Localization of the fibronexus at the surface of granulation tissue myofibroblasts using double-label immunogold electron microscopy on ultrathin frozen sections

Abstract

An intimate transmembrane complex of fibronectin-containing extracellular fibers and actin microfilaments termed the fibronexus has been observed at the adhesive surface of fibroblasts in vitro [19] and along the plasmalemma of myofibroblasts in vivo [22]. Although the observation of coincident actin and fibronectin immunofluorescence patterns in the latter work strongly suggested that the fibronexus is localized at the myofibroblast surface, we only obtained morphological evidence for its existence with electron microscopy. Therefore, in the present study, we have utilized a double-label immunoelectron microscopic technique to localize fibronectin and actin simultaneously in the putative fibronexuses of myofibroblasts within guinea pig granulation tissue, formed 7 to 9 days after skin wounding. This method employed rabbit antifibronectin and mouse anti-actin antibodies, followed by species-specific secondary antibodies conjugated to colloidal gold particles of different sizes. These probes were applied to the surfaces of ultrathin frozen sections mounted on grids. We found that fibronectin and actin were specifically localized on the respective external and internal components of myofibroblast fibronexuses. Our results suggest that specific transmembranous fibronectin-cytoskeletal complexes play an important role in the cohesion of granulation tissue.