Identification of fibronectin fragments that bind to carboxy-group-modified proteins.

Limited proteolysis of human plasma fibronectin with chymotrypsin, trypsin or thermolysin has been used to localize binding sites responsible for binding [Vuento, Korkolainen & Stenman (1982) Biochem. J. 205, 303-311] of fibronectin to carboxy-group-modified proteins. These bindings sites are different from those mediating binding of fibronectin to gelatin or heparin. They are located close to the C-terminus of the polypeptide chains of fibronectin, and apparently overlap with the C-terminal fibrin binding site.     

Association of fibronectin with the platelet cytoskeleton

Triton-insoluble cytoskeletons prepared from either normal or thrombasthenic platelets were found to contain approximately 1.3 micrograms of fibronectin/10(9) platelets as measured by a radioimmunoassay. Total endogenous platelet fibronectin was quantitatively retained on the platelet cytoskeleton, whereas 70% of exogenously added fibronectin that bound the surface of thrombin-activated platelets was recovered with the Triton-insoluble cytoskeleton. The exogenously added fibronectin specifically bound platelets and cytoskeletons with the same affinity giving an apparent binding constant of 1.47 X 10(-7) M. The possibility that fibrin associated with the platelet cytoskeleton could serve as the fibronectin receptor was investigated by measuring the binding constant of fibronectin for polymerizing fibrin and by measuring the amount of fibronectin associated with cytoskeletons of thrombasthenic platelets which contain 4-fold less fibrin than controls. The binding constant of fibronectin for polymerizing fibrin was 14-fold lower than that for cytoskeletons and cytoskeletons prepared from thrombasthenic platelets contained approximately the same amount of fibronectin as controls. Therefore, it is unlikely that fibrin is the platelet fibronectin receptor. These results support the hypothesis that platelet fibronectin is released from platelet alpha granules upon thrombin stimulation and becomes bound to the platelet surface and cytoskeleton either directly or through some intermediate protein that spans the membrane and interacts both with fibronectin and the internal cell cytoskeleton.     

Association of fibronectin with the microfibrils of connective tissue

The association of fibronectin with the microfibrils of connective tissue was examined in the zonular fibers of the mouse eye by immunohistochemical methods at the light and electron microscopic level. Mouse eyes fixed in formaldehyde were embedded either in paraffin for immunostaining by the peroxidase-antiperoxidase (PAP) method or in Lowicryl for immunolabeling by antirabbit globulin antibodies bound to 5 or 15 nm gold particles. Ultrastructural studies were also carried out after glutaraldehyde perfusion. Both the PAP and immunogold procedures demonstrated the association of fibronectin with microfibrils. After immunolabeling with 5 nm gold particles, examination at high magnification localized fibronectin to fine filaments that appeared to be attached to the surface of microfibrils. The filaments extended outward singly or formed loose aggregates. Their diameter ranged from 1.2 to 3 nm, with a mean of 1.5 nm. Because of their similarity to the fibronectin molecules previously described after rotary shadowing, the filaments were likely to be fibronectin molecules themselves. Since fibronectin is known to have high affinity for the amyloid P component, a model is presented in which fibronectin filaments are bound to the amyloid P component making up the tubular core of microfibrils in mice. Evidence is presented that fibronectin filaments may link microfibrils to one another and thus insure the continuity and strength of zonular fibers. More generally, it is likely that connective-tissue microfibrils, whether or not inserted into elastic fibers, are bonded through fibronectin to surrounding cells, collagen fibrils, or proteoglycans, and thus insure cohesion among connective tissue elements.     

Differential interaction of Xenopus embryonic cells with fibronectin in vitro

Two distinct cell types from the amphibian gastrula were compared with regard to their interactions in vitro with fibronectin (FN). Xenopus embryonic endoderm cells attach to FN substrates in a way characteristic of most cell types studied so far; that is, adhesion increases abruptly at a certain threshold concentration of FN, and maximal binding of cells already occurs at low FN concentrations (10 micrograms/ml). In contrast, embryonic ectodermal cells bind maximally to FN substrates only at unusually high concentrations of FN (200 micrograms/ml). This peculiar mode of attachment to FN has been characterized more closely. It is shown that the adhesion of ectodermal cells is modified by their interaction with a heparin-binding domain of the FN molecule. Furthermore, ectodermal cell adhesion increases very slowly with increasing FN concentrations. Despite these characteristic differences, both ectodermal and endodermal cells attach to the normal RGD cell-binding site of FN, as can be shown by competitive inhibition of adhesion by a hexapeptide containing the RGD sequence of amino acids.     

Redistribution of fibronectin and cytoskeletal proteins during the differentiation of rat mammary tumor cells in vitro

The tumorigenic mammary epithelial stem cell line, Rama 25, is capable of synthesizing and secreting fibronectin but incorporates only small amounts of fibronectin into pericellular material localised in regions of cell-cell and cell-substratum contact. Under certain culture conditions, Rama 25 differentiates into a non-tumorigenic myoepithelial-like cell line, Rama 29, which is capable of retaining fibronectin on the cell surface in characteristic fibrillar formation. The redistribution of fibronectin is accompanied by a reorientation of the cytoskeleton from circular bundles in Rama 25 to parallel arrays of filaments in Rama 29. In vivo, fibronectin is found in the basement membrane of the mammary gland and our in vitro studies lead us to suggest that the mammary myoepithelial cell in vivo synthesizes much of the basement membrane fibronectin.     

Cross-linking of fibronectin to collagenous proteins

Summary Attempts were made to cross-link several collagenous proteins to fibronectin with Factor XIII_a (plasma transglutaminase). Cross-linking was demonstrated with type I collagen, type II collagen, type III collagen, type V or AB collagen, and 1(I)-CB7 and 1(I)-CB8 cyanogen bromide fragments of type I collagen. Cross-linking was not demonstrated with type IV collagen, Clq, and cyanogen bromide fragment a 1(I)-CB6. The pH optimum for cross-linking of 1(I)-CB7 to fibronectin was 8.5 to 9.6. Cross-linking of 1(I)-CB7 to fibronectin was somewhat enhanced at lower than physiological ionic strength.     

Disulfide-bonded dimerization of fibronectin in vitro.

Human plasma fibronectin was denatured with 8 M urea and reduced with dithiothreitol. Dialysis or dilution of the solution led to formation of fibronectin dimers which migrated in non-reducing SDS/PAGE similarly to untreated control protein. When the redimerized fibronectin was reduced and re-electrophoresed it formed a doublet of alpha and beta chains of equal intensity indicating that it was a heterodimer. Low concentrations (less than 1 mM) of Fe3+ enhanced the redimerization of fibronectin, suggesting that metal ions may mediate oxidative reactions in the formation of the disulfides. Consequently, redimerization of fibronectin was completely prevented by deferoxamine, an iron chelator. Dimerization of fibronectin took place most effectively at pH greater than or equal to 8.8 but decreased strongly at lower pH, representing more unfavourable conditions for the action of the thiolate anion in the thiol/disulfide exchange reaction. Redimerized fibronectin, however, lost many of its binding properties to macromolecular ligands, suggesting that the disulfide bonding did not entirely regenerate the proper conformation of the protein. Pulse/chase experiments of fibroblast cultures showed that the initially monomeric fibronectin was rapidly and quantitatively dimerized under conditions representing natural pH and environment. SDS/PAGE analysis of the dialyzed urea-denatured/reduced thrombin and plasmin digests of fibronectin revealed that the NH2-terminal 30-kDa fragment and other fragments that contained intrachain disulfides quantitatively regained their non-reduced electrophoretic mobility. The results show that the dimerization and formation of intrachain disulfides of fibronectin may occur, in part, spontaneously, based on the amino acid sequence information of the protein. However, complete disulfide formation may also need other factors, present only in living cells, as suggested by pulse/chase experiments in fibroblasts.     

Association of calcineurin with mitochondrial proteins

Using mouse hearts from Swiss Webster mice, calcineurin was immunoprecipitated using commercially available anti-calcineurin antibody and the resulting complex analyzed by using sodium dodecyl sulfate-gel electrophoresis with silver staining. Distinct proteins were observed and subjected to in situ trypsin digestion followed by extraction of the resulting peptides. Peptides from each protein band were loaded onto a target for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and analyzed. The resulting peptide mass spectrum was compared with the Mascot and Protein Prospector databases and resulted in the specific identification of heart mitochondrial proteins, specifically Mn-superoxide dismutase (SOD), aconitase (ACN), and malate dehydrogenase (MDH). Each of the three mitochondrial enzymes was identified with approximately 15-25% sequence coverage and all with statistical significance (P < 0.05) according to the Mascot database search engine. Tandem mass spectrometry analysis of the peptide fragmentation spectra confirmed the identification of these protein partners and also yielded the identification of mitochondrial isocitrate dehydrogenase (ICDH) as another protein in the immunoprecipitated complex. Using antibody preparations against Mn-SOD, ACN, and ICDH showed the presence of calcineurin and each of the three proteins in the immunoprecipitated complex by Western slot blotting. The activity of ACN, but not MDH or ICDH, was enhanced after incubation with calcineurin indicating one possible regulatory function for the complex. The mitochondrial forms of Mn-SOD, ACN, MDH, and ICDH were identified as partner proteins of calcineurin with all the proteins present in a single multiprotein complex.     

Extracellular matrix microfibrils are composed of core proteins coated with fibronectin

Extracellular proteins of cultured calf aortic smooth muscle cells consist predominantly of microfibrils 10-20 nm in diameter typical of "elastin-associated" microfibrils described in many tissues. Chemical and immunochemical evidence is presented that microfibrils consist of at least two proteins: core protein and fibronectin. Insoluble proteins of the microfibrils were obtained in the form of a pellet and antibodies raised in rabbits against these components. The antisera reacted with the insoluble microfibrillar proteins and with soluble fibronectin in enzyme-linked immunosorbent assay, and immunostained the extracellular microfibrils in cultured cells. An immunoglobulin (Ig) fraction was prepared and absorbed with fibronectin. The absorbed IgG retained its reactivity with the microfibrillar proteins but was no longer reactive with soluble fibronectin. Immunofluorescence studies were carried out using the absorbed IgG and IgG to soluble fibronectin. Both antibodies showed immunoreactive microfibrils in the extracellular matrix of cells in log phase. However, with increasing time in culture, as the cells reached confluence, the immunofluorescence of microfibrils reacting with the absorbed IgG became less intense, whereas that of microfibrils reacting with IgG to fibronectin increased; in confluent cells, essentially no staining was detected with the absorbed IgG, and a dense network of intensely stained microfibrils was seen with IgG to fibronectin. Treatment of these cultures with urea led to partial dissociation of the fibronectin and increased visualization of the microfibrils with the absorbed IgG; double-label immunofluorescence showed that both proteins occurred on the same microfibrils. The localization of immunoreactive sites to the extracellular microfibrils was confirmed by immunoelectron microscopy. Nearly quantitative cleavage with CNBr failed to dissociate the antigenically active fragments of fibronectin from the CNBr fragments of the core proteins of the microfibrils. It was concluded that microfibrils contain core proteins and fibronectin that are codistributed in insoluble, possibly covalently cross-linked, aggregates. The core proteins are first deposited by the cell and, as a function of time in culture, fibronectin gradually coats their surface.     

Association of adenovirus type 2 early proteins with a soluble complex that synthesizes adenovirus DNA in vitro

A soluble Ad2 DNA synthesizing complex was prepared from Ad2-infected KB cell nuclei and purified by exclusion chromatography on a BioGel A-50m column. The purified complex was able to synthesize DNA from all regions of the virus genome, as indicated by $\text{Eco}$RI restriction endonuclease analysis of $\text{in vitro}$ labeled DNA. Experiments were performed to identify Ad2-induced early polypeptides present in the complex. Ad2-infected and mock-infected cells were labeled with [³⁵S]methionine 7–10 h postinfection, then incubated for 8 h to allow the ³⁵S-labeled early polypeptides to become associated with the complex. The polypeptides in the purified complex and each of the cell fractions were identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis and autoradiography. The major components of the purified complex were the 73K DNA binding phosphoprotein and 11K, two adenovirus 2-induced early polypeptides. The 11K has a preferred nuclear location. Small quantities of other Ad2-induced early proteins, 21K, 15K, and possibly 8.3K were also associated with the complex.     

Microbial adhesion to fibronectin in vitro correlates with production of endocarditis in rabbits

Microbial adhesion to the constituents of nonbacterial thrombotic endocarditis (NBTE) is an important early event in the pathogenesis of infective endocarditis. Fibronectin is a ubiquitous mammalian glycoprotein with diverse functions which binds to certain bacteria but not to others. In this study, we determined that fibronectin is present on the surface of NBTE (after catheter-induced aortic valve trauma) but not on normal rabbit cardiac valvular endothelium. The adhesion of various bacteria and yeasts to human fibronectin in tissue culture wells was then measured. Microorganisms with a high isolation frequency from endocarditis cases (Staphylococcus aureus, Candida tropicalis, C. albicans, Streptococcus faecalis, S. sanguis) bound significantly better (P less than 0.01) to fibronectin in vitro than other organisms (Escherichia coli, C. krusei, Pseudomonas aeruginosa) rarely implicated in this disease. Microbial adhesion to fibronectin correlated closely with the propensity of each organism to produce endocarditis in rabbits (e.g., ID50) with preexistent NBTE. A similar distribution was noted after binding of soluble radiolabeled fibronectin to bacteria in suspension. The results suggest that fibronectin, expressed on the surface of NBTE, may mediate microbial adhesion of circulating organisms to initiate colonization during the early pathogenesis of infective endocarditis.     

Interaction of human plasma fibronectin with viral proteins of human immunodeficiency virus

Abstract Fibronectin (FN) is present in soluble and matrix forms in various body fluids and tissues, and has been shown to bind to several pathogens, including viruses. The interaction of FN with viral proteins of human immunodeficiency virus (HIV-1) was investigated by immunofluorescence technique using a cell line chronically infected with HIV-1 (H9-V). The results of this study showed that FN binds to HIV-1 infected cells. especially at FN concentration of 5 μg/ml. In addition, FN-pentapeptide has shown the ability to bind to HIV-1 infected cells. On the other hand, preincubation with antibodies against FN abolished the binding of FN to HIV-1 infected cells. Finally, FN has shown to bind to HIV-1 glycoproteins, including gp41 and pg120. In contrast, no binding to HIV-1 core proteins, including p15 and p24, was noted. We suggest that FN, in binding HIV-1 particles, may reduce viremia and thus may be involved in the clearance of viral proteins from the cells.     

Association of motor proteins with membranes.

The motility of intracellular components, and of the cell as a whole, is mediated by cytoplasmic motor proteins. This review discusses what is known about the binding of cytoplasmic motors to membranes. Mechanisms by which these interactions may result in observed motile phenomena are proposed.     

Association of ribosomes with in vitro assembled microtubules

Microtubules were purified from unfertilized eggs of the sea urchins Arbacia punctulata, Lytechinus pictus, Lytechinus variegatus, and Strongylocentrotus purpuratus. Numerous densely stained particles (24 x 26 nm) are associated with microtubules isolated from each of these sea urchins. The most striking aspect of this structure is an extended, slightly curved arm that appears to attach the particles to the microtubule. Morphologically similar particles are associated with microtubules of the isolated first cleavage mitotic apparatus. The particles are attached to the microtubules by ionic interactions and contain large amounts of extractable RNA. Based upon their size and density, RNA and protein composition, and sedimentation in sucrose gradients, the microtubule-associated particles are identified as ribosomes.     

Haptotactic activity of fibronectin on lymphocyte migration in vitro

In addition to mediating cell adhesion, fibronectin (FN) also affects the migration of different cell types. However, the role of FN in lymphocyte migration is unclear. In this study, we examined the effects of FN on the in vitro migration of lymphocytes. Using the checkerboard analysis in a blind-well microchemotaxis assay, soluble FN was determined to have neither a chemotactic nor chemokinetic effect on spleen or thymus lymphocytes. However, when the nitrocellulose filter was coated unidirectionally with FN, the migration of both spleen and thymus lymphocytes into the filter was enhanced, indicating that FN is haptotactic for lymphocytes. When the filter was coated bidirectionally, no enhancement in migration was observed, indicating that FN is not haptokinetic for lymphocytes. When the FN cell-binding domain and the heparin-binding domain were tested, the cell-binding domain was haptotactic for both spleen and thymus lymphocytes, whereas the heparin-binding domain was only haptotactic for spleen lymphocytes. Because the heparin-binding domain can mediate strong adhesion of thymus lymphocytes, the lack of haptotactic activity is likely to be the result of excessive binding that prevents cell motility.     

Direct association of fibronectin and actin molecules in vitro

Affinity chromatography with actin-Sepharose conjugates of purified human fibronectin, normal human plasma, or serum-free culture fluid from human fibroblasts showed that fibronectin molecules can directly bind to actin. A quantitative recovery of soluble human fibronectin was accomplished by chromatography on actin immobilized on Sepharose beads. Human fibronectin molecules bound to actin-Sepharose were eluted with 0.25--0.35 M potassium bromide, and these molecules competed in a species-specific radioimmunoassay for human fibronectin. The subunits of fibronectin isolated by actin-Sepharose chromatography comigrated in SDS polyacrylamide gel electrophoresis with those of electrophoretically homogeneous fibronectin purified by conventional procedures. The efficient direct binding of fibronectin to actin suggests that interactions between these proteins might also take place in vivo but further studies are needed to elucidate the biological significance of this affinity.     

Association of fibronectin and vinculin with focal contacts and stress fibers in stationary hamster fibroblasts

We have recently observed a transmembrane association between extracellular fibronectin (FN) fibers and elongated focal patches or fibers of vinculin (VN) in G1-arrested stationary Nil 8 hamster fibroblasts, with double-label immunofluorescence microscopy (Singer and Paradiso, 1981, Cell. 24:481-492). We hypothesized that these FN-VN complexes might correspond to focal contacts, the membrane sites that are probably mainly responsible for attaching cells to their substrata, because vinculin is often localized in focal contacts. However, because fibronectin-vinculin associations may not be restricted to the substrate adhesive surface of the cell, it became necessary to determine whether some or all of the various kinds of FN-VN complexes which we described are in proximity to the substrate. Using interference reflection optics and double-label immunofluorescence microscopy for fibronectin and vinculin, many elongated (up to 38 micrometer) FN-VN associations were found to be strikingly coincident with focal contacts in the perinuclear area of extremely flattened arrested Nil 8 fibroblasts in 0.3% fetal bovine serum (FBS). In addition, the long FN-VN adhesion complexes were precisely aligned with the major phase-dense stress fibers observed at the ventral surfaces of these stationary cells with phase contrast microscopy. Fibronectin was neither associated with vinculin-containing focal contacts of Nil 8 cells cultured in medium with 5% FBS nor with vinculin-negative focal contacts located at the extreme edges of stationary cells arrested in 0.3 FBS. Our time-course experiments suggest that early FN-VN lacking- focal contacts, which form at the cellular margins, develop into mature substrate adhesion complexes containing both fibronectin and vinculin, localized in the major stress fibers at the centers of sessile fibroblasts.