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.     

Altered beta-actin gene expression in phorbol myristate acetate-treated chondrocytes and fibroblasts.

Phorbol-12-myristate-13-acetate (PMA), a potent tumor promoter, was shown to have opposite effects on the cellular morphology and steady-state levels of beta-actin mRNA in embryonic chicken muscle fibroblasts and sternal chondrocytes. When fibroblasts were treated with PMA, they formed foci of densely packed cells, ceased to adhere to culture plates, and had significantly reduced levels of beta-actin mRNA and protein. Conversely, when treated with PMA, floating chondrocytes attached to culture dishes, spread out, and began to accumulate high levels of beta-actin mRNA and proteins. In the sternal chondrocytes the stimulation of the beta-actin mRNA production was accompanied by increased steady-state levels of fibronectin mRNAs and protein. These alterations were concomitant with a fivefold reduction in type II collagen mRNA and a cessation in its protein production. After fibronectin and actin mRNAs and proteins reached their maximal levels, type I collagen mRNA and protein synthesis were turned on. Removal of PMA resulted in reduced beta-actin mRNA levels in chondrocytes and in a further alteration in the cell morphology. These observed correlations between changes in cell adhesion and morphology and beta-actin expression suggest that the effect of PMA on cell shape and adhesion may result in changes in the microfilament organization of the cytoskeleton which ultimately lead to changes in the extracellular matrix produced by the cells.     

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.     

Regulation of cell attachment and cell number by fibronectin and laminin

We have examined the effect of laminin and fibronectin on the attachment and growth on type IV collagen of a line of mouse epithelial cells and a strain of adult human fibroblasts. Laminin stimulated attachment of the epidermal cells and fibronectin stimulated fibroblast attachment. At high concentrations (100 micrograms/ml), the attachment proteins altered the growth of cells in culture. The epidermal cells grew better in media containing fibronectin-free serum supplemented with laminin. Fibroblasts, on the other hand, grew best in media containing serum supplemented with fibronectin. These data suggest that laminin promotes epithelial cell growth whereas fibronectin promotes fibroblast growth. This observation was confirmed when these cells were cocultured in the presence of the attachment proteins or of their respective antibodies. The mouse epidermal cells grew best when laminin was added to cocultures of fibroblasts and epithelial cells. Fibroblasts grew best in the presence of antibody to laminin and poorly in the presence of antibody to fibronectin. Thus, fibronectin and laminin may participate in the regulation of cell populations in vivo and may be involved in epithelial-mesenchymal interactions.     

Fibrillar organization of fibronectin is expressed coordinately with cell surface gangliosides in a variant murine fibroblast

NCTC 2071A cells, a line of transformed murine fibroblasts, grow in serum-free medium, are deficient in gangliosides, synthesize fibronectin, but do not retain and organize it on the cell surface. When the cells are exposed to exogenous gangliosides, fibrillar strands of fibronectin become attached to the cell surface. A morphologically distinct variant of NCTC 2071A cells was observed to both retain cell surface fibronectin and organize it into a fibrillar network when the cells were stained with anti-fibronectin antibodies and a fluorescent second antibody. A revertant cell type appeared to resemble the parental NCTC 2071A cells in terms of morphology and fibronectin organization. All three cell types were subjected to mild NaIO4 oxidation and reduction with KB3H4 of very high specific radioactivity in order to label the sialic acid residues of surface gangliosides. The variant had much more surface gangliosides than the parental, particularly more complex gangliosides corresponding to GM1 and GD1a. The surface gangliosides of the revertant were intermediate between the parental and the variant. By using sialidase, which hydrolyzes GD1a to GM1, and 125I-labeled cholera toxin, which binds specifically to GM1, the identity and levels of these gangliosides were confirmed in the three cell types. When variant cells were exposed to sialidase for 2 d, there appeared to be little change in fibronectin organization. Concomitant treatment of the cells with the B subunit of cholera toxin, which bound to all the surface GM1 including that generated by the sialidase, however, eliminated the fibrillar network of fibronectin. In addition, exposure of the variant cells to a 70,000-mol-wt fragment of fibronectin, which lacks the cell attachment domain but contains a matrix assembly domain, inhibited the formation of fibers. Finally, all three cell types were assayed for their ability to attach to and spread on fibronectin-coated surfaces; no significant differences were found. Our results further establish that the ability of a cell to organize fibronectin into an extracellular matrix is dependent on certain gangliosides, but they also indicate that cell adhesion to fibronectin is independent of these gangliosides. We suggest that matrix organization and cell attachment and spreading are based on separate mechanisms and that these functions are associated with different cell surface "receptors."     

The adhesive glycoprotein laminin is an agglutinin

The glycoprotein laminin appears to function in the attachment of various epithelial cells to basement membranes. We examined whether its putative cell-adhesive activity could be analyzed in a simple, one-component model system—the agglutination of erythrocytes. Laminin is a potent agglutinin of aldehyde-fixed sheep and human erythrocytes, with half-maximal agglutination of 0.8 μg/ml in a standard hemagglutination assay. Inhibitors of this hemagglutinating activity include gangliosides and certain charged phospholipids. The spectrum of molecules is similar but not identical to inhibitors of the hemagglutinating activity of the adhesive glycoprotein fibronectin. Laminin is much less biologically active in three other assays for fibronectin biological activity involving cell spreading on tissue culture substrates, attachment of fibroblastic cells to type I collagen, and restoration of normal morphology to transformed fibroblasts. The adhesive glycoproteins laminin and fibronectin therefore differ markedly in biological activities in several specific adhesion assays; however, they resemble one another in binding to heparin, collagen, and cell surfaces and in their agglutinin activity.     

Adhesion of Human Glioma Cell Lines to Fibronectin, Laminin, Vitronectin and Collagen I Is Modulated by Gangliosides in vitro

Adhesion of eight cell lines, derived from human gliomas of different histological types, to fibronectin, collagen I, vitronectin, and laminin was investigated in vitro. The glioma cell lines were found to attach to these substrates to different extents. Interestingly, all cell lines strongly attached to laminin. In addition, glioma cell adhesion was found to be dose dependent. Moreover, adhesion of three cell lines to fibronectin and collagen I was partially inhibited and to vitronectin completely prevented by GRGDTP peptide, indicating the involvement of integrin receptors in glioma cell adhesion. We have demonstrated, recently, that gangliosides play an important role in promoting glioma cell invasion of the reconstituted basement membrane, Matrigel, in vitro. In order to study the mechanism of action of gangliosides in this process, the role of six gangliosides (GM1, GM3, GD3, GD1a, GD1b, and GT1b) in cell adhesion to the four proteins was investigated in three cell lines. Although all gangliosides, with the exception of GM3, were found to enhance cell adhesion to these proteins to different extents, GD3 proved to be the most effective adhesion-promoting ganglioside in all three cell lines. GM3 was found to inhibit cell adhesion to the four proteins in one cell line but enhanced cell adhesion in two other cell lines. The three cell lines were found to express both GD3 and gangliosides recognised by the A2B5 antibody. Furthermore, adhesion of the three cell lines to fibronectin, vitronectin, laminin, and collagen I was inhibited by incubation with A2B5, demonstrating the involvement of intrinsic cell membrane gangliosides in adhesion of glioma cells to these proteins. Taken together with the observation that gangliosides modulate integrin receptor function, these data suggest that gangliosides may play a central role in the control of the adhesive and invasive properties of human glioma cells.     

Analysis of heterogeneity of human keratinocytes interacting with immobilized fibronectin and collagenes of types I and IV

Basing on the natural affinity of skin keratinocytes toward extracellular matrix proteins, we have attempted to dissect the population of these cells by varying the time of their adhesion to substrates from fibronectin and collagen of types I and IV. After selection for 10, 20, and 30 min, the keratinocytes were cultivated for 24 h under standard conditions. The area of cell projection on the substrate and the spreading coefficient were measured. Statistically significant morphological differences between cells selected on different substrates were found. The size of cells growing on type-I collagen was twice as large as that of the cells cultivated on collagen type-IV or on fibronectin. Independent of the substratum, up to 60–65% of the cells had a round shape. Keratinocytes cultivated on collagens revealed heterogeneity both in the control and after selection in their adhesion times, while the cells grown on fibronectin behaved as a homogeneous population. These results suggest that, contrary to fibronectin, collagens stabilize some physiological states of keratinocytes corresponding to their interactions with extracellular matrix proteins in the organism. Original Russian Text O.G. Spichkina, G.P. Pinaev, Y.P. Petrov, 2008, published in Tsitologiya, Vol. 50, No. 2, 2008.     

Involvement of gangliosides and glycoprotein fibronectin receptors in cellular adhesion to fibronectin

We have used a rat neural cell line, B65, to investigate the relative contributions of gangliosides and glycoprotein receptors in adhesion to fibronectin. Monoclonal antibodies against two neuroectoderm-associated gangliosides, D1.1 and GD3, inhibit the rate of B65 attachment to fibronectin, suggesting that these gangliosides are involved in the adhesion process. Adhesion to fibronectin is not affected by a third monoclonal antibody against a separate, unidentified cell-surface component of B65 cells. Furthermore, B65 cells lacking D1.1 adhere to fibronectin at a slower rate than B65 cells that express D1.1. The involvement of glycoprotein receptors in adhesion is demonstrated by the ability of antibodies against human fibronectin receptor to inhibit B65 attachment to fibronectin. In addition, adhesion is blocked by a hexapeptide containing the Arg-Gly-Asp fibronectin sequence which is necessary for binding to the receptor. Trypsin treatment of B65 cells in the absence of divalent cations results in proteolysis of the fibronectin receptor with an accompanying loss of ability of the cells to attach to fibronectin. D1.1 and GD3 expression is not affected by this trypsinization, indicating that the gangliosides alone are incapable of mediating attachment. The glycoprotein receptors must be primarily responsible for adhesion to fibronectin with the gangliosides playing a secondary role as enhancers or modulators.     

Latent TGF-beta binding protein LTBP-2 decreases fibroblast adhesion to fibronectin

We have analyzed the effects of latent TGF-beta binding protein 2 (LTBP-2) and its fragments on lung fibroblast adhesion. Quantitative cell adhesion assays indicated that fibroblasts do not adhere to full-length LTBP-2. Interestingly, LTBP-2 had dominant disrupting effects on the morphology of fibroblasts adhering to fibronectin (FN). Fibroblasts plated on LTBP-2 and FN substratum exhibited less adherent morphology and displayed clearly decreased actin stress fibers than cells plated on FN. These cells formed, instead, extensive membrane ruffles. LTBP-2 had no effects on cells adhering to collagen type I. Fibroblasts adhered weakly to the NH2-terminal fragment of LTBP-2. Unlike FN, this fragment did not augment actin stress fiber formation. Interestingly, the adhesion-mediating and cytoskeleton-disrupting effects were localized to the same NH2-terminal proline-rich region of LTBP-2. LTBP-2 and its antiadhesive fragment bound to FN in vitro, and the antiadhesive fragment associated with the extracellular matrix FN fibrils. These observations reveal a potentially important role for LTBP-2 as an antiadhesive matrix component.     

Role of laminin and fibronectin in selecting myogenic versus fibrogenic cells from skeletal muscle cells in vitro

Growth of embryonic skeletal muscle occurs by fusion of multinucleated myotubes with differentiated, fusion-capable myoblasts. Selective recognition seems to prevent fusion of myotubes with nonmyogenic cells such as muscle fibroblasts, endothelial cells, or nerve cells, but the nature of the signal is as yet unknown. Here we provide evidence that one of the selection mechanisms may be the enhanced affinity for laminin of myogenic cells as compared to fibrogenic cells. Growing myotubes in myoblast cultures accumulate laminin and type IV collagen on their surface in patches and strands as the first step in assembling a continuous basal lamina on mature myofibers (U. Kühl, R. Timpl, and K. von der Mark (1982), Dev. Biol. 93, 344-359). Fibronectin, on the other hand, assembles into an intercellular fibrous meshwork not associated with the free myotube surface. Over a brief time period (10-20 min) myoblasts from embryonic mouse thigh muscle adhere faster to laminin than do fibroblasts from the same tissue; these adhere faster to fibronectin. When a mixture of the cells is plated for 20 min on laminin/type IV collagen substrates, only myogenic cells adhere, giving rise to cultures with more than 90% fusion after 2 weeks; on fibronectin/type I collagen in the same time primarily fibroblastic cells adhere, giving rise to cultures with less than 10% nuclei in myotubes. The differential affinities of myoblasts for basement membrane constituents and of fibroblasts for interstitial connective tissue components may play a role in sorting out myoblasts from fibroblasts in skeletal muscle development.     

High molecular weight,cell surface-associated glycoprotein (fibronectin) lost in maglinant 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 many 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 cultutred cells and for some of the malignant behavior of neoplastic cells in vivo.     

Analysis of PC12 cell adhesion to muscle and non-muscle cells and components of the extracellular matrix

The relative strength of PC12 cell adhesion to monolayers of myoblasts and fibroblasts as well as to purified components of the extracellular matrix has been determined. PC12 cell adhesion to myoblasts and fibroblasts was dependent on temperature, and the relative strength of adhesion to both increased over a 4-h period. The strength of adhesion to myoblasts was consistently found to be greater than that to fibroblasts. Whereas coating tissue culture plastic with purified collagen and laminin increased its ability to support PC12 cell adhesion, coating with plasma fibronectin or gelatin reduced cell adhesion. Trypsin treatment of PC12 cells was without effect on their adhesion to collagen and laminin and only partially reduced the strength of adhesion to myoblasts. In contrast trypsinized cells showed a greatly reduced ability to adhere to fibroblast monolayers. Pretreatment of PC12 cells with NGF had no obvious effect on the ability of the cells to adhere to any of the above substrata.     

Fluorescent gangliosides as probes for the retention and organization of fibronectin by ganglioside-deficient mouse cells

Ganglioside-deficient transformed mouse fibroblasts (NCTC 2071A cells), which grow in serum-free medium, synthesize fibronectin but do not retain it on the cell surface. When fluorescent derivatives of gangliosides, containing either rhodamine or Lucifer yellow CH attached to the sialic acid residues, were added to the culture medium, the cells incorporated the derivatives and their surfaces became highly fluorescent. When the cells were stained with anti-fibronectin antibodies and a fluorescent second antibody, fibrillar strands of fibronectin were observed to be attached to the cell surface, with partial coincidence of the patterns of direct ganglioside fluorescence and indirect fibronectin immunofluorescence at the cell surface. When the cells were exposed to bacterial neuraminidase during the time of ganglioside insertion, similar patterns of fluorescence were observed. Because the fluorescent gangliosides are resistant to the enzyme, these results suggest that neuraminidase-sensitive endogenous glycoconjugates were not involved in the ganglioside-mediated retention and organization of endogenous fibronectin. After cells were exposed to exogenous chicken fibronectin, most of the fibronectin was attached to the substratum and only a few fibrils were attached to the cells. When exogenous gangliosides were included in the incubation, there was a striking increase in cell-associated exogenous fibronectin, which was highly organized into a fibrillar network. Conversely, cells incubated for 18 h with exogenous unmodified gangliosides exhibited a highly organized network of endogenously derived fibronectin. Upon further incubation of the cells for 2 h with fluorescent gangliosides, there was considerable co-distribution of the fluorescent gangliosides with the fibronectin network as revealed by immunofluorescence. Our results support the concept that gangliosides can mediate the attachment of fibronectin to the cell surface and its organization into a fibrillar network.     

Endothelial cell adhesion, signaling, and morphogenesis in fibroblast-derived matrix

Extracellular matrix plays a critical role in cellular development by providing signaling cues that direct morphogenesis. In order to study both the cues that natural matrix provides and endothelial cell responses to that information, human fetal lung fibroblasts were used to produce a fibrous three-dimensional matrix. Following the removal of the fibroblasts by detergent extraction, protein and proteoglycan constituents of the remaining matrix were identified by immunofluorescence and immunoblotting. Matrix components included fibronectin, tenascin-C, collagen I, collagen IV, collagen VI, versican, and decorin. Colocalization analysis suggested that fibronectin was a uniquely distributed matrix protein. Morphology, three-dimensional matrix adhesions, and integrin-mediated signaling during vasculogenesis were then studied in human endothelial cells seeded onto the fibroblast-derived matrix. Elongated morphology and decreased cell area were noted, as compared with cells on fibronectin-coated coverslips. Cell-matrix adhesions contained vinculin, pY397-FAK, and pY410-p130Cas, and all of these colocalized more with fibronectin than tenascin-C, collagen I, or collagen VI. Additionally, the endothelial cells remodeled the fibroblast-derived matrix and formed networks of tubes with demonstrable lumens. Matrix adhesions in these tubes also predominantly colocalized with fibronectin. The pattern of membrane type 1 matrix metalloprotease expression in the endothelial cells suggested its involvement in the matrix remodeling that occurred during tubulogenesis. These results indicated that information in fibroblast-derived matrix promoted vasculogenic behavior.     

Relationship between neuronal migration and cell-substratum adhesion: laminin and merosin promote olfactory neuronal migration but are anti- adhesive

Regulation by the extracellular matrix (ECM) of migration, motility, and adhesion of olfactory neurons and their precursors was studied in vitro. Neuronal cells of the embryonic olfactory epithelium (OE), which undergo extensive migration in the central nervous system during normal development, were shown to be highly migratory in culture as well. Migration of OE neuronal cells was strongly dependent on substratum- bound ECM molecules, being specifically stimulated and guided by laminin (or the laminin-related molecule merosin) in preference to fibronectin, type I collagen, or type IV collagen. Motility of OE neuronal cells, examined by time-lapse video microscopy, was high on laminin-containing substrata, but negligible on fibronectin substrata. Quantitative assays of adhesion of OE neuronal cells to substrata treated with different ECM molecules demonstrated no correlation, either positive or negative, between the migratory preferences of cells and the strength of cell-substratum adhesion. Moreover, measurements of cell adhesion to substrata containing combinations of ECM proteins revealed that laminin and merosin are anti-adhesive for OE neuronal cells, i.e., cause these cells to adhere poorly to substrata that would otherwise be strongly adhesive. The evidence suggests that the anti- adhesive effect of laminin is not the result of interactions between laminin and other ECM molecules, but rather an effect of laminin on cells, which alters the way in which cells adhere. Consistent with this view, laminin was found to interfere strongly with the formation of focal contacts by OE neuronal cells.     

Laminin responsiveness is associated with changes in fibroblast morphology,motility, and anchorage-independent growth: Cell system for examining the interaction between laminin and EGF signaling pathways

Laminin can influence the adhesion, differentiation, and motility of motility of several cell types, including epithelial and neural cells. In addition, laminin, which contains an epidermal growth factor (EGF)-like motif, can stimulate DNA synthesis in fibroblasts possessing the EGF receptor, but laminin does not compete for EGF binding. To further investigate laminin action in fibroblasts, and the relationship between laminin and EGF receptor function, we have developed a system wherein cells containing laminin-binding activity were cloned from a mouse fibroblast cell line (B82L-wt) that cannot adhere to laminin but that have been transfected with the wild-type human EGF receptor. Although only the isolated clones can efficiently attach to laminin-coated plates, all the cells can adhere to plastic, fibronectin, and collagen I, and all exhibit comparable levels of cell surface-associated laminin. Ligand-binding assays showed that the cells with laminin attachement activity possess high-affinity EGF binding (Kd ～ 0.4 nM), and all express a similar level of the human EGF receptor. However, when compared to the B82L-wt cells, the cells with laminin-binding activity exhibit altered morphology, anchorage-independent growth, and motility. Specifically, the morphology of the fibroblasts possessing laminin binding activity appears more elongated and they spread more-extensively on plastic plates. Analysis of their growth in soft agar revealed that the clones have a 2-5-fold increase in colony formation in comparison to the B82L-wt cells. The cells possessing laminin attachment ability also exhibit laminin-induced motility, and this movement is directional (chemotaxis) rather than random (chemokinesis), indicating functional laminin receptors and signaling pathways. To examine the specific laminin receptors involved in these effects, the influence of anti-integrin subunit antibodies on cell adhesion and migration was evaluated. These studies showed that an anti-α₆ integrin antibody can completely inhibit the clonal cells' attachment and migration to laminin, and anti-α₆ immunoblots revealed that only the clones express measurable levels of α₆. These data indicate that α₆-containing integrins contribute to the lamininmediated attachment and motility of these clones and that this system may also influence the morphology and anchorage-independent growth of these fibroblasts. In addition, these cells provide a unique system for examining the interaction between EGF and laminin receptor action. © 1995 Wiley-Liss, Inc.     

Extracellular matrix proteins of human epidermal keratinocytes and feeder 3T3 cells

Cultures of human epidermal keratinocytes obtained from adult epidermis were initiated using irradiated BALB/3T3 cells as feeder layers. At different stages of confluence of the epidermal islands, feeder cells were removed and the extracellular matrix proteins of both pure component cells and cocultures were analyzed biochemically and by immunochemical methods and compared to those of skin fibroblasts of the same donors. The keratinocytes synthesized and secreted fibronectin and small amounts of laminin and type IV collagen. In addition, a nondisulfide-linked collagenous polypeptide (Mr = 120,000) was synthesized by the keratinocytes and was confined to the cell layers. Collagenous polypeptides with Mr = 120,000 were also synthesized by organ cultures of epidermal tissue and were detected in its acid or detergent extracts but again no secretion to culture medium was found. The Mr = 120,000 collagen had biochemical and immunological properties distinct from those of types I-V collagens. In immunofluorescence of keratinocyte cultures, fibronectin staining was prominent in the lining marginal cells of the expanding periphery of the epidermal cell islands but was not detected in the terminally differentiating cells in the upper layers of stratified colonies. Very little type IV collagen was found deposited in pericellular matrix form by the keratinocytes. In contrast, the mouse 3T3 feeder cells were found to produce both type IV collagen and laminin in addition to the previously identified connective tissue glycoproteins of fibroblasts, interstitial procollagens, and fibronectin. Basement membrane collagen of the 3T3 cells was found deposited as apparently unprocessed procollagen alpha 1(IV) and alpha 2(IV) chains. The production in culture conditions of basal lamina glycoproteins by the fibroblastic feeder cells may promote the attachment and growth of the cocultured keratinocytes.     

Stimulated single‐cell force spectroscopy to quantify cell adhesion receptor crosstalk

To control their attachment to substrates and other cells, cells regulate their adhesion receptors. One regulatory process is receptor crosstalk, where the binding of one type of cell adhesion molecule influences the activity of another type. To identify such crosstalk and gain insight into their mechanisms, we developed the stimulated single‐cell force spectroscopy assay. In this assay, the influence of a cells adhesion to one substrate on the strength of its adhesion to a second substrate is examined. The assay quantifies the adhesion of the cell and the contributions of specific adhesion receptors. This allows mechanisms by which the adhesion is regulated to be determined. Using the assay we identified crosstalk between collagen‐binding integrin α₁β₁ and fibronectin‐binding integrin α₅β₁ in HeLa cells. This crosstalk was unidirectional, from integrin α₁β₁ to integrin α₅β₁, and functioned by regulating the endocytosis of integrin α₅β₁. The single‐cell assay should be expandable for the screening and quantification of crosstalk between various cell adhesion molecules and other cell surface receptors.