In vivo co-distribution of fibronectin and actin fibers in granulation tissue: immunofluorescence and electron microscope studies of the fibronexus at the myofibroblast surface.

Abstract

The fibronexus (FNX), a very close transmembrane association of individual extracellular fibronectin fibers and actin microfilaments, was found previously at the substratebinding surface of fibroblasts in tissue culture (Singer, I . I ., 1979, Cell, 16 :675-685) . To determine whether the fibronexus might be involved in fibroblast adhesion during wound healing in vivo, we looked for co-localization of actin and fibronectin in granulation tissue formed within full-thickness guinea pig skin wounds . At 7-9 d, most of the actin fibers were observed to be coincident with congruent fibronectin fibers using double-label immunofluorescence microscopy . These fibronectin and actin fibers were co-localized at the myofibroblast surface surrounding the nucleus, and along attenuated myofibroblast processes which extended deeply into the extracellular matrix. This conspicuous co-distribution of fibronectin and actin fibers prompted us to look for fibronexuses at the myofibroblast surface with electron microscopy . We observed three kinds of FNXs: (a) tandem associations between the termini of individual extracellular fibronectin fibers and actin microfilament bundles at the tips of elongate myofibroblast processes, (b) plaque-like and, (c) track-like FNXs, in which parallel fibronectin and actin fibers were connected by perpendicular transmembranous fibrils . Coniometric studies on the external and internal components of these cross-linking fibrils showed that their membrane-associated ends are probably co-axial . Using immunoelectron microscopy on ultrathin cryosections, we confirmed that the densely staining external portion of these various FNXs does indeed contain fibronectin . The finding that these FNXs appear to connect Collagen fibers to intracellular bundles of actin microfilaments is particularly significant. Our studies strongly suggest that the fibronexus is an important in vivo cell surface adhesion site functioning in wound repair, and perhaps within fibronectin-rich tissues during embryogenesis, tumor growth, and inflammation .The in vitro enhancement of fibroblast adhesion and spreading by fibronectin has been well documented (20,29,55,56), whereas the importance of the cytoskeleton in these processes has only recently been realized (23,26,27,42,43,46). The observation that actin microfilament bundles (stress fibers) are highly developed in well-spread stationary fibroblasts in vitro (23,27,35,43,46) transformed cells, strongly suggests a dominant role for the cytoskeleton in the fibroblast-to-substrate adhesion mechanism. A fascinating aspect ofthis phenomenon is the coincident distribution of fibronectin-containing extracellular matrix fibers and bundles of actin microfilaments on a global scale at the substrate adhesive surface (22,26,27). Electron microscopic studies of this substrate-binding plasma mem-

Fibroblasts, myofibroblasts, and wound contraction

“…During granulation tissue formation, migrating fibroblasts differentiate into myofibroblasts (Darby et al, 1990;Welch et al, 1990). Judging from the presence of fibronexus junctions, myofibroblasts form very tight adhesions to the surrounding fibronectin-rich granulation tissue (Singer et al, 1984;Tomasek and Haaksma, 1991). The timing of the switch from fibroblast to myofibroblast phenotype appears to depend at least in part on the extent to which the wound resists contraction (Darby et al, 1990;Welch et al, 1990).…”

Modulation of Fibroblast Morphology and Adhesion during Collagen Matrix Remodeling

“…Focal adhesions and the in vivo equivalent fibronexus junctions (Singer et al, 1984) are the paradigmatic example of integrin connection between the extracellular matrix and the internal cell cytoskeleton (Hynes, 1992;Burridge and ChrzanowskaWodnicka, 1996). The precise role of focal adhesions in cell migration has been somewhat enigmatic, however, because their presence typically limits the capacity of cells to migrate (Burridge et al, 1988).…”

Myofibroblasts in interstitial lung diseases show diverse electron microscopic and invasive features

“…[4][5][6] The increased cytoplasmic contractility of myofibroblasts has been attributed to the presence of alpha-smooth muscle actin (a-SMA)-positive filaments. 7,8 The fibronexus (FNX) structure is a characteristic marker for the myofibroblast when the cells are examined in the transmission electron microscope (TEM), although its detailed molecular composition has not yet been fully characterized.…”