Basement membrane-like matrix of teratocarcinoma-derived endodermal cells: presence of laminin and heparan sulfate in the matrix at points of attachment to cells

Teratocarcinoma-derived endodermal PYS-2 cells are known to synthesize an extracellular matrix containing the basement membrane molecules laminin, type IV collagen, and heparan sulfate proteoglycan as major constituents (I. Leivo, K. Alitalo, L. Risteli, A. Vaheri, R. Timpl, J. Wartiovaara, Exp Cell Res 137:15-23, 1982). Immunoferritin techniques with specific antibodies were used in the present study to define the ultrastructural localization of the above constituents in the fibrillar network. Laminin was detected in matrix network adjacent to the basal cell membrane and in protruding matrix fibrils that connect the matrix to the cell membrane. Ruthenium red-stainable heparinase-sensitive 10- to 20-nm particles were often present at the junction of the attachment fibrils and the matrix network, or along the attachment fibrils. A corresponding distribution of ferritin label was observed for basement membrane heparan sulfate proteoglycan. Type IV collagen was found in the matrix network but not in the attachment fibrils. The results suggest that the PYS-2 cells are connected to their pericellular matrix by fibrils containing laminin associated with heparan sulfate-containing particles. These results may also have relevance for the attachment of epithelial cells to basement membranes.     

Transmembrane control of laminin, lectin receptors and surface villi in teratocarcinoma-derived endodermal cells

Distribution of laminin on the surface of teratocarcinoma-derived parietal endoderm cells was studied by immuno-histochemical staining of the fixed specimen using affinity-purified anti-laminin antibody. Laminin was distributed on the basal surface of the cells, while treatment either with colchicine or with cytochalasin D (CD) resulted in a severely polarized distribution; laminin was seen only at one end of the cell. Treatment with both the reagents did not cause the severe polarization. Receptors for lectins and cell surface villi were polarized by treatment with CD but not by treatment with colchicine. These results suggest that laminin--or its cell surface receptor--is linked to both microfilament and microtubules and that the mode of transmembrane control for laminin is different from certain other cell surface components of the cells.     

Galectin binding to Mgat5-modified N-glycans regulates fibronectin matrix remodeling in tumor cells

Oncogenic signaling stimulates the dynamic remodeling of actin microfilaments and substrate adhesions, essential for cell spreading and motility. Transformation is associated with increased expression of beta1,6GlcNAc-branched N-glycans, products of Golgi beta1,6-acetylglucosaminyltransferase V (Mgat5) and the favored ligand for galectins. Herein we report that fibronectin fibrillogenesis and fibronectin-dependent cell spreading are deficient in Mgat5(-/-) mammary epithelial tumor cells and inhibited in Mgat5(+/+) cells by blocking Golgi N-glycan processing with swainsonine or by competitive inhibition of galectin binding. At an optimum dosage, exogenous galectin-3 added to Mgat5(+/+) cells activates focal adhesion kinase (FAK) and phosphatidylinositol 3-kinase (PI3K), recruits conformationally active alpha5beta1-integrin to fibrillar adhesions, and increases F-actin turnover. RGD peptide inhibits PI3K-dependent fibronectin matrix remodeling and fibronectin-dependent cell motility, while galectin-3 stimulates and overrides the inhibitory effects of RGD. Antibodies to the galectin-3 N-terminal oligomerization domain stimulate alpha5beta1 activation and recruitment to fibrillar adhesions in Mgat5(+/+) cells, an effect that is blocked by disrupting galectin-glycan binding. Our results demonstrate that fibronectin polymerization and tumor cell motility are regulated by galectin-3 binding to branched N-glycan ligands that stimulate focal adhesion remodeling, FAK and PI3K activation, local F-actin instability, and alpha5beta1 translocation to fibrillar adhesions.     

Laminin-like glycoproteins in extracellular matrix of endodermal cells

Extracellular matrix from a mouse endodermal cell line consisted mainly of two polypeptides with molecular weights of about 200,000 (200K) and 400,000 (400K). Both poly-peptides incorporated radioactivity from [³H]proline and [³H]glucosamine and were solubilized from the matrix by treatment with bacterial collagenase or 0.5 m sodium chloride. These polypeptides appeared similar to those of laminin (R. Timpl, H. Rohde, P. G. Robey, S. I. Rennard, J.-M. Foidart, and G. R. Martin, 1979, J. Biol. Chem., 254, 9933–9937) in polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate but the laminin polypeptides seemed slightly larger than the 200K and 400K polypeptides, respectively. The amino acid compositions of the isolated 200K and 400K polypeptides resembled one another and the previously published amino acid composition of laminin. Antibodies prepared against the solubilized extracellular matrix protein (mixture of 200K and 400K components) as well as those against the isolated 400K component precipitated both the 400K component and the 200K component from culture media. These antisera and antisera to laminin showed identical reactivities in immunodiffusion and in immunofluorescence of tissue sections where they stained basement membranes. The immunofluorescent staining pattern was similar to that obtained with antifibronectin except in the liver where antifibronectin stained the biliary ducts and the liver sinusoids, while laminin-like immunoreactivity was not present in the sinusoidal areas. Such differences in distribution of matrix components could be involved in generation of signals for differentiation and growth of the adjacent cells.     

Binding of hyaluronan to plasma fibronectin increases the attachment of corneal epithelial cells to a fibronectin matrix

We wished to determine whether hyaluronan would affect the attachment of epithelial cells to extracellular matrix proteins. Multiwell tissue culture plates were coated with human plasma fibronectin, laminin, or collagen type IV (0.01–10.0 μg/ml). Single-cell suspensions of rabbit corneal epithelial cells were placed in the wells, and after 45 minutes incubation the cells adhering to the matrix proteins were stained and counted. Cells attached to all three types of proteins. Preincubation of the matrix proteins with hyaluronan (0.1–1.0 mg/ml) significantly increased the number of cells attached to the fibronectin matrix, but it did not increase the numbers of cells attached to laminin or collagen type IV. Hyaluronidase inhibited this stimulatory effect. Glycosaminoglcyans other than hyaluronan (chondroitin sulfate, keratan sulfate, or heparan sulfate) failed to increase the numbers of attached cells. Treatment of the fibronectin matrix with monoclonal antibodies against the cell-binding domain of fibronectin (FN12–8 or FN30–8, 0.03–0.3 mg/ml, for 1 hour), before or after hyaluronan treatment, significantly decreased the numbers of attached cells. Monoclonal antibody against the fibrin- and heparin-binding domain at the N-terminal (FN9–1), however, significantly decreased the number of attached cells only when this antibody treatment preceded the hyaluronan treatment. Preincubation of the cells with hyaluronan had no effect; preincubation with GRGDSP (1 mg/ml), a synthetic peptide that blocks the cell surface receptor for fibronectin, significantly decreased cell attachment whether the fibronectin matrix was treated with hyaluronan or not. Further studies demonstrated that monoclonal antibody against the fibrin- and heparin-binding domain at the N-terminal of plasma fibronectin prevented radiolabeled hyaluronan from binding to fibronectin; likewise, the isolated N-terminal fragment, coupled with Sepharose 4B, bound to hyaluronan in columns. We conclude that hyaluronan binds to a fibrin- and heparin-binding domain at the N-terminal of plasma fibronectin and facilitates the attachment of epithelial cells. © 1994 wiley-Liss, Inc.     

Basal lamina glycoproteins laminin and type IV collagen are assembled into a fine-fibered matrix in cultures of a teratocarcinoma-derived endodermal cell line

Extracellular matrix glycoproteins synthesized and deposited by a mouse teratocarcinoma-derived endodermal cell line (PYS-2) in culture were analysed by metabolic labelling and immunochemical methods, and the matrix structure was studied by immunofluorescence and electron microscopy. PYS-2 cells secreted two major high-molecular weight glycoproteins, laminin and type IV collagen, which were deposited in apparently unprocessed form under the cells into a lamellar matrix composed of a loose network of fine fibrils and attached dense grains. The cells did not synthesize detectable amounts of fibronectin, but the matrix was found to bind fibronectin from the culture medium. The matrix structure was sensitive to bacterial collagenase indicating a role for type IV collagen in matrix integrity. The PYS-2 matrix which contains defined basal lamina glycoproteins provides possibilities for in vitro studies on the organization of deposited basal lamina components.     

The influence of exogenous fibronectin on blood granulocyte adherence to vascular endothelium in vitro

Fibronectin is a major cell surface and extracellular matrix glycoprotein. It binds to a variety of substrata and supports the attachment and spreading of a number of cell types. We have found that purified human plasma fibronectin can also support blood granulocyte adhesion to cultured human umbilical vein endothelial cells. This activity is protected by treatment of the fibronectin with a sulphhydryl-containing agent. The effect of granulocyte attachment was observed at fibronectin concentration of 100 ng/ml with maximum effect at a concentration of 10 μg/ml. The attached granulocytes retained a rounded appearance, compared with the flattening that occurs on attachment to plastic. Granulocytes attached poorly to cultured human vascular smooth muscle cells and no enhancement occurred when fibronectin was added. Immunofluorescence microscopy using monospecific rabbit anti-human fibronectin demonstrated that the sulphhydryl-treated fibronectin accumulated on the endothelial cell surface, forming aggregates on the apical surface by 3 h of continued incubation. Washed, cultured endothelial cells not exposed to fibronectin or exposed to untreated purified plasma fibronectin did not demonstrate an aggregation of cell-surface fibronectin.     

Carbohydrate specific binding of fibronectin to Vibrio cholerae cells

Cells of 10 strains of Vibrio cholerae were grown on Trypticase Soy Broth and were tested for different surface porperties such as expression of surface haemagglutinins, cell-surface hydrophobicity and binding to 3 connective tissue proteins: fibronectin, type II collagen and fibrinogen.All strains bound fibronectin and one selected strain was shown to bind in a time-dependent and saturable manner.The binding of ¹²⁵I-labelled fibronectin could be completely inhibited by unlabelled fibronectin, and also partly by some other glycoproteins. Mannose inhibited binding of fibronectin up to 60%. The data indicate that carbohydrate structures within the 40 kDa (gelatin binding) and 105 kDa (cell binding) fragments of fibronectin are recognized by lectins on V. cholerae. The binding of collagen or fibrinogen was low or negligible.     

Basement membrane formation and lung cell differentiation in vitro

In high density cultures of mouse fetal lung cells, so-called "mass cultures", development of organoid structures, formation of a basement membrane (BM), and differentiation of pneumocytes type II occur accompanied by synthesis and secretion of lamellar bodies. The relationship between the formation of a BM, on the one hand, and morphogenesis as well as differentiation of pneumocytes type II, on the other hand, has been investigated by use of antibodies against BM components in the lung mass culture. It is shown here that anti-laminin antibodies prevented BM formation, but morphogenesis and pneumocyte differentiation occurred as in untreated cultures. Short-term treatment with the antibody revealed that the BM is formed only during the first 2 to 3 days in vitro. Already formed BM could not be removed by anti-laminin. Anti-collagen type IV antibodies showed no effect in the lung mass culture except for a stronger staining of the BM. Anti-BM-1 antibodies caused no changes in morphogenesis, cell differentiation and BM formation either, but the mesenchymal intercellular space exhibited a dark staining, which is probably due to antigen-antibody complexes. The results obtained with anti-laminin antibodies indicate that a BM is not necessary for lung cell differentiation in vitro.     

Syndecan-4 binding to the high affinity heparin-binding domain of fibronectin drives focal adhesion formation in fibroblasts

Cell adhesion to extracellular matrix involves signaling mechanisms which control attachment, spreading and the formation of focal adhesions and stress fibers. Fibronectin can provide sufficient signals for all three processes, even when protein synthesis is prevented by cycloheximide. Primary fibroblasts attach and spread following integrin ligation, but do not form focal adhesions unless treated with a heparin-binding fragment of fibronectin (HepII), a peptide from this domain, or phorbol esters to activate protein kinase C. Syndecan-4 heparan sulfate proteoglycan is a transmembrane component present together with integrins in focal adhesions. Syndecan-4 binds and activates protein kinase Calpha, whose activity is needed for focal adhesion formation. We now report that the glycosaminoglycan chains of syndecan-4 bind recombinant HepII and it is incorporated into forming focal adhesions.     

Basement membrane proteoglycans: from cellar to ceiling

The biology of basement membrane proteoglycans extends far beyond the original notion of anionic filters. These complex molecules have dual roles as structural constituents of basement membranes and functional regulators of several growth-factor signalling pathways. As such, they are involved in angiogenesis and, consequently, in tumour progression and their partial or total absence causes several congenital defects that affect the musculoskeletal, cardiovascular and nervous systems. New findings indicate a potential functional coupling between the intricate make-up of basement membrane proteoglycans and their ability to control important biological processes.     

Force-induced unfolding of fibronectin in the extracellular matrix of living cells

Whether mechanically unfolded fibronectin (Fn) is present within native extracellular matrix fibrils is controversial. Fn extensibility under the influence of cell traction forces has been proposed to originate either from the force-induced lengthening of an initially compact, folded quaternary structure as is found in solution (quaternary structure model, where the dimeric arms of Fn cross each other), or from the force-induced unfolding of type III modules (unfolding model). Clarification of this issue is central to our understanding of the structural arrangement of Fn within fibrils, the mechanism of fibrillogenesis, and whether cryptic sites, which are exposed by partial protein unfolding, can be exposed by cell-derived force. In order to differentiate between these two models, two fluorescence resonance energy transfer schemes to label plasma Fn were applied, with sensitivity to either compact-to-extended conformation (arm separation) without loss of secondary structure or compact-to-unfolded conformation. Fluorescence resonance energy transfer studies revealed that a significant fraction of fibrillar Fn within a three-dimensional human fibroblast matrix is partially unfolded. Complete relaxation of Fn fibrils led to a refolding of Fn. The compactly folded quaternary structure with crossed Fn arms, however, was never detected within extracellular matrix fibrils. We conclude that the resting state of Fn fibrils does not contain Fn molecules with crossed-over arms, and that the several-fold extensibility of Fn fibrils involves the unfolding of type III modules. This could imply that Fn might play a significant role in mechanotransduction processes.     

Extracellular matrix fibers containing fibronectin and basement membrane heparan sulfate proteoglycan coalign with focal contacts and microfilament bundles in stationary fibroblasts

Double-label immunofluorescence microscopy and immunoelectron microscopy were performed on stationary cultures of Nil 8 fibroblasts to determine if fibronectin and basement membrane heparan sulfate proteoglycans play coordinated roles in cell-to-substrate adhesion. Relationships between subcellular matrix fibers containing fibronectin plus proteoglycan, and focal contacts associated with microfilament bundles, were studied simultaneously using interference reflection microscopy, differential interference contrast microscopy, and immunofluorescence microscopy. Cells maintained in 0.3% FBS were doubly stained with monospecific anti-fibronectin IgG and antibodies against a basement membrane proteoglycan purified from the EHS (Engelbreth-Holm-Swarm) tumor. Coincident patterns of fibronectin and proteoglycan-containing fibers were found to codistribute with focal contacts and microfilament bundles in both early (6-h) and late (24-h) cultures. The early cells showed doubly-stained fibers colinear with substrate adhesion sites in 43% of the sample, while 100% of the later cells exhibited these coaligned matrix-cytoskeletal attachment complexes. Immunoelectron microscopy showed that both of these antigens were situated in the same type of extracellular matrix fiber that appeared to be loosely associated with the cell surface membrane. We hypothesize that the appearance of proteoglycan in subcellular matrix fibers of these fibroblasts might stabilize fibronectin-containing cell-to-substrate contacts.     

The role of integrin binding sites in fibronectin matrix assembly in vivo

The extracellular matrix (ECM) glycoprotein fibronectin (FN) requires the help of cells to assemble into a functional fibrillar matrix, which then orchestrates the assembly of other ECM proteins and promotes cell adhesion, migration and signalling. Fibrillogenesis is initiated and governed by cell surface integrins that bind to specific sites in the FN molecule. Recent studies identified novel integrin binding sites in FN that can also participate in FN fibril formation and in morphogenetic events during development.     

Intracellular localization of basement membrane precursors in the endodermal cells of the rat parietal yolk sac. I. Ultrastructure and phosphatase activity of endodermal cells

The parietal layer of the rat yolk sac includes a 5 microliter thick sheet known as Reichert's membrane that exhibits properties of basement membranes. Its inner side is lined by a single layer of loosely distributed cells referred to as endodermal cells. Both Reichert's membrane and endodermal cells were examined at 13-14 days' gestation with emphasis on the ultrastructure of the Golgi apparatus, the identification of its component parts by specific phosphatase activities, and its possible role in the cells' secretory process. Reichert's membrane is composed of a series of stacked layers similar to basal laminae and composed of a network of fibrils with a diameter of 2-8 nm along which dots are located at irregular intervals. The endodermal cells contain the usual organelles, including interconnected rough endoplasmic reticulum (rER) cisternae and a prominent Golgi apparatus. With the help of phosphatase reactions, the stacks of Golgi saccules were divided into a) "phosphatase-free" saccules, the first ones on the cis or forming side, b) one or two "intermediate" saccules in the middle of the stacks, containing nicotinamide adenine dinucleotide phosphatase activity, c) one or two "last" saccules rich in thiamine pyrophosphatase activity on the trans or mature side, and d) continuing beyond the trans side, the GERL element displaying acid phosphatase activity. The latter is associated with profiles equally rich in acid phosphatase and tentatively considered to be prosecretory granules. Finally, the ectoplasm adjacent to Reichert's membrane displays large, acid phosphatase-containing structures tentatively considered to be secretory granules. Thus, the extensive rER network, the well-compartmentalized Golgi apparatus, and the presence of structures which may be prosecretory and secretory granules indicate that the endodermal cells are well-equipped for the secretion of the components of Reichert's membrane.     

Embryonal carcinoma cells adhere preferentially to fibronectin and laminin but their endodermal differentiation leads to a reduced adherence to laminin

F9 and PC13 embryonal carcinoma (EC) cells adhered rapidly to growth substrata coated with fibronectin or laminin. When F9 cells were induced to differentiate into visceral or parietal endoderm-like cells, their ability to adhere to laminin diminished, but their adherence to fibronectin remained unchanged. Correspondingly, permanently differentiated teratocarcinoma-derived endoderm cells (PYS-2 and PSA-5e) adhered markedly less efficiently to laminin than to fibronectin. F9 cells adhered to proteolytic fibronectin fragments containing the cell-binding site but not to fragments containing gelatin- or heparin-binding sites. They also adhered slowly to gelatin, but this adhesion was completely blocked by cycloheximide. The results show that the teratocarcinoma stem cells may have specific mechanisms mediating adhesion to fibronectin and laminin and that endodermal differentiation leads to a reduction in their capacity to adhere to laminin but not to fibronectin.     

Assembly of exogenous fibronectin by fibronectin-null cells is dependent on the adhesive substrate

The role of endogenously synthesized fibronectin (FN) in assembly was studied with cells lacking or expressing FN. Cells were cultured as homogeneous or mixed populations on surfaces coated with different matrix proteins. Compared with FN-expressing cells, FN-null cells poorly assembled exogenous plasma FN (pFN) when adhered to vitronectin or the recombinant cell-binding domain (III(7-10)) of FN. Vitronectin had a suppressive effect that was overcome by co-adsorbed pFN or laminin-1 but not by soluble FN. In co-cultures of FN-expressing cells and FN-null cells, endogenous FN was preferentially assembled around FN-expressing cells regardless of the adhesive ligand. If the adhesive ligand was vitronectin, exogenous pFN assembled preferentially around cells expressing cellular FN or recombinant EDa- or EDa+ FN. In co-cultures on vitronectin of FN-null cells and beta(1) integrin subunit-null cells, fibrils of cellular FN and pFN were preferentially deposited by FN-null (beta(1)-expressing) cells immediately adjacent to (FN-secreting) beta(1)-null cells. In co-cultures on vitronectin of FN-null cells and beta(1)-null cells expressing a chimera with the extracellular domain of beta(1) and the cytoplasmic domain of beta(3), preferential assembly was by the chimera-expressing cells. These results indicate that the adhesive ligand is a determinant of FN assembly by cells not secreting endogenous FN (suppressive if vitronectin, non-suppressive but non-supportive if III(7-10), supportive if pFN or laminin-1) and suggest that efficient interaction of freshly secreted cellular FN with a beta(1) integrin, presumably alpha(5)beta(1), substitutes for integrin-mediated adherence to a preformed matrix of laminin-1 or pFN to support assembly of FN.     

Recombinant 70-kDa protein from the amino-terminal region of rat fibronectin inhibits binding of fibronectin to cells and bacteria

Binding of fibronectin to substrate-attached cells and to Staphylococcus aureus is mediated by the amino-terminal 70-kDa portion of fibronectin. The 70-kDa amino-terminus is composed of nine type I and two type II internal homology units, each containing two intrachain disulfide bonds. The exact structural features of the 70-kDa amino-terminus that are necessary for binding to cells and bacteria are not known. We characterized a recombinant 70-kDa protein from the amino-terminus of rat fibronectin using a baculovirus expression system. Recombinant 70-kDa (r70kDa) protein was easily purified in high amounts from the conditioned medium by affinity chromatography on gelatin-agarose. Secretion was much less when N-linked glycosylation was blocked by tunicamycin. Like the native fragment, the r70kDa protein contains intrachain disulfide bonds. In addition, the r70kDa protein was indistinguishable from the nonrecombinant 70-kDa fragment in its ability to compete for binding sites on fibroblasts and S. aureus. Thus, the r70kDa protein retains the important functional characteristics of the native fragment. This expression system is well adapted to studying the structural features important for the interaction of 70-kDa protein with cells.