and provides a substrate on which peripheral epidermal cells migrate to close the wound (Donaldson et al, 1985(Donaldson et al, , 1987. This process is initiated by cells at the wound edge extending lamellipodia and actively moving across the wound, and more proximal cells similarly extending lamellipodia behind these lead cells (Mahan and Donaldson, 1986;Dungan et al, 2002).…”