Inhibition of fibronectin receptor function by antibodies against baby hamster kidney cell wheat germ agglutinin receptors

Previous studies suggest that the baby hamster kidney (BHK) cell fibronectin receptor is also a wheat germ agglutinin receptor (WGA-R). To analyze this possibility further, IgG and Fab fragments of antibodies produced against a BHK cell WGA-R preparation were tested to determine their effects on cell adhesion mediated by fibronectin, wheat germ agglutinin, concanavalin A, and polycationic ferritin. The WGA-R preparation was isolated by octylglucoside extraction of BHK cells followed by chromatography of the extract on WGA-agarose. The antibodies against the WGA-R preparation reacted primarily with polypeptides of molecular weights 48, 61, 83, 105, 120, 165, 210, and 230 kilodaltons (kdaltons). It was concluded that the antibodies interfered with BHK cell fibronectin receptors on the basis of the ability of anti-WGA-R IgG or Fab fragments to (a) inhibit cell spreading on fibronectin-coated substrata; (b) cause rounding and detachment of cells previously spread on fibronectin-coated substrata; and (c) inhibit binding of fibronectin-coated latex beads to the cells. Antibody activity was blocked by treatment of anti-WGA-R with the WGA-R preparation or by absorption of anti-WGA-R with intact BHK cells. The antibodies also appeared to prevent coupling of ligand-receptor complexes (involving concanavalin A or polycationic ferritin) with the cytoskeleton. Finally, cell rounding and detachment caused by the antibodies were found to require metabolic energy since it did not occur in the presence of azide or at 4 degrees C.     

Role of integrin receptors in manganese-dependent BHK cell spreading on albumin-coated substrata.

In manganese-containing medium, tissue cells can spread on albumin and other substrata typically nonadhesive for cells in calcium/magnesium-containing medium. To learn whether integrin receptors play a role in Mn-dependent adhesion, we tested the effects of RGD peptides and polyclonal anti-fibronectin receptor antibodies on BHK cell spreading on fibronectin and albumin-coated substrata. In Ca/Mg-containing medium on fibronectin substrata, the RGD-related peptides GRG-DSP and GRGDS but not RGDS inhibited cell spreading. In Mn-containing medium, spreading on albumin was inhibited by GRGDSP and GRGDS and also by RGDS. GRGESP, on the other hand, did not inhibit cell spreading under any condition tested. Antibodies directed against fibronectin receptors also inhibited Mn-dependent cell spreading on albumin substrata, but higher levels of antibody were required than were necessary to inhibit Ca/Mg-dependent spreading on fibronectin. On the basis of these results, we suggest that integrin receptors, but probably not fibronectin receptors, mediate Mn-dependent BHK cell spreading on albumin.     

Effects of plant lectins on the adhesive properties of baby hamster kidney cells

We studied the effects of different lectins on the adhesive properties of baby hamster kidney (BHK) cells. The purpose of these studies was to learn more about the cell surface receptors involved in cell adhesion. Three adhesive phenomena were analyzed: 1) the adhesion of BHK cells to lectin-coated substrata; 2) the effects of lectins on the adhesion of cells to substrata coated by plasma fibronectin (pFN); and 3) the effects of lectins on the binding of pFN-coated beads to cells. Initial experiments with fluorescein-conjugated lectins indicated that concanavalin A (Con A), ricinus communis agglutinin I (RCA I), and wheat germ agglutinin (WGA) bound to BHK cells but peanut agglutinin (PNA), soybean agglutinin (SBA), and ulex europaeus agglutinin I (UEA I) dod not bind. All three of the lectins which bound to the cells promoted cell spreading on lectin substrata, and the morphology of the spread cells was similar to that observed with cells spread on pFN substrata. Protease treatment of the cells, however, was found to inhibit cell spreading on pFN substrata or WGA substrata more than on Con A substrata or RCA I substrata. In the experiment of cells with Con A or WGA inhibited cell spreading on pFN substrata, but RCA I treatment had no effect. Finally, treatment of cells with WGA inhibited binding to cells of pFN beads, but neither Con A nor RCA I affected this interaction. These results indicate that the lectins modify cellular adhesion in different ways, probably by interacting with different surface receptors. The possibility that the pFN receptor is a WGA receptor is discussed.     

Induction of cell spreading by substratum-adsorbed ligands directed against the cell surface

Studies were carried out to compare the spreading of baby hamster kidney (BHK) cells, which occurs by an interaction between the cells and a specific serum glycoprotein (ASF) adsorbed onto the substratum surface, with the spreading of BHK cells that occurs by an interaction between the cells and substrata coated with ligands directed at various cell surface determinants. The ligands tested were polycationic ferritin, concanavalin A (ConA) and antibody directed against BHK plasma membranes. Cell spreading onto ASF and ligand-coated substrata were similar even though different cell surface components were apparently involved. The similarities were:

1. 1. The shape of the spread cells.

2. 2. The inhibition of cell spreading by conditions that interfere with metabolic activity, block free sulfhydryl groups, or interfere with microtubules and microfilaments.

3. 3. The similar reorganization of certain cell surface antigenic determinants during cell spreading onto any of the substrata.

The results indicate that cell spreading is a general cellular response to specific cell-substratum interactions but does not depend upon binding between a unique cell surface receptor and the substratum.     

Collagen can modulate cell interactions with fibronectin

We have examined the effects of soluble collagen on the function of fibronectin in baby hamster kidney (BHK) cells. Collagen and its purified alpha1(l) chain noncompetitively inhibited cell spreading on substrates precoated with fibronectin or a 75,000-D cell-binding fragment of fibronectin. Neither preincubation of cells with collagen followed by washing nor the addition of collagen to previously spread cells had any inhibitory effect on cell spreading, which indicates a requirement for the concurrent presence of collagen during the process of spreading. Treatment of collagen or alpha1(l) chain with collagenase abolished the inhibitory effect on fibronectin-mediated cell spreading. However, direct attachment of BHK cells to fibronectin-coated or 75,000-D fragment-coated substrates was not inhibited by collagen or by the alpha1(l) chain. Moreover, the binding of [3H]fibronectin or the 3'-75,000-D fragment to cell surfaces was not inhibited by the presence of soluble collagen, whereas soluble fibronectin inhibited binding. Although the binding of [3H]fibronectin-coated beads to BHK cell surfaces was also not inhibited by collagen, the phagocytosis of such beads was inhibited by the presence of collagen. On the other hand, soluble fibronectin partially inhibited the binding of fibronectin-coated beads but did not inhibit phagocytosis of the beads that did bind. The mechanism of the inhibition of fibronectin function by collagen and the possible interactions of two different kinds of receptors on the cell surface are discussed.     

STUDIES ON CELL ADHESION : III. Adhesion of Baby Hamster Kidney Cells

Normal and transformed baby hamster kidney (BHK) cells attach to Falcon polystyrene with the same first order rate constant. The longer the cells are attached to the bottles, the more difficult they are to remove. Sulfhydryl (—SH) binding reagents inhibit both the attachment of BHK cells and the increase in adhesive strength of attached cells. Attached BHK cells bind fewer molecules of [1-¹⁴C]N-ethylamleimide (an —SH binding reagent) than do suspended cells. Incubation of cells with high concentrations of trypsin results in a reversible loss of cell adhesiveness. The recovery of adhesiveness of trypsin-treated cells is inhibited by cycloheximide.     

Morphology of epidermoid carcinoma A431 cells spread on immobilized ligands

Cell interaction with extracellular matrix is a multi-step process characterized by cell attachment to substrata with subsequent cell spreading accompanied by actin cytoskeleton and cellular membrane receptor reorganization. It has been shown elsewhere that epidermoid carcinoma A431 cells, spread on solid substrata coated with fibronectin, laminin-2/4 or antibodies to EGF receptor, form specific actin filament structures typical for each particular ligand. Here quantitative analysis of heterogeneous A431 cell population spread on the above ligands has been reported. Cells were subdivided into morphological classes, according to their shape and actin filament structure, and the relationship among classes under various experimental conditions were quantitatively estimated for every ligand. We studied the influence of cell detachment pattern, short-term and long-term starvation, and cell incubation in suspended state in the medium before plating on the cell population composition. It was possible to recognize the modal morphological class of cells with typical actin cytoskeleton structure dominating for the ligand in the population. Long-term starvation and incubation in suspension before cell spreading are considered as the crucial experimental parameters leading to dramatic changes in cell population.     

Binding of plasma fibronectin to the surfaces of BHK cells in suspension at 4 °C

Plasma fibronectin is shown by several different criteria to bind to suspended BHK cells if the binding incubations are carried out at 4 °C. In indirect immunofluorescence experiments, fibronectin bound to suspended BHK cells at 4 °C in a punctate distribution over the entire cell surfaces. Little binding, however, was detected on cells incubated with fibronectin at 37 °C. The fibronectin bound to the cells at 4 °C was functionally active, since these cells subsequently were able to spread on tissue culture dishes in protein-free medium, unlike cells preincubated with fibronectin at 37 °C or in the absence of fibronectin. Also, the cell surface receptors for soluble fibronectin and fibronectin-coated beads appeared to be similar, since cells preincubated with fibronectin at 4 °C subsequently bound fewer fibronectin-coated beads than control cells. In biochemical studies with radiolabeled fibronectin, binding of fibronectin to the cells was shown to increase with incubation time up to 4 h. In competition experiments with unlabeled fibronectin, 30% of the binding of radiolabeled fibronectin could be inhibited.     

Functional differences in the interactions of glycosylation-deficient cell lines with fibronectin, laminin, and type IV collagen

Fibronectin isolated from the conditioned medium of monolayer cultures of baby hamster kidney (BHK) cells and several ricin-resistant (Ric) mutants derived from them express differences in N-glycosylation. The asparagine-linked oligosaccharides of BHK cell-derived fibronectin consist largely of complex chains, whereas hybrid and/or high-mannose chains are present in the fibronectins of mutant cell lines. The fibronectins exhibiting different glycosylation patterns are incorporated to similar extents into the cell-layer of human skin fibroblasts. In contrast, mutant cells retain significantly less endogenously produced fibronectin than BHK cells and also incorporate less human cellular fibronectin into a pericellular matrix. In vitro adhesion assays show that mutant cells attach to and spread relatively poorly on fibronectin-or type IV collagen-coated substrata but interact as well as do BHK cells with a laminin substratum. These results indicate that asparagine-linked oligosaccharides of fibronectin are not required for the binding and incorporation of the molecule into cell layers, but, as constituents of other cellular glycoproteins, they do modulate the ability of BHK cells to interact with some matrix components.     

Fibroblast spreading and phagocytosis: similar cell responses to different-sized substrata

Experiments were carried out to test the hypothesis that cell spreading and phagocytosis are similar cell responses to different-sized substrata. The following morphological and biochemical studies provided evidence for this supposition. Cells phagocytosed 1.09-micron and 5.7-micron latex beads, but were unable to completely ingest 15.8-micron or 25.7-micron beads. With the larger beads, the cells spread around the bead surfaces with an appearance typical of cells spread on culture dishes. Biochemical studies with cytochalasin D, azide, and iodoacetate, as well as temperature-dependence studies, demonstrated similar responses of cell spreading and phagocytosis to these treatments. Similar cell surface receptors were involved in cell spreading and phagocytosis based upon experiments using antibodies to baby hamster kidney (BHK) cell wheat germ agglutinin receptors. And finally, BHK cell variants with defective plasma fibronectin (pFN) receptors were unable to spread on pFN-coated dishes or ingest pFN-coated beads. Evidence also is presented concerning the "contact" process in cell adhesion. It was found that azide and low temperature inhibited cell attachment per se but did not block fibronectin-receptor interactions based upon cell binding of pFN-coated beads. A possible explanation for the contact process is presented based upon the resistance of cells and beads to shear forces.     

Degree of cell spreading on a substrate as the factor determining the nature of cell microrelief in suspension

Quantitative ratio of various types of cell surface microrelief was determined in suspensions prepared from mouse monolayer cultures of embryo fibroblasts grown on different solid substrates: with high (Falcon) or low poly(2-hydroxyethylmethacrylate) adhesiveness; with flat or cylindrical (53-mu curvature radius) surfaces (polyvinylchloride). The electron microscopy revealed that poorly spread cells (on low adhesive or cylindrical substrata) in suspensions had the microvillous surface relief much more often than the cells brought to suspension from highly adhesive or flat substrata. Thus, the lower the degree of cell spreading on the substratum, the higher the probability for the cell to acquire the microvillous relief in suspended state. The microvillous relief of transformed cells in suspensions is, probably, due to their poor spreading on substrata in the monolayer cultures.     

Inhibition of neutrophil spreading during adhesion to fibronectin reveals formation of long tubulovesicular cell extensions (cytonemes)

Human neutrophils developed long thin tubulovesicular extensions (cytonemes) upon adhesion to fibronectin-coated substrata, when spreading was blocked. We observed extension formation when neutrophils were plated to fibronectin-coated substrata in Na(+)-free extracellular medium or in the presence of drugs capable of inhibiting spreading: 4-bromophenacyl bromide, N-ethylmaleimide, 7-chloro-4-nitrobenz-2-oxa-1,3-diazole, and cytochalasin D. Addition of Na(+) ions or washing of inhibitors restored neutrophil spreading. Phase-contrast and scanning electron microscopy revealed two types of extensions: (1) highly dynamic, flexible tubulovesicular extensions with unattached tips 0.2-0.4 microm in diameter, which can achieve 70-80 microm in length during 20 min, and (2) thinner straight extensions with flattened tips, which were formed in the presence of phorbol 12-myristate 13-acetate and connected cells to substratum or to the neighboring cells several cell diameters away. The latter may have derived from the former through tension after attachment of the tips. Spreading and extension formation may represent two states of the cell adhesive and communicative mechanism.     

Metabolic regulation of neutrophil spreading, membrane tubulovesicular extensions (cytonemes) formation and intracellular pH upon adhesion to fibronectin

Circulating leukocytes have a round cell shape and roll along vessel walls. However, metabolic disorders can lead them to adhere to the endothelium and spread (flatten). We studied the metabolic regulation of adhesion, spreading and intracellular pH (pHi) of neutrophils (polymorphonuclear leukocytes) upon adhesion to fibronectin-coated substrata. Resting neutrophils adhered and spread on fibronectin. An increase in pHi accompanied neutrophil spreading. Inhibition of oxidative phosphorylation or inhibition of P- and F-type ATPases affected neither neutrophil spreading nor pHi. Inhibition of glucose metabolism or V-ATPase impaired neutrophil spreading, blocked the increase in the pHi and induced extrusion of membrane tubulovesicular extensions (cytonemes), anchoring cells to substrata. Omission of extracellular Na(+) and inhibition of chloride channels caused a similar effect. We propose that these tubulovesicular extensions represent protrusions of exocytotic trafficking, supplying the plasma membrane of neutrophils with ion exchange mechanisms and additional membrane for spreading. Glucose metabolism and V-type ATPase could affect fusion of exocytotic trafficking with the plasma membrane, thus controlling neutrophil adhesive state and pHi. Cl(-) efflux through chloride channels and Na(+) influx seem to be involved in the regulation of the V-ATPase by carrying out charge compensation for the proton-pumping activity and through V-ATPase in regulation of neutrophil spreading and pHi.     

Gangliosides as receptors for fibronectin?: Comparison of cell spreading on a ganglioside-specific ligand with that on fibronectin

We have examined the possibility that gangliosides act as the cell surface receptor for fibronectin, as previously suggested by the data of Kleinman et al. (Proc natl acad sci US 76 (1979) 3367), using three different approaches.

1. 1. Gangliosides inhibited the spreading of both CHO and BHK cells on fibronectin-coated substrates. 50% inhibition of cell spreading was produced by 1.0 and 3.5 × 10⁻⁵ M di- and trisialogangliosides respectively, although monosialogangliosides were less effective inhibitors. The inhibition was apparently due to an interaction of gangliosides with fibronectin and not due to a direct effect of gangliosides on the cells.

2. 2. Using anti-fibronectin antibodies, ¹²⁵I-labelled protein A, and gangliosides adsorbed to polystyrene tubes, we have provided direct evidence that fibronectin will bind to gangliosides. However, the interaction is apparently of low affinity compared with binding of cholera toxin to gangliosides.

3. 3. We have compared the ability of BALB/c 3T3 cells to spread on fibronectin with that on substrates coated with a ganglioside-specific ligand, cholera toxin B-subunit. Cells plated onto fibronectin-coated substrates rapidly (within 60 min) adopted a well spread morphology, whereas spreading on substrates coated with the toxin B-subunit was less extensive even after 2 h. In addition, the organization of F-actin within cells spread on the two types of substrate was also quite different.

We conclude that the interaction of cells with fibronectin may well be influenced by membrane-bound gangliosides. It is unlikely, however, that binding of fibronectin to such gangliosides can lead to the cytoskeletal reorganization which is characteristic of cells spread on fibronectin.     

Activation of macrophage-like cells by multiple grooved substrata. Topographical control of cell behaviour.

We studied the influence of substrata topography on the behaviour of murine P388D1 macrophage cell line. Cells were plated on plain fused silica substrata or substrata with microfabricated grooves of varying depth and width. Cell spread area, elongation, orientation and F-actin content were measured on plain substratum and 6 sets of gratings. The speed and persistence of cell movement were also studied. We found that patterned substrata substantially activated cell spreading and elongation and significantly increased the persistence and speed of cell movement, shallow grooves being more effective than deep ones. The contact of cells with micropatterned substrata significantly increased the F-actin content in cells. The sensitivity of LPS (lipopolisaccharide) stimulated and unstimulated macrophages to topographical cues was also compared.     

Cell spreading factor. Occurrence and specificity of action.

A factor required for spreading of substratum-attached baby hamster kidney cells (BHK), Chinese hamster ovary (CHO) cells, HeLa cells, and L cells has been isolated and purified from fetal calf serum. A similar factor has also been found in calf, porcine, human, rabbit, and chicken sera. The spreading factor was active when adsorbed to the substratum and prior adsorption of other proteins prevented cell spreading, regardless of the addition of spreading factor or unfractionated serum to the incubation medium. Antibody against the fetal calf spreading factor inhibited the spreading activity associated with unfractionated fetal calf serum and also the spreading activity associated with calf serum and porcine serum. In model system studies it was found that antibody against BHK cell surfaces induced cell spreading when the antibody was adsorbed to the substratum; when it was present in the incubation medium as well as on the substratum, cell spreading was not observed. The data are discussed in terms of the hypothesis that there is a specific serum factor which adsorbs to the substratum surface and is thereby activated, and which then forms the target for certain cell surface receptors. Interaction between adsorbed-activated factor and cell surface receptors leads to cell spreading.     

Glycosphingolipids in detergent-insoluble substrate attachment matrix (DISAM) prepared from substrate attachment material (SAM) : Their possible role in regulating cell adhesion

The glycosphingolipids isolated from the detergent-insoluble material (DIM) of whole cells as well as from a similar detergent-insoluble substrate attachment matrix (DISAM) have been investigated in comparison with the glycosphingolipids of whole cells. The proportion of glycolipids in the total lipid extract was enriched in the DISAM as well as DIM fractions as compared to whole cells. The ratio of ganglioside (GM₃) to neutral glycolipids was also higher in the DISAM fractions than in whole cells. The radioactivity incorporated into DISAM glycolipids of BHK cells, metabolically labeled with radioactive glucosamine, was greater in confluent cells than in sparsely growing cells; however, label incorporation into glycolipids of the DISAM fraction of BHKpy cells was 2–3-fold higher than that of confluent BHK cells, although the chemical quantity of GM₃ in whole cells was much lower in BHKpy cells than in BHK cells. In order to confirm the enhanced label in DISAM glycolipids of BHKpy cells by other procedures, the labeled cells were detached by EGTA, washed, and reattached on plates. The amount of label in DISAM glycolipids of the reattached matrix of BHKpy cells was much higher than that of BHK cells.Cell spreading and cell attachment on plastic plate were inhibited by inclusion of GM₃ in the medium. These data suggest that: (i) glycolipids, particularly GM₃, at the cell attachment site have different metabolic activity from those of whole cells; the label in glycolipids goes preferentially into cell attachment sites, and may have some functional role in regulating cell attachment of BHK cells; (ii) metabolic activity and turnover of GM₃ in cell attachment sites of confluent cells are higher than actively growing cells, yet those of transformed cells are much higher than any state of non-transformed cells.     

Spreading and orientation of epithelial cells on grooved substrata

The spreading and orientation of epithelial (E) cells was studied on titanium-coated grooved substrata by light, transmission (TEM) and scanning electron microscopy (SEM). Vertical-walled grooves and V-shaped grooves, 3-60 microns deep, were produced in silicon wafers by micromachining, a process which was developed for the fabrication of micro-electronic components, and the grooved substrata were replicated in Epon. Photolithography was used to prepare photoresist-based and silicon dioxide-silicon substrata with grooves of approximately 2 and approximately 0.5 micron deep, respectively. Cell clusters were markedly oriented by all the grooved substrata examined, with the orientation index being highest for substrata with grooves of the smallest repeat spacing. Time-lapse cinemicrography showed that the grooves directed the migration of E cells, but the control was not absolute, as some cells crossed over the ridges and descended into the grooves. The 0.5 micron grooves appeared less effective than the deeper grooves in directing cell locomotion. SEM and TEM of E cells spreading on the grooved substrata demonstrated that cell processes, including lamellae and filopodia, were capable of bending around and closely adapting to groove edges. E cells did not flatten as extensively on a substratum with 22 microns deep V-shaped grooves as on a smooth surface, although some cells were markedly elongated. One mechanism proposed to explain contact guidance of fibroblasts is that linear elements of the locomotory system, such as microfilament bundles, are unable to operate when bent. The observed flexibility of epithelial cell processes and the ability of substrata with shallow grooves to orient E cells indicate that contact guidance of E cells on micromachined substrata cannot be explained by the mechanical stiffness of long linear cytoskeletal elements.     

Interaction of bovine epithelial lens (BEL) cells with extracellular matrix (ECM) and eye-derived growth factor (EDGF): I. Effects on short-term adhesiveness and on long-term organization of the culture

A growth factor (EDGF) derived from the retina controls the proliferation and shape of adult bovine epithelial lens (BEL) cells in vitro as well as extracellular matrix (ECM) assembly. In order to analyse this mechanism and the specificity of the interactions between BEL cells and the extracellular matrix we have investigated the adhesion and growth of BEL cells on various substrata (fibronectin, laminin, ECM). BEL cells treated with EDGF adhered more slowly to plastic Petri dishes than untreated cells, in part due to EDGF inhibition of fibronectin deposition. The untreated BEL cells spread less well on ECM or laminin than on fibronectin-coated plastic. The preferential adhesiveness of BEL cells on fibronectin vs laminin was confirmed by attachment experiments performed on replicas of SDS-PAGE of these proteins. However, in long-term cultures, 8 days after seeding, BEL cells were very differently arranged on plastic or on ECM. ECM by itself did not increase the proliferation rate but helped to restore an organized cell monolayer. BEL cells stimulated to grow on ECM by treatment with EDGF exhibited at least transiently contact inhibition producing a perfectly organized epithelium similar to the one observed in vivo. These results suggest specific interactions between ECM or ECM components with BEL cell that restrain excessive cell spreading and restore an original polarized phenotype of the cells seen in vivo.     

Topographical control of cell behaviour: II. Multiple grooved substrata

Electronics miniaturization techniques have been used to fabricate substrata to study contact guidance of cells. Topographical guidance of three cell types (BHK, MDCK and chick embryo cerebral neurones) was examined on grooved substrata of varying dimensions (4-24 microns repeat, 0.2-1.9 microns depth). Alignment to within 10 degrees of groove direction was used as our criterion for guidance. It was found that repeat spacing had a small effect (alignment is inversely proportional to spacing) but that groove depth proved to be much more important in determining cell alignment, which increased with depth. Measurements of cell alignment and examination by scanning electron microscopy showed that BHK cells and MDCK cells interacted differently with grooved substrata, and also that the response of MDCK cells depended on whether or not the cells were isolated or part of an epithelial cell island. Guidance by a multiple topographical cue is greater than could be predicted from cells' reactions to a single cue (Clark et al. Development 99: 439-448, 1987). Substratum topography is considered to be an important cue in many developmental processes. Cellular properties such as cytoskeletal organisation, cell adhesion and the interaction with other cells are discussed as being factors determining a cells susceptibility to topography.