A rapid and convenient preparation of [4-3H]NADP and stereospecifically tritiated NADP3H

The glass-binding properties of a number of purified glycoproteins capable of promoting attachment and spreading of a variety of types of animal cells in culture have been examined. Two such factors in human serum, fibronectin and serum spreading factor, exhibited strong affinities for glass beads and could be eluted from glass-bead columns under similar conditions. A number of other glycoproteins of human serum that do not promote cell adhesion did not bind to glass beads under conditions that resulted in binding of serum spreading factor or fibronectin. At a sufficiently low ratio of serum volume to glass-bead volume, human serum could be simultaneously depleted of serum spreading factor, fibronectin, and cell spreading-promoting activity by glass-bead affinity chromatography. Laminin, another cell spreading-promoting glycoprotein, possessed glass-binding properties similar to those of serum spreading factor and fibronectin while chondronectin, a fourth cell spreading-promoting factor of more limited specificity of biological activity and distribution in vivo, did not exhibit a strong interaction with glass beads under the same conditions. These observations suggest that glass-bead column affinity chromatography may prove useful as a general method for isolation and study of glycoprotein factors promoting attachment and spreading of cells in culture.     

Role of thrombospondin in the adhesion of human endothelial cells in primary culture

Summary The role of thrombospondin on the adhesion of endothelial cells in primary culture was studied using a serum-free defined medium or thrombospondin-depleted fetal bovine serum. Under these conditions, only 6% of the cells adhered to gelatin-coated dishes, whereas cells adhering to gelatin in the presence of normal fetal bovine serum were considered as 100% adhesion. The percentage of cells attached to fibronectin or thrombospondin-coated dishes in thrombospondin-depleted serum was 66 and 32%, respectively. The addition of purified platelet thrombospondin to thrombospondin-depleted serum increased the adhesion of endothelial cells to gelatin and to thrombospondin, up to 32 and 59%, respectively, and restored the attachment to fibronectin to the same extent as that observed in the presence of normal serum. In contrast to the attachment, the spreading of the adhering cells was not further influenced by the addition of soluble thrombospondin. Subcultured cells did not require any protein for adhering to gelatin substrata. These observations indicate that thrombospondin plays a major role in the adhesion of endothelial cells in primary culture.     

Tissue transglutaminase is an integrin-binding adhesion coreceptor for fibronectin

The protein cross-linking enzyme tissue transglutaminase binds in vitro with high affinity to fibronectin via its 42-kD gelatin-binding domain. Here we report that cell surface transglutaminase mediates adhesion and spreading of cells on the 42-kD fibronectin fragment, which lacks integrin-binding motifs. Overexpression of tissue transglutaminase increases its amount on the cell surface, enhances adhesion and spreading on fibronectin and its 42-kD fragment, enlarges focal adhesions, and amplifies adhesion-dependent phosphorylation of focal adhesion kinase. These effects are specific for tissue transglutaminase and are not shared by its functional homologue, a catalytic subunit of factor XIII. Adhesive function of tissue transglutaminase does not require its cross-linking activity but depends on its stable noncovalent association with integrins. Transglutaminase interacts directly with multiple integrins of beta1 and beta3 subfamilies, but not with beta2 integrins. Complexes of transglutaminase with integrins are formed inside the cell during biosynthesis and accumulate on the surface and in focal adhesions. Together our results demonstrate that tissue transglutaminase mediates the interaction of integrins with fibronectin, thereby acting as an integrin-associated coreceptor to promote cell adhesion and spreading.     

HeLa cell adhesion to microcarriers in high shear conditions: evidence for membrane receptors for collagen but not laminin or fibronectin

HeLa-S3 cells were analyzed for their ability to attach and spread on cell culture microcarriers that were made either positively or negatively charged with polymeric plastics or were coated with BSA, gelatin, fibronectin or laminin. The cells stuck to all microcarriers under low shear, i.e. low stirring conditions with similar rates of attachment. Except in the case of gelatin microcarriers where cells fully spread, cells did not or only partially spread on the others. Under high shear, cells attached with the following rates: positive = negative = gelatin = BSA greater than laminin greater than fibronectin. Cells detached from all but the gelatin and BSA coated beads. However, the cells did not fully spread on BSA beads. The observation that cells not only attached but also spread on gelatin beads indicated that gelatin could be a specific substratum adhesion protein while the other surfaces were 'non-specific'. It should be noted that neither antibodies to laminin nor fibronectin interfered with attachment to gelatin. Protein synthesis inhibitors reduced the attachment and spreading on gelatin beads under high but not low shear conditions. With low shear, attachment and spreading appeared normal. We concluded that the density of the cell surface attachment proteins was reduced by the protein synthesis inhibitors and there were not enough present to facilitate attachment under high shear. The results also indicated that protein synthesis was not essential for cell spreading. Proteolysis of the cell surface with low concentrations of trypsin abolished the attachment of cells to gelatin-coated beads. The reappearance of attachment ability took several hours and was inhibited by actinomycin-D.     

The adhesion modulating properties of tenascin-W

Tenascins are extracellular matrix glycoproteins associated with cell motility, proliferation and differentiation. Tenascin-C inhibits cell spreading by binding to fibronectin; tenascin-R and tenascin-X also have anti-adhesive properties in vitro. Here we have studied the adhesion modulating properties of the most recently characterized tenascin, tenascin-W. C2C12 cells, a murine myoblast cell line, will form broad lamellipodia with stress fibers and focal adhesion complexes after culture on fibronectin. In contrast, C2C12 cells cultured on tenascin-W fail to spread and form stress fibers or focal adhesion complexes, and instead acquire a multipolar shape with short, actin-tipped pseudopodia. The same stellate morphology is observed when C2C12 cells are cultured on a mixture of fibronectin and tenascin-W, or on fibronectin in the presence of soluble tenascin-W. Tenascin-W combined with fibronectin also inhibits the spreading of mouse embryo fibroblasts when compared with cells cultured on fibronectin alone. The similarity between the adhesion modulating effects of tenascin-W and tenascin-C in vitro led us to study the possibility of tenascin-W compensating for tenascin-C in tenascin-C knockout mice, especially during epidermal wound healing. Dermal fibroblasts harvested from a tenascin-C knockout mouse express tenascin-W, but dermal fibroblasts taken from a wild type mouse do not. However, there is no upregulation of tenascin-W in the dermis of tenascin-C knockout mice, or in the granulation tissue of skin wounds in tenascin-C knockout animals. Similarly, tenascin-X is not upregulated in early wound granulation tissue in the tenascin-C knockout mice. Thus, tenascin-W is able to inhibit cell spreading in vitro and it is upregulated in dermal fibroblasts taken from the tenascin-C knockout mouse, but neither it nor tenascin-X are likely to compensate for missing tenascin-C during wound healing.     

Alternative splicing during chondrogenesis: modulation of fibronectin exon EIIIA splicing by SR proteins

The alternative exon EIIIA of the fibronectin gene is included in mRNAs produced in undifferentiated mesenchymal cells but excluded from differentiated chondrocytes. As members of the SR protein family of splicing factors have been demonstrated to be involved in the alternative splicing of other mRNAs, the role of SR proteins in chondrogenesis-associated EIIIA splicing was investigated. SR proteins interacted with chick exon EIIIA sequences that are required for exon inclusion in a gel mobility shift assay. Addition of SR proteins to in vitro splicing reactions increased the rate and extent of exon EIIIA inclusion. Co-transfection studies employing cDNAs encoding individual SR proteins revealed that SRp20 decreased mRNA accumulation in HeLa cells, which make A+ mRNA, apparently by interfering with pre-mRNA splicing. Co-transfection studies also demonstrated that SRp40 increased exon EIIIA inclusion in chondrocytes, but not in HeLa cells, suggesting the importance of cellular context for SR protein activity. Immunoblot analysis did not reveal a relative depletion of SRp40 in chondrocytic cells. Possible mechanisms for regulation of EIIIA splicing in particular, and chondrogenesis associated splicing in general, are discussed.     

Different integrins mediate cell spreading,haptotaxis and lateral migration of HaCaT keratinocytes on fibronectin

Collaborative role of various fibronectin-binding integrins (α5β1, αvβ1 and αvβ6) as mediators of cell adhesion and migration on fibronectin was studied using cultured HaCaT keratinocytes. This cell line spontaneously expressed all three fibronectin-binding integrins. In addition, the expression of αvβ6 integrin was strongly and specifically upregulated by transforming growth factor-β1 (TGFβ1) whereas the amount of other integrins remained practically unchanged on the cell surface. Adhesion, spreading and motility of HaCaT keratinocytes on fibronectin were promoted by TGFβ1. Based on antibody blocking experiments, both untreated and TGFβ1-treated HaCaT cells used αvβ6 integrin as their main fibronectin receptor for cell spreading. In contrast to TGFβ1-treated cells, the untreated cells also needed α5β1 integrin for maximal cell spreading on fibronectin. Combinations of antibodies blocking both of these receptors totally prevented spreading of both untreated and TGFβ1-treated cells. Haptotactic motility of individual HaCaT cells through fibronectin-coated membranes was again mainly dependent on αvβ6 integrin, while αvβ1 and α5β1 integrins played a lesser role both in untreated and TGFβ1-treated HaCaT cells. However, unlike haptotaxis, lateral migration of HaCaT cell sheet was mainly mediated by β1 integrins, and αvβ6 integrin showed a minor role. The migration process appeared to involve a number of β1 integrins that could adaptively replace each other when blocking antibodies were present. Thus, keratinocytes appear to use different fibronectin receptors for different functions, such as cell spreading, haptotaxis and lateral migration. The cells can also adapt to a situation where one receptor is unfunctional by switching to another receptor of the same ligand.     

Multiple mechanisms of dissociated epidermal cell spreading

To test the possibility that epidermal cells use a common basement membrane protein whenever they spread, in vitro experiments were conducted using trypsin-dissociated guinea pig epidermal cells and the following proteins: human serum, bovine serum albumin, serum fibronectin, Type IV collagen, laminin, and epibolin (a recently described serum glycoprotein which supports epidermal cell spreading; Stenn, K.S., 1981, Proc. Natl. Acad. Sci. U.S.A. 78:6907.). When the cells were added to media containing the specific proteins, all the tested proteins, except for serum albumin, supported cell spreading. Added to protein-coated substrates in defined media, the cells spread on fibronectin, epibolin, and laminin-Type IV collagen, but not on albumin or whole serum. In none of these experiments were the results qualitatively affected by the presence of cycloheximide. Antibodies to a specific protein blocked cell spreading on that protein but not on the other active proteins, e.g. whereas antibodies to epibolin blocked cell spreading on epibolin, they did not affect spreading on fibronectin, collagen, or laminin. In a second assay in which the cells were allowed to adhere to tissue culture plastic before the protein-containing medium was added, the cells spread only if the medium contained epibolin. Moreover, under these conditions the spreading activity of whole serum and plasma was neutralized by antiepibolin antibodies. These results support the conclusion that dissociated epidermal cells possess multiple spreading modes which depend, in part, on the proteins of the substrate, proteins of the medium, and the sequence of cell adhesion and protein exposure.     

Effect of serum,fibronectin, and laminin on adhesion of rabbit intestinal epithelial cells in culture

Rabbit intestinal epithelial cells, obtained after a limited hyaluronidase digestion, were incubated in medium with or without calf serum, on bacteriological plastic dishes. The dishes, either plain or coated with an air-dried type I collagen film, were pretreated with medium alone or with medium containing purified laminin or purified fibronectin. Cells did not attach in significant numbers to untreated bacteriological plastic, even in the presence of serum. Cells did attach to collagen-coated dishes, and were judged viable on the basis of their incorporation of radiolabeled leucine into cell protein. Cell adhesion to the collagen substrate increased in proportion to the concentration of serum in the medium, with maximal attachment at 5% serum or greater. Pretreatment of plain or collagen-coated dishes with increasing amounts of fibronectin enhanced cell adhesion in a concentration-dependent manner. Either serum, or fibronectin-free serum in the medium enhanced cell attachment to substrates pretreated with cither fibronectin or laminin. Thus, intestinal epithelial cells appear to possess surface receptors for both laminin and fibronectin. The evidence further suggests that calf serum may contain factors, other than fibronectin, capable of enhancing intestinal epithelial cell attachment to collagen substrates.     

Exogenous fibronectin is not required for organogenesis in vitro

Summary The biological effect of plasma fibronectin on the differentiation of embryonic mouse kidney and tooth was studied in organ cultures. Transferrin (50μg/ml) was a strong mitogen for kidney cells, whereas, the addition of soluble fibronectin (50 to 250 μg/ml) had no detectable effect on differentiation or proliferation. The same serum-free, transferrin-containing medium did not support tooth differentiation. however, fibronectin was not a necessary serum component because fibronectin-free serum supported tooth development. It was demonstrated with antibodies specific for human fibronectin that the exogenously added human fibronectin at 50 μg/ml did not become incorporated to the cultured organs. Only minimal incorporation to the kidney basement membrane area was observed when fibronectin concentration was 250μg/ml. The mesenchymal stroma and the basement membranes of the kidney and tooth rudiments cultured in fibronectin-free media stained intensely with conventional fibronectin antibodies, indicating endogenous production of fibronectin. Outgrowing epithelial cells from isolated kidney tubules produced fibronectin as well as laminin. The results suggest that the fibronectin found in the stroma and basement membranes is an endogenous product of the developing tissues and that plasma fibronectin is not required for in vitro organogenesis. The results also indicate that it is difficult to study the effect of fibronectin on morphogenetic processes because it may not penetrate the organ explants in vitro. This work was supported by grants from the Sigrid Jusélius Foundation, Finska L?kares?llskapet, Finnish Life Insurance Companies and Grant CA 28896 and Cancer Center Support Grant CA 30199 from the National Cancer Institute, Department of Health and Human Services, Washington, D.C.     

Human spreading factor: synthesis and response by HepG2 hepatoma cells in culture

Human serum spreading factor (SF) is a cell adhesion and spreading-promoting glycoprotein purified from serum or plasma that mediates effects in a wide variety of animal cell culture systems. HepG2 human hepatoma cells were found to synthesize and secrete SF into culture medium. Quantitative immunoassay of the protein indicated a concentration of about 1 microgram/ml in 48 hr-conditioned medium from confluent cultures. Although fibronectin also was synthesized and secreted into the culture medium, HepG2 cell spreading was observed in response to human serum SF, but not in response to human plasma fibronectin. Immunoprecipitation of SF from culture medium of cells metabolically-labeled with leucine, fucose or glucosamine identified a single form of the molecule of approximately 70,000 daltons. Treatment of cultures with tunicamycin inhibited incorporation of fucose and glucosamine into immunoprecipitated SF, but did not prevent synthesis and secretion of the protein. Electrophoretic analysis and cell spreading assays showed that SF secreted by tunicamycin-treated HepG2 cells was of molecular weight (mw) approximately 60,000, and was biologically active.     

Different effects of fibronectin on the phagocytosis of Staphylococcus aureus and coagulase-negative staphylococci by murine peritoneal macrophages

Fibronectin is known to be an important factor in colonization by Staphylococcus aureus of host tissues as well as other extracellular matrix proteins such as collagen and laminin. We investigated the effect of fibronectin on the phagocytosis of the S. aureus Cowan I strain by macrophages and of coagulase-negative staphylococci (CNS) strains for comparison. Fibronectin-reduced serum in place of normal serum lowered the phagocytic activity of the macrophages on the Cowan I strain. Purified fibronectin enhanced the phagocytic activity of the strain in a dose-dependent manner. On the other hand, fibronectin did not show any opsonic effect on the ingestion of CNS strains, though the binding of fibronectin occurred equally well in CNS strains and the Cowan I strain. Fibronectin-binding protein (FnBP), the specific fibronectin receptor on the surface on S. aureus, was detected in both the Cowan I strain and CNS strains. Polymerase chain reaction confirmed that not only the Cowan I strain, but also CNS strains possessed the FnBP gene. These results indicate that fibronectin shows an opsonic effect on the S. aureus, Cowan I strain but not on CNS strains, and suggest that the binding of fibronectin to FnBP is not sufficient for efficient phagocytosis of the staphylococci strains by macrophages.     

Serum starvation induces abnormal spindle location,RhoA delocalization,and extension of intercellular bridge with the midbody

Serum starvation induces binucleation in HeLa cells, but the effects of serum starvation on mitosis and the significance of binucleation remain unknown. We investigated the effect of serum starvation on mitosis and analyzed the growth of binucleated cells. The frequency of binucleation caused by cytokinesis failure in DMEM without FBS (0% medium) was higher than that in DMEM with FBS (10% medium). In 0% medium, the metaphase spindle location was off-center, and RhoA localization significantly lacked symmetry. The frequency of the extension of intercellular bridge with the midbody in 0% medium was significantly higher than that in 10% medium. Moreover, all mononucleated mitotic cells caused bipolar mitosis and produced only mononucleated daughter cells, but binucleated cells produced various nucleated cells by multipolar mitosis in 0% medium. These results suggest that serum starvation may have various effects on mitosis, and binucleated cells may be related to formation of aneuploidy.     

The fibronectin-binding integrins alpha5beta1 and alphavbeta3 differentially modulate RhoA-GTP loading,organization of cell matrix adhesions,and fibronectin fibrillogenesis

We have studied the formation of different types of cell matrix adhesions in cells that bind to fibronectin via either alpha5beta1 or alphavbeta3. In both cases, cell adhesion to fibronectin leads to a rapid decrease in RhoA activity. However, alpha5beta1 but not alphavbeta3 supports high levels of RhoA activity at later stages of cell spreading, which are associated with a translocation of focal contacts to peripheral cell protrusions, recruitment of tensin into fibrillar adhesions, and fibronectin fibrillogenesis. Expression of an activated mutant of RhoA stimulates alphavbeta3-mediated fibrillogenesis. Despite the fact that alpha5beta1-mediated adhesion to the central cell-binding domain of fibronectin supports activation of RhoA, other regions of fibronectin are required for the development of alpha5beta1-mediated but not alphavbeta3-mediated focal contacts. Using chimeras of beta1 and beta3 subunits, we find that the extracellular domain of beta1 controls RhoA activity. By expressing both beta1 and beta3 at high levels, we show that beta1-mediated control of the levels of beta3 is important for the distribution of focal contacts. Our findings demonstrate that the pattern of fibronectin receptors expressed on a cell dictates the ability of fibronectin to stimulate RhoA-mediated organization of cell matrix adhesions.     

Spreading of HeLa cells on a collagen substratum requires a second messenger formed by the lipoxygenase metabolism of arachidonic acid released by collagen receptor clustering.

HeLa cells attach to a variety of substrata but spread only on collagen or gelatin. Spreading is dependent on collagen-receptor upregulation, clustering, and binding to the cytoskeleton. This study examines whether second messengers are involved in initiating the spreading process on gelatin. The levels of cytosolic free calcium ([Ca++]i), cAMP, and cytoplasmic pH (pHi) do not change during cell attachment and spreading. However, a basal level of [Ca++]i and an alkaline pH(i) are required for spreading. There is an activation of protein kinase C (PKC) and a release of arachidonic acid (AA) on attachment and before cell spreading. Inhibition of PKC does not block cell spreading, indicating that PKC activation is not essential for spreading. Inhibition of phospholipase A2 blocks cell spreading, whereas addition of exogeneous AA overcomes this inhibitory effect. Among AA metabolic pathways, inhibitors of lipoxygenase (LOX) block cell spreading, suggesting that a LOX product(s) formed from AA initiates spreading. Clustering receptors for collagen with polyclonal antibodies, or with anti-collagen-receptor antigen-binding fragments (Fab) in combination with a secondary antibody, induce AA release. Also, AA is released when cells attach to either immobilized gelatin or immobilized Arg-Gly-Asp (RGD) peptide. Thus, AA is released whenever receptor clustering is observed. Receptor occupancy is not sufficient to release AA; when cells are treated with gelatin or RGD peptide in solution or anti-collagen-receptor Fab fragments without secondary antibody, conditions where receptor clustering is not observed, AA is not released. Thus, a LOX metabolite(s) of AA formed by collagen-receptor clustering is a second messenger(s) that initiates HeLa cell spreading. LOX inhibitors also block the spreading of bovine aortic endothelial cells, chicken embryo fibroblasts, and CV-1 fibroblasts on gelatin or fibronectin, indicating that other cells might use the same second messenger system in initiating cell-substratum adhesion.     

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 cytoskeletal 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.     

Cellular and metabolic specificity in the interaction of adhesion proteins with collagen and with cells

Fibronectin mediates the adhesion of fibroblasts to collagen substrates, binding first to the collagen and then to the cells. We report here that the interaction of the cells with the fibronectin-collagen complex is blocked by specific gangliosides, GD_{1 a} and GT₁, and that the sugar moieties of these gangliosides contain the inhibitory activity. The gangliosides act by binding to fibronectin, suggesting that they may be the cell surface receptor for fibronectin. Evidence is presented that other adhesion proteins or mechanisms of attachment exist for chondrocytes, epidermal cells, and transformed tumorigenic cells, since adhesion of these cells is not stimulated by fibronectin. Chondrocytes adhere via a serum factor that is more temperature-sensitive and less basic than fibronectin. Unlike that of fibroblasts chondrocyte adhesion is stimulated by low levels of gangliosides. Epidermal cells adhere preferentially to type IV (basement membrane) collagen but at a much slower rate than fibroblasts or chondrocytes. This suggests that these epidermal cells synthesize their own specific adhesion factor. Metastatic cells cultured from the T241 fibrosarcoma adhere rapidly to type IV collagen in the absence of fibronectin and do not synthesize significant amounts of collagen or fibronectin. Their growth, in contrast to that of normal fibroblasts, is unaffected by a specific inhibitor of collagen synthesis. These data indicate the importance of specific collagens and adhesion proteins in the adhesion of certain cells and suggest that a reduction in the synthesis of collagen and of fibronectin is related to some of the abnormalities observed in transformed cells.     

Adhesion, orientation, and movement of cells cultured on ultrathin fibronectin fibers

Summary This study examined the behavior of rat tendon fibroblasts, baby hamster kidney fibroblasts, macrophage-like P388D1 cells, and neurons from rat dorsal root ganglia, cultured on fibronectin strands 0.2–5 μm in diameter. We investigated cell spreading, orientation, formation of focal contacts, the speed of cell movement, and the speed of neurite outgrowth in cells cultured on fibronectin strands, glass covered with fibronectin, and plain, nontreated glass. Fibronectin strands significantly promoted cell spreading and caused a marked alignment of all kinds of cells to the direction of the fiber. The fibers caused the alignment of actin filaments in fibroblasts and focal contacts in fibroblasts and macrophages and increased polymerization of F-actin in cells. Fibronectin fibers also increased the speed and persistence of cell movement and the rate of neurite outgrowth. Macrophages grown on fibronectin fibers produced numerous actin-rich microspikes and adopted a polarized, migratory phenotype. These findings indicate that fibronectin strands, resembling natural components of the extracellular matrix, are more effective in activating various types of cells than two-dimensional, fibronectin-covered substrata. The results also confirm the suitability of the three-dimensionally oriented fibronectin form for use in clinical practice.     

Evidence for involvement of more than one class of glycoprotein in cell interactions with fibronectin

The receptor mechanism by which cells attach to fibronectin has been investigated by a combined immunologic and electrophoretic approach. One particular antiserum directed against 3T3 cell plasma membranes was found to contain antibodies that blocked spreading of these murine cells on fibronectin but not on laminin or serum spreading factor (vitronectin). Proteolysis experiments confirmed that this cell line has calcium-protected polypeptides necessary for cell spreading on fibronectin. Consequently, protein antigens were fractionated according to size by SDS gel electrophoresis, and antigens that could block the inhibitory activity of the polyclonal antibody were identified. One class of blocking antigen appeared to correspond to the 140,000-dalton complexes favored by several laboratories as fibronectin receptor candidates, but a second class of 45,000 daltons was also apparent. This 45,000-dalton antigen was a major absorbing activity from 3T3 cell membranes and the predominant activity from L929 membranes. By isoelectric focusing and two-dimensional gel electrophoresis, it was found to exist as a set of isoelectric point variants with pK = 5.3 to 6.2. Our results indicate that current models postulating a simple, unimolecular receptor mechanism for fibronectin may be oversimplified and that fibronectin may instead interact with more than one protein receptor component on the fibroblast cell surface.