Assembly of epithelial cell fibrillins.

Fibrillins are large structural macromolecules that are components of connective tissue microfibrils. Fibrillin microfibrils have been found in association with basement membranes, where microfibrils appear to insert directly into the lamina densa. It is unknown whether fibrillins are limited to these sites of microfibril insertion or are present throughout the lamina densa. In this study, electron microscopic immunolocalization demonstrated the presence of fibrillin-1 throughout the lamina densa in the dermal-- epidermal junction. In order to investigate whether fibrillin microfibrils might be present in the lamina densa, epithelial cell cultures (WISH, HaCaT, and primary keratinocytes) were analyzed by immunofluorescence, immunoblotting, and extraction of microfibrils followed by rotary shadowing electron microscopy and compared to mesenchymal cell cultures (dermal fibroblasts and MG63 osteosarcoma). In contrast to mesenchymal cells, which elaborate a fibrillin fibril network, epithelial cells primarily deposit fibrillin into the extracellular matrix in a nonfibrillar form. Coculture experiments using human epithelial cells and mouse fibroblasts implicated the cells themselves in the assembly of fibrillin. The importance of the cell in this process was further underscored by novel data demonstrating that keratinocytes selectively secrete fibrillin-1 into the matrix and not into the medium and can differentiate between fibrillin-1 and fibrillin-2.

Fibrillin-1 and fibulin-2 interact and are colocalized in some tissues.

Microfibrils 10-12 nm in diameter are found in elastic and non-elastic tissues with fibrillin as a major component. Little is known about the supramolecular structure of these microfibrils and the protein interactions it is based on. To identify protein binding ligands of fibrillin-1, we tested binding of recombinant fibrillin-1 peptides to different extracellular matrix proteins in solid phase assays. Among the proteins tested, only fibulin-2 showed significant binding to rF11, the N-terminal half of fibrillin-1, in a calcium-dependent manner. Surface plasmon resonance demonstrated high affinity binding with a Kd = 56 nM. With overlapping recombinant fibrillin-1 peptides, the binding site for fibulin-2 was narrowed down to the N terminus of fibrillin-1 (amino acid positions 45-450). Immunofluorescence in tissues demonstrated colocalization of fibrillin and fibulin-2 in skin, perichondrium, elastic intima of blood vessels, and kidney glomerulus. Fibulin-2 was not present in ocular ciliary zonules, tendon, and the connective tissue around kidney tubules and lung alveoli, which all contain fibrillin. Immunogold labeling of fibulin-2 on microfibrils in skin was found preferentially at the interface between microfibrils and the amorphous elastin core, suggesting that in vivo the interaction between fibrillin-1 and fibulin-2 is regulated by cellular expression and deposition as well as by protein-protein interactions.

Substrate-specific binding of the amino terminus of fibronectin to an integrin complex in focal adhesions.

The assembly of fibronectin fibrils involves the amino-terminal and cell adhesion domains of fibronectin as well as alpha 5 beta 1 integrins. Efficient binding of biotinylated or radioiodinated 70-kDa amino-terminal fragments occurred only if fibroblasts were plated on fibronectin or on 180- or 85-kDa cell adhesion fragments of fibronectin. On an 11.5-kDa fragment of fibronectin that included the Arg-Gly-Asp (RGD) sequence, but not the synergy site, binding was reduced 50-fold. Conformation of the 180-kDa fragment was important for direct binding interactions with the amino terminus of fibronectin. No binding was seen if cells were plated on type I collagen, vitronectin, RGD peptides or antibodies to alpha 5 beta 1 integrins. High affinity interactions between invasin and alpha 5 beta 1 integrin promoted low levels of binding. Monoclonal antibodies that blocked the function of either the RGD or the synergy site inhibited binding of 125I-labeled 70-kDa fragments to cells by approximately 60%. By fluorescence and interference reflection microscopy, biotinylated 70-kDa fragments were shown to co-localize with alpha 5 beta 1 integrins in focal adhesions. We propose that cell-mediated binding of the amino terminus of fibronectin involves interactions with both fibronectin and its alpha 5 beta 1 integrin receptor in an activated complex.

Fibronectin binding site in type I collagen regulates fibronectin fibril formation.

Mov13 fibroblasts, which do not express endogenous alpha 1(I) collagen chains due to a retroviral insertion, were used to study the role of type I collagen in the process of fibronectin fibrillogenesis. While Mov13 cells produced a sparse matrix containing short fibronectin fibrils, transfection with a wild type pro alpha 1(I) collagen gene resulted in the production of an extensive matrix containing fibronectin fibrils of normal length. To study the amino acids involved in the fibronectin-collagen interaction, mutations were introduced into the known fibronectin binding region of the pro alpha 1(I) collagen gene. Substitution of Gln and Ala at positions 774 and 777 of the alpha 1(I) chain for Pro resulted in the formation of short fibronectin fibrils similar to what was observed in untransfected Mov13 cells. Type I collagen carrying these substitutions bound weakly to fibronectin-sepharose and could be eluted off with 1 M urea. The effect of this mutation on fibronectin fibrillogenesis could be rescued by adding either type I collagen or a peptide fragment (CB.7) which contained the wild type fibronectin binding region of the alpha 1(I) chain to the cell culture. These results suggest that fibronectin fibrillogenesis in tissue culture is dependent on type I collagen synthesis, and define an important role for the fibronectin binding site in this process.

Arrangement of cellular fibronectin in noncollagenous fibrils in human fibroblast cultures.

The assembly of fibronectin into fibrils was examined by high-voltage immunoelectron microscopy in subconfluent cultures of ascorbate-treated human skin fibroblasts. Cells grown in the presence of ascorbic acid for 24, 48 or 72 h were labeled with Ist-9, a monoclonal antibody specific for the EIIIA site in fibronectin, and polyclonal antibodies to type I collagen. Cells were then labeled with goat anti-mouse IgG and goat anti-rabbit IgG coupled to 5 or 18 nm colloidal gold beads. Our results show that by 24 h, fibronectin is observed in fibrils in the extracellular matrix. The majority of fibronectin in fibrils does not co-localize with type I collagen. Morphometric analysis of the distance between EIIIA sites in fibronectin fibrils (less than 12 nm in diameter) show that the EIIIA sites appear to be spaced approximately 84 nm apart. The distance of 84 nm suggests that fibronectin is fully extended in fibrils and that the amino termini of adjacent fibronectin dimers overlap by 20 nm. As fibronectin fibrils become thicker, the average distance between EIIIA sites in fibronectin dimers decreases to 42 nm. This decrease in the distance between EIIIA sites may be due to a staggering of fibronectin dimers within the fibril as the fibril matures.