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At least 19 recordsLinked to original sources
PubMed1979

Distribution of fetal bovine serum fibronectin and endogenous rat cell fibronectin in extracellular matrix.

Normal rat kidney cells were cultured in medium supplemented with normal fetal bovine serum (FBS) or FBS depleted of fibronectin. The cell surface fibronectin of these cultures was visualized by indirect immunofluorescence using species-specific antisera for either rat fibronectin or bovine fibronectin. Anti-rat-fibronectin revealed fibrillar structures on the cells grown in either normal medium or fibronectin-depleted medium. Anti-bovine fibronectin revealed similar fibrillar networks, but only on the cells grown in medium containing bovine fibronectin. Staining in each case was abolished by absorption with the homologous antigen. It appears that exogenous fibronectin was incorporated into the same structures as endogenous fibronectin. This finding suggests that circulating fibronectin may serve as a building block for the assembly of extracellular matrix, possibly by cells which are incapable of synthesizing it.

Animals↗
PubMed1978

Synthesis of fibronectin by cultured human endothelial cells.

Plasma fibronectin is probably the major nonimmune particulate opsonin in blood and is cross-linked to fibrin during the final stage of blood coagulation. Fibronectin also occurs in an insoluble form in basement membranes especially those underlying endothelial cells and in loose connective tissue. Fibronectin was demonstrated in cultured human endothelial cells and in the surrounding extracellular matrix by immunofluorescence microscopy by using antibody to human plasma fibronectin. Cultured human endothelial cells released fibronectin into the culture medium which was immunologically identical to the fibronectin in human plasma. Cultured human endothelial cells were labeled with [3H] leucine. The radioactive fibronectin present in the endothelial postculture medium and in urea extracts of cellular monolayers was isolated with either anti-fibronectin coupled to Protein A-Sepharose or double antibody immunoprecipitation and characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. When reduced, the [3H] fibronectin synthesized by cultured endothelial cells had the same mol wt (approximately 200,000) as plasma fibronectin. Unreduced, the [3H] fibronectin synthesized by endothelial cells migrated as a dimer, as did plasma fibronectin. Fibronectin accounted for approximately 15% of the protein synthesized and released by endothelial cells into the culture medium. Thus, cultured endothelial cells synthesize fibronectin, secrete it into the culture medium, and incorporate it into extracellular matrix. The results suggest that the endothelial cell is potentially a major site of synthesis of circulating plasma fibronectin. In addition, fibronectin derived from endothelial cells may be an important structural component of the subendothelium.

Cells, Cultured↗
PubMed1978

High molecular weight, cell surface-associated glycoprotein (fibronectin) lost in malignant transformation.

Fibronectin is a polymorphic glycoprotein found in blood and tissues of vertebrates and in cultures of adherent vertebrate cells. There are several forms of fibronectin is composed of two high molecular weight subunits held together by forms found in tissues and on and around the surfaces of cultured cells. Soluble fibronectin is composed of two high molecular weight subunits held together by disulfide bonds. Insoluble fibronectin may be covalently cross-linked in larger complexes. Fibronectin has affinities for collagen, fibrin, heparin, and cell surfaces. In culture, fibronectin in growth medium may mediate attachment of cells to substratum, and fibronectin synthesized by cells may mediate adhesion to substratum. The widespread occurrence of fibronectin in basal lamina indicates that may different cell types in vivo abut against a fibronectin-containing matrix. Cultured transformed cells usually lack cell-surface fibronectin, also called large, external transformation-sensitive (LETS) protein. The failure of transformed cells to synthesize or bind fibronectin is paralleled (at least in some systems) by failures to synthesize or bind collagen and proteoglycans. Abnormal synthesis of fibronectin and other matrix components and abnormal interactions with the tissue matrix may account for several phenotypic characteristics of transformed cultured cells and for some of the malignant behavior of neoplastic cells in vivo.

Amino Acids↗
PubMed1979

Cross-linking of fibronectin to collagen by blood coagulation Factor XIIIa.

Soluble fibronectin is found in body fluids and media of adherent cultured cells and binds to fibrin and collagen. Insoluble fibronectin is found in tissue stroma and in extracellular matrices of cultured cells. Fibronectin is a substrate for Factor XIIIa (plasma transglutaminase) and can be cross-linked by Factor XIIIa to itself and the the alpha-chain of fibrin. We used sodium dodecyl sulfate-polyacrylamide gel electrophoresis to investigate Factor XIIIa-mediated crosslinking of fibronectin to collagen. At O degrees or 37 degrees C, fibronectin could be cross-linked to iodinated cyanogen bromide fragment 7 of the alpha 1(I) chain. At 22 degrees or 37 degrees C, fibronectin could be cross-linked to isolated alpha 1(I) chains of type I collagen. Fibronectin could also be crosslinked to types I and III collagen, but only at 37 degrees C. alpha 1(I)-CB7, alpha 1(I) collagen chains, type I collagen, type III collagen, and fibrin all blocked cross-linking between 125I-alpha 1 (I)-CB7 and fibronectin. alpha 1(I)-CB7 blocked cross-linking between fibronectin and fibrin. These results indicate that the determinants of fibronectin-fibrin and fibronectin-collagen binding and cross-linking are similar. Cross-linking of fibronectin to collagen likely occurs in vivo and may be important for normal wound healing, collagen fibrillogenesis, and embryogenesis.

Binding Sites↗
PubMed1979

Initial adhesion of human fibroblasts in serum-free medium: possible role of secreted fibronectin.

Experiments were carried out to test the hypothesis that the initial attachment and spreading of human fibroblasts in serum-free medium occurs to cell fibronectin which has been secretd spread on tissue culture substrata in serum-free medium in 60 min. When potential protein adsorption sites on the substratum were covered with bovine serum albumin before initial human fibroblasts attachment, their subsequent attachment to the substratum was prevented. When substratum adsorption sites were covered immediately after initial attachment, subsequent cell spreading was prevented. The distribution of fibronectin on human fibroblast surfaces during initial attachment and spreading was studied by indirect immunofluorescence analysis using a monospecific anti-cold-insoluble globulin antiserum. The initial appearance (10 min) of fibronectin was in spots over the entire cell surface. Concomitant with human fibroblast spreading, the random distribution of sites disappeared, and most fibronectin was subsequently observed in spots at the cell substratum interface (60 min). A fibrillar pattern of fibronectin appeared later (2-8 hr). The sites beneath the cells could be visualized as footprints on the substratum following treatment of the attached human fibroblasts with 0.1 M NaOH. A second fluorescence pattern of fibronectin secreted on the substratum was characterized by a diffuse halo around the cells and a very faint, diffuse staining elsewhere on the substratum. Another cell type (baby hamster kideny cells) was used to assay biologically for the presence or absence of the factor secreted by human fibroblasts on the substratum. Human fibroblasts were found to secrete an adhesion factor for baby hamster kidney cells into the substratum in a time- and temperature-dependent fashion, and immunological studies indicated that the factor secreted by human fibroblasts was cross-reactive with cold-in-soluble globulin, the plasma form of fibronectin. The conditioning factor secreted by the human fibroblasts was also found to be an attachment and spreading factor for human fibroblasts in experiments measuring human fibroblast adhesion to fibronectin footprints of human fibroblasts. Substratum-adsorbed cold-insoluble globulin was also found to be an attachment and spreading factor for human fibroblasts. Based upon the timing of appearance of conditioning factors on the substratum and the immunofluorescence patterns, it seems that the diffusely organized fibronectin on the substratum constitutes the sites to which cell attachment occurs. The bright spots of fibronectin that appear beneath the cells may represent fibronectin reorganization during cell spreading.

Animals↗
PubMed1977

Changes in the distribution of a major fibroblast protein, fibronectin, during mitosis and interphase.

The distribution of a major fibroblast protein, fibronectin, was studied by immunofluorescence and immunoscanning electron microscopy in cultures of human and chicken fibroblasts during different phases of the cell cycle. The main findings were: (a) In interphase cells, the intensity of surface-associated fibronectin fluorescence correlated with that of intracellular fibronectin fluorescence. (b) The intensity of the fluorescence of both surface-associated and intracellular fibronectins was not changed in cells that were synthesizing DNA. (c) Mitotic cells had reduced amounts of surface-associated but not of intracellular fibronectin. The surface fibronectin that remained on meta-, ana-, or telophase cells had a distinct punctate distribution and was also localized to strands attaching the cells to the substratum. Fibronectin strands first reappeared on the surface of flattening cytoplasmic parts of telophase cells. (d) Fibronectin was also detected in extracellular fibrillar material on the growth substratum, particularly around dividing cells. Thus, surface-associated fibrillar fibronectin was present during G(1), S, and G(2) but in cells undergoing mitosis the distribution was altered and the amount appeared to be reduced. The observations on the distribution of surface-associated fibronectin suggest that rather than being involved in growth control this fibronectin plays a structural role in interactions of cells with the environment.

Animals↗
PubMed1978

Synthesis and extracellular deposition of fibronectin in chondrocyte cultures. Response to the removal of extracellular cartilage matrix.

Fibronectin, the major cell surface glycoprotein of fibroblasts, is absent from differentiated cartilage matrix and chondrocytes in situ. However, dissociation of embryonic chick sternal cartilage with collagenase and trypsin, followed by inoculation in vitro reinitiates fibronectin synthesis by chondrocytes. Immunofluorescence microscopy with antibodies prepared against plasma fibronectin (cold insoluble globulin [CIG]) reveals fibronectin associated with the chondrocyte surface. Synthesis and secretion of fibronectin into the medium are shown by anabolic labeling with [35S]methionine or [3H]glycine, and identification of the secreted proteins by immunoprecipitation and sodium dodecyl sulfate (SDS)-disc gel electrophoresis. When chondrocytes are plated onto tissue culture dishes, the pattern of surface-associated fibronectin changes from a patchy into a strandlike appearance. Where epithelioid clones of polygonal chondrocytes develop, only short strands of fibronectin appear preferentially at cellular interfaces. This pattern is observed as long as cells continue to produce type II collagen that fails to precipitate as extracellular collagen fibers for some time in culture. Using the immunofluorescence double-labeling technique, we demonstrate that fibroblasts as well as chondrocytes which synthesize type I collagen and deposit this collagen as extracellular fibers show a different pattern of extracellular fibronectin that codistributes in large parts with collagen fibers. Where chondrocytes begin to accumulate extracellular cartilage matrix, fibronectin strands disappear. From these observations, we conclude (a) that chondrocytes synthesize fibronectin only in the absence of extracellular cartilage matrix, and (b) that fibronectin forms only short intercellular "stitches" in the absence of extracellular collagen fibers in vitro.

Cartilage↗
PubMed1978

Fibronectin and the pericellular matrix of normal and transformed adherent cells.

Fibronectin is a major glycoprotein component of normal fibroblasts in culture. External fibronectin is predominantly present in a pericellular fibrillar matrix that mediates distant cell-cell and cell-substratum contacts. A small proportion of external fibronectin is closely associated with the plasma membrane. In the matrix, fibronectin is partially disulfide bonded into complexes. Plasma transglutaminase, activated by thrombin, also cross-links external fibronectin into high-molecular-weight covalent complexes. In cultures of normal fibroblasts, pericellular matrix fibronectin displays extensive codistribution with (pro)collagens types I and III. Transformed adherent cells show decreased formation of the fibronectin-collagen matrix. The deficient synthesis of fibronectin and other matrix components and abnormal interactions with the matrix may account for several phenotypic characteristics of transformed cells. The pericellular matrix structure has been prepared by use of deoxycholate and hypotonic medium to solubilize the cells. The matrix contains glycosaminoglycans, procollagens, and fibronectin. The fibronectin codistributes with the procollagens. The matrix may be considered to be an in vitro equivalent of the connective tissue matrix and basal laminae found in vivo. Human sarcoma cells spread rapidly on the prepared matrix and assume an elongated morphology characteristic of normal fibroblasts. The prepared matrix may provide a general tool to study the effects of matrix on cellular behavior and differentiation.

Animals↗
PubMed1979

Fibronectin and proteoglycans as determinants of cell-substratum adhesion.

When normal or SV40-transformed Balb/c 3T3 cells are treated with the Ca++-specific chelator EGTA, they round up and pull away from their footpad adhesion sites to the serum-coated tissue culture substrate, as shown by scanning electron microscope studies. Elastic membranous retraction fibers break upon culture agitation, leaving adhesion sites as substrate-attached material (SAM) (Cells leave "footprints" of substrate adhesion sites during movement by a very similar process.) SAM contains 1-2% of the cell's total protein and phospholipid content and 5-10% of its glucosamine-radiolabeled polysaccharide, most of which is glycosaminoglycan (GAG). By one- and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis, there is considerable enrichment in SAM for specific GAGs; for the glycoprotein fibronectin; and for the cytoskeletal proteins actin, myosin, and the subunit protein of the 10 nm-diameter filaments. Fibrillar fibronectin of cellular origin and substratum-bound fibronectin of serum origin (cold-insoluble globulin, CIg) have been visualized by immunofluorescence microscopy. The GAG composition in SAM has been examined under different cellular growth and attachment conditions. Heparan sulfate content correlates with glycopeptide content (derived from glycoprotein). Newly attaching cells deposit SAM with principally heparan sulfate and fibronectin and little of the other GAGs. Hyaluronate and chrondroitin proteoglycans are coordinately deposited in SAM as cells begin spreading and movement over the substrate. Cells attaching to serum-coated or CIg-coated substrates deposited SAM with identical compositions. The proteoglycan nature of the GAGs in SAM has been examined, as well as the ability of proteoglycans to form two classes of reversibly dissociable "supramolecular complexes" - one class with heparan sulfate and glycopeptide-containing material and the second with hyaluronate-chondroitin complexes. Enzymatic digestion of "intact" SAM with trypsin or testicular hyaluronidase indicates that (1) only a small portion of long-term radiolabeled fibronectin and cyto-skeletal protein is bound to the substrate via hyaluronate or chondroitin classes of GAG; (2) most of the fibronectin, cytoskeletal protein and heparan sulfate coordinately resist solubilization; and (3) newly synthesized fibronectin, which is metabolically labile in SAM, is linked to SAM by hyaluronate- and/or chondroitin-dependent binding. All of our studies indicate that heparan sulfate is a direct mediator of adhesion of cells to the substrate, possibly by binding to both cell-surface fibronectin and substrate-bound CIg in the serum coating; hyaluronate-chondroitin complexes in SAM appear to be most important in motility of cells by binding and labilizing fibronectin at the periphery of footpad adhesions, with subsequent cytoskeletal disorganization.

Animals↗
PubMed1979

Cross-linking of fibronectin to sulfated proteoglycans at the cell surface.

Fibronectin is a major surface protein of normal animal cells but is absent from many transformed cells. Addition of fibronectin to transformed cells causes increased cell substrate adhesion and changes in the morphology and cytoskeleton of the cells. We have coupled fibronectin to photoactivable chemical cross-linkers and have added it to cells to identify those molecules to which it binds. In this way, fibronectin can be cross-linked to sulfated proteoglycans at the cell surface. The cross-linking is specific for fibronectin. The fibronectin-proteoglycan complex is sensitive to chondroitinase ABC and AC and to trypsin. Addition of fibronectin also affects binding of hyaluronic acid to the cells. These results suggest that fibronectin interacts with proteoglycans at the cell surface. The existence of such interactions may have implications for the role of fibronectin and proteoglycans in cell adhesion.

Azides↗
PubMed1978

Fibronectin synthesis by epithelial crypt cells of rat small intestine.

Synthesis of fibronectin in an epithelial cell line (IEC-6) established from rat small intestine was demonstrated by using immunofluorescence, radioimmunoassay, and collagen-binding. Internally labeled radioactive fibronectin isolated from the IEC-6 cells gave a single main band in sodium dodecyl sulfate/polyacrylamide gel electrophoresis under reducing conditions. Fibronectin isolated from rat plasma gave two closely spaced bands. The slower one had the same mobility as the epithelial cell fibronectin. The distribution of fibronectin in IEC-6 cells as detected by immunofluorescence was different from that described for fibroblasts and other cell types; fibronectin was present exclusively in regions of cell-to-cell contact. No fluorescence was detected on the surface membrane facing the culture medium or underneath the cells. This suggests that fibronectin may not be involved in the adhesion of the epithelial cells to the growth surface but could mediate cell-to-cell contacts. In microscopic sections of the small intestine, immunofluorescent staining with antifibronectin serum was strong in the basement membrane underlying the epithelial cells in the crypts. The in vitro synthesis of fibronectin by the crypt cells and its abundant presence in the basement membrane underlying the same cells in vivo suggests that fibronectin is a structural component of the basement membrane, and that it may be, at least in part, synthesized and deposited by the intestinal epithelium.

Animals↗
PubMed1979

Fibronectin associated with the glial component of embryonic brain cell cultures.

In the basic approach to investigations of neuronal--glial interactions during both normal brain development and its pathogenesis, embryonic brain cell populations were fractionated into purified neuronal and glial components. Using separation procedures based on differential adhesion and cytotoxicity, the isolated neuronal and glial phenotypes could be identified by distinct morphological and biochemical characteristics, including the visualization of glial fibrillary acid protein (GFA) within glial cells in immunohistochemical assays with monospecific anti-GFA serum. When unfractionated cerebrum cells dissociated from 10-day chick or 14-day mouse embryos were plated as monolayers and cultured for 1--14 days, monospecific antiserum against fibronectin (LETS glycoprotein) was found to react with many, but not all, of the cells as revealed by indirect immunofluorescence microscopy. The isolated neuronal and glial components of these populations were used to determine whether the appearance of membrane-associated fibronectin was characteristic of one cell type or the other, or both, and if neuronal--glial cell interaction was required for its expression. It was found that the surfaces of glial cells, completely isolated from neurons, showed an intense fluorescent reaction to the anti-fibronectin serum. In contrast, the purified neuronal cultures showed no fluorescence with either the anti-GFA or anti-fibronectin sera. These results demonstrate fibronectin as a cell surface protein associated primarily with glial cells and independent of neuronal--glial cell interaction for its expression. Furthermore, the results indicate that the fibronectin observed on glial cell surfaces in these cultures is produced endogenously and is not due to the preferential binding of fibronectin present in the culture medium. The role of fibronectin as an adhesive molecule in neuronal--glial interactions is discussed.

Animals↗
PubMed1979

Studies on fibronectin in the skin. III. Indirect immunofluorescence studies in lichen planus.

Fibronectin is a glycoprotein which is responsible for a varity of functions in the human organism, such as mediation of contact between cells and between cells and fibres, opsonic qualities, interaction in the stabilization of fibrin, etc. Fibronectin is an important constituent of the ground substance having a special affinity to collagen. In indirect immunofluorescence studies its presence has been abundantly demonstrated in normal human skin, in collagen-rich structures such as the basement membranes, the papillary and reticular dermis, and in the vascular and neural structures, demonstrable by its characteristic staining patterns. Fibronectin is not found in the epidermis. In lichen planus, the distribution in unaffected skin is identical with that in normal skin, whereas in affected skin, changes in the pattern of fibronectin are found. The basement membrane zone becomes broader and hazy, later undergoing disintegration and destruction, concomitant with swelling and homogenization of the reticular distribution of fibronectin in the papillary dermis. Globular structures containing fibronectin are found in the basement membrane area, together with an intensified immunofluorescence in the vascular system. Fibronectin has certain adhesional properties and changes in the distribution of this glycoprotein may result in loss of tissue stability. The pathophysiological significance of the changes of fibronectin in lichen planus is, however, difficult to evaluate at present.

Adult↗
PubMed1978

Relationships between fibronectin (LETS protein) and actin.

Double label immunofluorescence was used to study the distribution of fibronectin (LETS protein), actin and intermediate filaments in cultured cells. No relationship was observed between fibronectin and intermediated filaments, but fibronectin and actin showed coincident staining in a large proportion of cells during spreading or when fully spread. The distributions of actin and fibronectin staining during the course of cell spreading progressed through a series of patterns. Certain actin patterns correlated with certain fibronectin patterns. When fibrillar patterns developed, there was correspondence between the two fibrillar arrays in 80--100% of the cells. These results suggest a transmembrane relationship between microfilament bundles and fibronectin. We propose that fibronectin may participate in the formation of attachment plaques and discuss the interrelationship between plaques, microfilament bundles and fibronectin in cell-substratum and cell-cell contacts.

Actins↗
PubMed1978

Codistribution of pericellular matrix proteins in cultured fibroblasts and loss in transformation: fibronectin and procollagen.

Antibodies to fibronectin and to distinct types of procollagens and collagens were used in immunofluorescent staining to localize these proteins in cell cultures. Normal human skin or lung fibroblasts produced a fibrillar pericellular matrix in which fibronectin and procollagen (types I and III) showed extensive codistribution. Fibronectin and procollagen were synthesized by the same cells as judged by double-stain immunofluorescence. Pericellular procollagen was specifically digested with collagenase without an effect on the fibrillar distribution of matrix fibronectin. Brief treatment with trypsin removed both matrix proteins. The human tumor cell lines HT-1080 (fibrosarcoma) and RD (rhabdomyosarcoma) produced little or no matrix fibronectin or procollagen. At sites of cell contact, simian virus 40-transformed lung fibroblasts (VA13) produced small amounts of pericellular fibrillar matrix fibronectin that codistributed with procollagen type I. Intracellular fibronectin and procollagen were visualized in all of these human sarcoma cell lines. When chicken embryo fibroblasts infected with a T class mutant (NY68) of Rous sarcoma virus temperature-sensitive for transformation were maintained at the nonpermissive temperature (41 degrees ) the cells had normal phenotype and a fibrillar matrix containing fibronectin and procollagen was present. At the permissive temperature (35 degrees ), the cells showed transformed phenotype and the matrix was lost. The failure to produce a pericellular fibronectin/collagen matrix may account for several phenotypic characteristics of transformed cultured fibroblasts.

Cell Line↗
PubMed1979

Fibronectin (cold-insoluble globulin), VI. Influence of heparin and hyaluronic acid on the binding of native collagen.

Fibronectin of human plasma associated readily with denatured collagen but gave only a weak reaction with the native protein. In the presence of heparin, however, solutions of native collagen type III, and fibronectin produced precipitates at an ionic strength of 0.2. In the presence of fibronectin and optimal additions of heparin, up to 60% of soluble native 125I-collagen type III, but only about 10% of native 125I-collagen type I, were insolubilized. Heparin also enhanced the formation of insoluble complexes from fibronectin and denatured collagen type I and type III. In the absence of collagen 125I-fibronectin was partially precipitated by heparin. Electron micrographs showed filamentous structures. Collagen did not increase the amount of 125I-fibronectin precipitated by heparin unless a critical collagen concentration was exceeded. It is suggested that heparin induced the transition of fibronectin from a globular to an elongated form, capable of forming filamentous precipitates which adsorb native collagen. Hyaluronic acid and putrescine prevented the insolubilization of native collagen type III, by fibronectin and heparin.

Collagen↗
PubMed1979

Production of fibronectin by human epithelial cells in culture.

Human epithelial cell lines derived from both carcinomatous and nomalignant tissues were characterized with respect to the presence and distribution of fibronectin by immunofluorescence microscopy. In cell lines derived from nonmalignant tissues or from primary carcinomas, fibronectin was found predominantly in an extracellular matrix. In contrast, cell lines derived from metastatic carcinomas displayed very little or no fibronectin. Metabolic labeling studies indicated that a positive line synthesized fibronectin de novo rather than absorbing the protein from the media. Negative lines neither synthesized fibronectin nor secreted it into the culture fluid, suggesting that they were not producing fibronectin. Evidence is presented that cells in culture change their properties after extensive subculture since a small amount of fibronectin in an extracellular matrix was observed after extensive subculture of two metastatic lines that were originally negative.

Cell Line↗