Fibronectins Connect Cells and ECM, Influencing Cell Shape, Differentiation, and Movement
Many different cell types synthesize fibronectin, an abundant multi-adhesive matrix protein found in all vertebrates. The discovery that fibronectin functions as an adhesion molecule stemmed from observations that it is present on the surfaces of normal fibroblasts, which adhere tightly to petri dishes in laboratory experiments, but is absent from the surfaces of tumorigenic (i.e., cancerous) cells, which adhere weakly. The 20 or so isoforms of fibronectin are generated by alternative splicing of the RNA transcript produced from a single gene (see Figure 5-16). Fibronectins are essential for the migration and differentiation of many cell types in embryogenesis. These proteins are also important for wound healing because they promote blood clotting and facilitate the migration of macrophages and other immune-system cells into the affected area.
Fibronectins help attach cells to the ECM by binding to other ECM components, particularly fibrillar collagens and heparan sulfate proteoglycans, and to adhesion receptors such as integrins (see Figure 20-2). Through their interactions with adhesion receptors, fibronectins influence the shape and movement of cells and the organization of the cytoskeleton. Conversely, by regulating their receptor-mediated attachments to fibronectin and other ECM components, cells can sculpt the immediate ECM environment to suit their needs.
Fibronectins are dimers of two similar polypeptides linked at their C-termini by two disulfide bonds; each chain is about 60–70 nm long and 2–3 nm thick. Partial digestion of fibronectin with small amounts of proteases and analysis of the fragments showed that each chain comprises several functional regions with different ligand-binding specificities (Figure 20-33a). Each region, in turn, contains multiple copies of certain domain-encoding sequences that can be classified into one of three types. These domains are designated fibronectin type I, II, and III repeats, on the basis of similarities in amino acid sequence, although the sequences of any two repeats of a given type are not identical. These linked repeats give the molecule the appearance of beads on a string. The combination of the different repeats composing the regions confers on fibronectin its ability to bind multiple ligands.
FIGURE 20-33 Organization of fibronectin and its binding to integrin. (a) Scale model of fibronectin is shown docked by two type III repeats to the extracellular domains of integrin. Only one of the two similar chains, which are linked by disulfide bonds near their C-termini, in the dimeric fibronectin molecule is shown. Each chain contains about 2446 amino acids and is composed of three types of repeating amino acid sequences (type I, II, or III repeats) or domains. The EIIIA, EIIIB—both type III repeats—and IIICS domain are variably spliced into the structure at locations indicated by arrows. Circulating fibronectin lacks EIIIA, EIIIB, or both. At least five different sequences may be present in the IIICS region as a result of alternative splicing (see Figure 5-16). Each chain contains several multi-repeat-containing regions, some of which contain specific binding sites for heparan sulfate, fibrin (a major constituent of blood clots), collagen, and cell-surface integrins. The integrin-binding domain is also known as the cell-binding domain. Structures of fibronectin’s domains were determined from fragments of the molecule. (b) A high-resolution structure shows that the RGD motif (red) extends outward in a loop from its compact type III domain on the same side of fibronectin as the synergy region (blue), which also contributes to high-affinity binding to integrins.
[Data from D. J. Leahy et al., 1996, Cell 84:155, PDB ID 1fnf.]
One of the type III repeats in the cell-binding region of fibronectin mediates binding to certain integrins. The results of studies with synthetic peptides corresponding to parts of this repeat identified the tripeptide sequence Arg-Gly-Asp, called the RGD motif, as the minimal sequence within this repeat required for recognition by those integrins. In one study, heptapeptides with and without the RGD motif were tested for their ability to mediate the adhesion of rat kidney cells to a culture dish. The results showed that heptapeptides containing the RGD motif mimicked intact fibronectin’s ability to stimulate integrin-mediated adhesion, whereas variant heptapeptides lacking this sequence were ineffective (Figure 20-34).
EXPERIMENTAL FIGURE 20-34 A specific tripeptide sequence (RGD) in the cell-binding region of fibronectin is required for adhesion of cells. The cell-binding region of fibronectin contains an integrin-binding hexapeptide sequence, GRGDSP in the single-letter amino acid code. Together with an additional C-terminal cysteine (C) residue, this heptapeptide and several variants were synthesized chemically. Different concentrations of each synthetic peptide were added to polystyrene dishes that had the protein immunoglobulin G (IgG) firmly attached to their surfaces; the peptides were then chemically cross-linked to the IgG. Subsequently, cultured normal rat kidney cells were added to the dishes and incubated for 30 minutes to allow adhesion. After the unbound cells were washed away, the relative amounts of cells that had adhered firmly were determined by staining the bound cells with a dye and measuring the intensity of the staining with a spectrophotometer. The results shown here indicate that cell adhesion increased above the background level with increasing peptide concentration for those peptides containing the RGD motif, but not for the variants lacking this sequence (modification underlined).
[Data from M. D. Pierschbacher and E. Ruoslahti, 1984, Proc. Natl. Acad. Sci. USA 81:5985.]
A three-dimensional model of fibronectin binding to integrin, based on partial structures of both fibronectin and integrin, has been assembled. In a high-resolution structure of the integrin-binding fibronectin type III repeat and its neighboring type III domain, the RGD motif is at the apex of a loop that protrudes outward from the molecule, in a position facilitating binding to integrins (Figure 20-33b). Although the RGD motif is required for binding to several different integrins, its affinity for integrins is substantially less than that of intact fibronectin or of the entire cell-binding region in fibronectin. Thus structural features near the RGD motif in fibronectins (e.g., parts of adjacent repeats, such as the synergy region; see Figure 20-33b) and in other RGD-containing proteins must enhance their binding to certain integrins. Moreover, the simple soluble dimeric forms of fibronectin produced by the liver or by fibroblasts are initially in a nonfunctional conformation that binds poorly to integrins because the RGD motif is not readily accessible. The adsorption of fibronectin onto a collagen matrix or basal lamina—or, experimentally, to a plastic tissue culture dish—results in a conformational change that enhances the ability of fibronectin to bind to cells. Possibly, this conformational change increases the accessibility of the RGD motif for integrin binding.
Microscopy and other experimental approaches (e.g., biochemical binding experiments) have demonstrated the role of integrins in cross-linking fibronectin and other ECM components to the cytoskeleton. For example, the colocalization of cytoskeletal actin filaments and integrins within cells can be visualized by fluorescence microscopy (Figure 20-35a). The binding of cell-surface integrins to fibronectin in the ECM induces the actin cytoskeleton–dependent movement of some integrin molecules in the plane of the plasma membrane. The ensuing mechanical tension due to the relative movement of different integrins bound to a single fibronectin dimer stretches the fibronectin (see Figure 20-9), a mechanosensor, and promotes self-association of fibronectins into multimeric fibrils.
EXPERIMENTAL FIGURE 20-35 Integrins mediate linkage between fibronectin in the ECM and the cytoskeleton. (a) Immunofluorescent micrograph of a fixed cultured fibroblast showing colocalization of the α5β1 integrin (green) and actin-containing stress fibers (red). The cell was incubated with two types of monoclonal antibodies: an integrin-specific antibody linked to a green-fluorescing dye and an actin-specific antibody linked to a red-fluorescing dye. Stress fibers are long bundles of actin microfilaments that radiate inward from points where the cell contacts a substratum. At the distal ends of these fibers, near the plasma membrane, the coincidence of actin (red) and fibronectin-binding integrin (green) produces a yellow fluorescence. (b) Electron micrograph of the junction of fibronectin and actin fibers in a cultured fibroblast. Individual actin-containing 7-nm microfilaments, components of a stress fiber, end at the obliquely sectioned cell membrane. The microfilaments appear aligned with and in close proximity to the thicker, densely stained fibronectin fibrils on the outside of the cell.
[Part (a) ©1988 Duband, J. et al., J. Cell Biol., 107:1385–1396. doi: 10.1083/jcb.107.4.1385; Cover. Part (b) republished by permission of Elsevier, from Singer, II, “The fibronexus: a transmembrane association of fibronectin-containing fibers and bundles of 5 nm microfilaments in hamster and human fibroblasts,” Cell, 1979, 16(3), 675–85; permission conveyed through the Copyright Clearance Center, Inc.]
The force needed to unfold and expose functional self-association sites in fibronectin is much less than that needed to disrupt fibronectin-integrin binding. Thus fibronectin molecules remain bound to integrin while cell-generated mechanical forces induce fibril formation. In effect, the integrins, through adapter proteins, transmit the intracellular forces generated by the actin cytoskeleton to extracellular fibronectin (inside-out signaling via mechanotransduction). Gradually, the initially formed fibronectin fibrils mature into highly stable matrix components by covalent cross-linking. In some electron micrographs, exterior fibronectin fibrils appear to be aligned in a seemingly continuous line with bundles of actin fibers within the cell (Figure 20-35b). These observations and the results from other studies provided the first example of a molecularly well-defined adhesion receptor forming a bridge between the intracellular cytoskeleton and the ECM components—a phenomenon now known to be widespread.