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.     

The interaction of fibronectin fragments with fibroblastic cells

We have examined the interaction of the purified cell-binding domain of fibronectin with fibroblastic baby hamster kidney cells. When the cell-binding region of fibronectin is part of a large 75,000-dalton fragment, the direct binding of the tritium-labeled fragment to cells in suspension can be observed. There is a single class of 10(5) sites/cell with an apparent dissociation constant of 4 X 10(-7) M. When the cell-binding region is part of a smaller 11,500-dalton fragment, an interaction with cells can only be observed indirectly via inhibition assays. The apparent affinity of this fragment for the cell surface fibronectin receptor is low. This 11,500-dalton fragment competitively inhibits both the direct binding of soluble [3H]fibronectin to cells in suspension and the spreading of cells on fibronectin-coated substrates, suggesting that the fragment binds to the same receptor site as intact fibronectin. Possible models describing the mechanism of the interaction of fibronectin with its receptor are proposed.     

Incorporation of fluorescent gangliosides into human fibroblasts: mobility, fate, and interaction with fibronectin

Rhodamine- and fluorescein-labeled gangliosides were used as probes to investigate the distribution, dynamics, and fate of plasma membrane-bound gangliosides on cultured human fibroblasts. When sparse cultures of fibroblasts were incubated with the fluorescent ganglioside derivatives, their surfaces became highly fluorescent. The fluorescent gangliosides were taken up by the cells in a time- and temperature-dependent manner and were not removed from the cell surface by trypsin or serum. Thus, the gangliosides appeared to be stably incorporated into the lipid bilayer of the plasma membrane. Fluorescent photobleaching recovery measurements showed that the inserted gangliosides were free to diffuse in the plane of the membrane with a high diffusion coefficient of approximately 10(-8) cm2/s. When the ganglioside-treated cells were washed and incubated in fresh medium, the surface gangliosides became internalized with time, and localized in the perinuclear region of the fibroblasts. In dense cultures of fibroblasts, a large fraction of the fluorescent gangliosides were organized in a fibrillar network and were immobile on the time scale of fluorescent photobleaching recovery measurements. Using antifibronectin antibodies and indirect immunofluorescence, these gangliosides were found to co-distribute with fibrillar fibronectin. Thus, exogenous gangliosides appear to be stably inserted into the lipid bilayer of the plasma membrane and to diffuse freely in its plane as well as form a less mobile state with the fibrillar networks of fibronectin associated with the cells.     

Differential interaction of Xenopus embryonic cells with fibronectin in vitro

Two distinct cell types from the amphibian gastrula were compared with regard to their interactions in vitro with fibronectin (FN). Xenopus embryonic endoderm cells attach to FN substrates in a way characteristic of most cell types studied so far; that is, adhesion increases abruptly at a certain threshold concentration of FN, and maximal binding of cells already occurs at low FN concentrations (10 micrograms/ml). In contrast, embryonic ectodermal cells bind maximally to FN substrates only at unusually high concentrations of FN (200 micrograms/ml). This peculiar mode of attachment to FN has been characterized more closely. It is shown that the adhesion of ectodermal cells is modified by their interaction with a heparin-binding domain of the FN molecule. Furthermore, ectodermal cell adhesion increases very slowly with increasing FN concentrations. Despite these characteristic differences, both ectodermal and endodermal cells attach to the normal RGD cell-binding site of FN, as can be shown by competitive inhibition of adhesion by a hexapeptide containing the RGD sequence of amino acids.     

Disialyl gangliosides enhance tumor phenotypes with differential modalities

Sialic acid-containing glycosphingolipids, gangliosides are highly expressed in human cancer cells and regulate cell signals transduced via membrane microdomains. Generally, disialyl gangliosides enhance tumor phenotypes, while monosialyl gangliosides suppress them. In particular, gangliosides GD3 and GD2 are highly expressed in melanomas and small cell lung cancer cells, and their expression cause increased cell growth and invasion. In osteosarcomas, expression of GD3 and GD2 also enhanced cell invasion and motility, and caused increased phosphorylation of focal adhesion kinase and paxillin. In addition to focal adhesion kinase, Lyn kinase was also activated by GD3/GD2 expression, leading to the phosphorylation of paxillin. In contrast with melanoma cells, osteosarcomas showed reduced cell adhesion with increased phosphorylation of paxillin. Thus, increased expression of GD3/GD2 caused enhanced activation of signaling molecules, leading to distinct phenotypes between melanomas and osteosarcomas, i.e. increased and decreased adhesion activity. Thus, whole features of glycolipid-enriched microdomain/rafts formed in the individual cancer types seem to determine the main signaling pathway and biological outcome.     

The interaction of plasma fibronectin with fibroblastic cells in suspension

We have examined the interaction of soluble plasma [3H]fibronectin with fibroblastic cells in suspension. Fibronectin labeled by reductive methylation binds to baby hamster kidney cells in serum-free medium in a time-dependent manner at 4, 22, and 37 degrees C, with half-maximal binding occurring in 12-15 min at 22 degrees C. The binding is saturable and reversible. At least 90% of the cell-associated fibronectin is external to the plasma membrane, as judged by trypsin susceptibility of the bound radioactivity. Scatchard analysis of the concentration dependence of binding indicates the presence of a single class of binding sites, even at low input concentrations of fibronectin. There are approximately 5 +/- 1 X 10(5) sites/cell with an apparent dissociation constant of 8.0 +/- 0.5 X 10(-7) M; thus, the binding of soluble fibronectin to these cells is of moderate affinity. This putative fibroblast fibronectin receptor is resistant to trypsin in the presence of physiological concentrations of divalent cations but is susceptible to trypsin in the presence of 5 mM EDTA. Binding of 0.1 mg/ml [3H]fibronectin is 60-80% inhibited by 8 mg/ml unlabeled fibronectin and 95% inhibited by 1 mg/ml purified 75-kDa fibronectin cell-binding domain, but is unaffected by 1 mg/ml 44-kDa collagen-binding domain or 5 mg/ml ovalbumin. The binding parameters determined in this study further define the fibroblast cell-surface fibronectin receptor.     

The effect of gangliosides on the adhesive interaction of Neisseria meningitidis with human cells

The influence of a number of gangliosides and sialic acid on the adhesive interaction of meningococci and human cells have been studied. Sialic acid has been found to produce no influence on adhesion, and the preliminary treatment of meningococci with gangliosides or their preparations suppresses the capacity of meningococci for attachment to epithelial cells and erythrocytes. At the same time the degree of the inhibition of adhesion depends on the kind and concentration of gangliosides. On the contrary, after the treatment of target cells with gangliosides (1.25 micrograms/ml) the adhesion indices of meningococci with respect to these cells increase 5- to 8-fold. These data are indicative of the participation of gangliosides in the adhesive interaction of meningococci and human cells.     

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.     

The role of gangliosides in the interaction of a growth inhibitor with mouse LM cells

We have isolated and characterized glycopeptides, derived from mouse and bovine cerebral cortex cells, that inhibit protein synthesis and cell growth of normal but not transformed cells. The inhibitor binds to target cell surfaces, and gangliosides have previously been shown to influence cell sensitivity to the glycopeptides. Preincubation with 3.0 micrograms/ml ganglioside GM1 at 0 degrees C for 3 hr sensitized the mouse L-cell line to the inhibitor, as determined by protein synthesis assays. Preincubation of LM cells with ganglioside GM1 alone did not affect protein synthesis rates. In addition, the gangliosides GD1a and GM3 also sensitized the LM cells to the protein synthesis inhibitory effect of the glycopeptide inhibitor. Binding experiments were performed with 3T3 (sensitive) and LM (insensitive) cells to determine if sensitivity to the glycopeptide inhibitor was reflected in binding of the inhibitor to these cells. Binding of 125I-labeled inhibitor to 3T3 cells was maximal after 60 min at 0 degrees C and saturable at approximately 1 X 10(4) molecules/cell. Furthermore, binding of the inhibitor was dose-dependent, with half-maximal binding at 1.5-2.0 nM and saturation at 8.0-10.0 nM. Scatchard plot analysis indicated that the Kd was about 1 X 10(-9) M and that there are 1 X 10(4) receptors/cell. Binding of the inhibitor to LM cells was maximal after 30 min at 0 degrees C and saturation occurred at 5 X 10(3) molecules/cell. We then examined the possibility that gangliosides are the cellular receptor or co-receptor for the glycopeptide inhibitor. Binding of the inhibitor to ganglioside GM1 was first examined after the ganglioside had been preadsorbed to polystyrene tubes. These experiments indicated that the ganglioside did not bind the inhibitor. Ganglioside-containing liposomes from phosphatidylcholine or LM cell membrane components were also prepared; these artificial membranes did not bind appreciable amounts of the iodinated inhibitor. Competition experiments showed that the gangliosides GM1 and GD1a did not neutralize the protein synthesis inhibitory activity of the glycopeptides, indicating that gangliosides do not directly interact with the glycopeptide inhibitor. In addition, binding of the inhibitor to LM cells preincubated with ganglioside GM1 was studied. Although the binding of the inhibitor to LM cells was one-half that observed for 3T3 cells, incorporation of exogenous gangliosides into LM cells did not result in increased binding of the inhibitor.(ABSTRACT TRUNCATED AT 400 WORDS)     

The interaction of calcium with gangliosides in bilayer membranes

We studied the binding of calcium to bilayer membranes formed from mixtures of phosphatidylcholine and mono-, di-, or trisialoganglioside by measuring its effect on the electrophoretic mobility of multilamellar vesicles and the conductance of planar bilayers. In 0.001 M monovalent salt solutions the surface potential of the membranes is large and micromolar concentrations of calcium have a significant effect on the mobility and conductance. In 0.1 M monovalent salt solutions the surface potential is small and millimolar concentrations of calcium are required to affect these parameters. The strong apparent binding of calcium we observed at low ionic strength could be due to the nonspecific accumulation of calcium in the electrical diffuse double layer. To distinguish between this nonspecific effect and binding of calcium to the membrane, we substituted dimethonium for calcium. Dimethonium is a divalent cation that screens negative charges but does not bind to lipids. We also examined the effect of replacing phosphatidylcholine by monoolein: calcium binds to phosphatidylcholine but not to monoolein. We describe our electrophoretic mobility results by combining the Poisson-Boltzmann and Navier-Stokes equations with the Langmuir adsorption isotherm. We conclude that calcium binds weakly to gangliosides with an intrinsic association constant of less than 100 M-1, which is similar to the association constant of calcium with phospholipids.     

Biochemical studies on the interaction of fibronectin with Ig

We have previously biochemically characterized three separate sites on the fibronectin (Fn) molecule that interact with IgG. These studies have been extended to examine the interaction of Fn with other classes and subclasses of Ig. By ELISA, a preferential quantitative binding order of Fn to the major Ig classes and subclasses was obtained as follows: IgG greater than IgM greater than IgA, IgG1 greater than IgG3 = IgG4 greater than IgG2, and IgA1 = IgA2. Using fragments of Fn obtained by subtilisin digestion followed by IgM and IgA affinity chromatography, immunoblot analysis using monospecific antisera to separate regions of the Fn molecule, and amino acid sequence analysis, these studies indicate that polyclonal IgA and IgM interact with Fn in the same three regions and under the same ionic conditions as previously described for IgG. Site 1 is a 22-kDa fragment that commences at residue 1 of the Fn molecule. Sites 2 (16 kDa) and 3 (26-29 kDa) begin at residues 588 and 1597, respectively. Under physiological conditions a monoclonal antibody that recognizes site 1 completely inhibited the interaction of intact Fn with IgG, IgM, and IgA. Therefore, this is the only physiologically active site in the intact molecule. Aggregated but not monomeric IgG competitively inhibited the binding of Fn to IgG-coated microtiter ELISA plates; thus, this interaction can take place in a fluid-phase system. These results indicate that Fn can potentially interact with immune complexes and aggregates of all Ig in the circulation and thus may play a significant role in both their clearance and deposition in Fn-containing tissues, such as occurs in immune-complex-related disorders.     

Binding of laminin to brain gangliosides and inhibition of laminin-neuron interaction by the gangliosides

Binding of laminin to glycolipids of neuronal membranes was studied with a thin-layer chromatography overlay assay. The major brain ganglioside GD1A was the main binding component, when chromatograms containing the same molar amount of the different brain gangliosides and the brain sulfatide were incubated with laminin at physiological ionic strength. The possible role of laminin binding to brain gangliosides in laminin-neuron interactions was studied with adhesion assays. It was found that binding of rat brain neurons to laminin is blocked by 10-40 microM brain gangliosides but not by sulfatide. The inhibition by the gangliosides is suggested to be due to competition with the cell surface interaction sites of laminin and not to binding of the gangliosides to the cells. Our findings support the idea that the adhesive and neurite-promoting effect of laminin is dependent on its interaction with gangliosides at the neuronal cell surfaces.     

The role of gangliosides in the interaction of human chorionic gonadotropin and cholera toxin with murine Leydig tumor cells

A clonal line of murine Leydig tumor cells (MLTC-1) bound both human chorionic gonadotropin (hCG) and cholera toxin (CT) with high affinity and accumulated cyclic AMP in response to either effector. The major cellular ganglioside was GM3 with small amounts of GM2, GM1, and GD1a. The gangliosides became labeled when the cells were grown in medium containing [3H] galactose or were exposed to galactose oxidase or NaIO4 followed by NaB3H4. CT specifically protected GM1 from surface labeling whereas hCG did not protect any gangliosides from being labeled. When the cells were exposed to sialidase, surface GD1a was eliminated, and GM1 increased with a corresponding increase in CT binding. When sialidase-treated cells were first incubated with the B component of CT, binding and action of CT was blocked. The cells, however, retained their ability to bind and respond to hCG. Addition of purified gangliosides to the medium effectively inhibited the binding and action of CT but not hCG. The cells incorporated the exogenous gangliosides and exhibited increased binding of and responsiveness to CT but not hCG. Both hCG- and CT-receptor complexes were extracted from the cells with nonionic detergent and analyzed by sucrose gradient centrifugation. The hCG-receptor complex had an apparent molecular weight of 190,000 whereas the CT-receptor complex sedimented only slightly faster than CT itself. MLTC-1 gangliosides were separated on thin layer chromatograms which were overlayed with either iodinated CT or hCG. The toxin bound to a ganglioside corresponding to GM1 whereas the hormone did not bind to any of the gangliosides. When the cells were incubated overnight with hCG, they lost their hCG receptors but exhibited an increase in CT binding and gangliosides. Our results indicate that GM1 is the specific receptor for CT whereas gangliosides are not involved in the binding and action of hCG.     

Calorimetric studies on the interaction of gangliosides with phospholipids and myelin basic protein

Differential scanning calorimetry was employed to investigate the interaction of GM1 gangliosides with phospholipids (phosphatidylethanolamine, phosphatidylserine or phosphatidylcholine). It was found that GM1 is completely miscible with phosphatidylethanolamine; however, the interaction with phosphatidylserine is minimal. Addition of excess Ca2+ to the interaction products of GM1 with phosphatidylcholine or phosphatidylethanolamine did not induce phase separation. The influence of myelin basic protein on the thermotropic behaviour of GM1 was also studied. It was found that basic protein has a very strong perturbing effect on GM1 micelles.     

Real-time monitoring of hematopoietic cell interaction with fibronectin fragment

Real-time cell analysis (RTCA) system based on measurement of electrical microimpedance has been introduced to monitor adherent cell cultures. We describe its use for real-time analysis of hematopoietic cell adhesion to bone marrow stroma proteins. Cells growing in suspension do not generate any significant change in the microimpedance signal until the surface with embedded microelectrodes is coated with a cell-binding protein. We show that in this case, the microimpedance signal specifically reflects cell binding to the coated surface. The optimized method was used to monitor the effect of two histone deacetylase inhibitors, suberoylanilide hydroxamic acid (SAHA) and tubastatin A, on JURL-MK1 cell adhesion to cell-binding fragment of fibronectin (FNF). Both compounds were used in non-toxic concentrations and induced an increase in the cell adhesivity. The kinetics of this increase was markedly slower for SAHA although tubulin hyperacetylation occurred rapidly for any of the two drugs. The strengthening of cell binding to FNF was paralleled with a decrease of Lyn kinase activity monitored using an anti-phospho-Src family antibody. The inhibition of Src kinase activity with PP2 accordingly enhanced JURL-MK1 cell interaction with FNF. Actin filaments were present at the proximity of the plasma membrane and in numerous membrane protrusions. In some cells, F-actin formed clusters at membrane regions interacting with the coated surface and these clusters colocalized with active Lyn kinase. Our results indicate that the role of Src kinases in the regulation of hematopoetic cell adhesion signaling is similar to that of c-Src in adherent cells.     

Quantitative study of cell interaction with collagen and fibronectin

The interaction of BHK-fibroblasts with collagen or fibronectin-collagen complex was investigated quantitatively. For that purpose an improved method for production of defined cell substrata was developed. The method permitted reproducible coupling of different ligands to glass via an amino or carboxyl group. BHK-cells grown on collagen required a minimum density of 15-20 ng collagen/cm2 for spreading. When grown on fibronectin adsorbed on collagen the cells were found to remove fibronectin from the substratum at a rate of 0.15 pg/(cell X h).     

Selective interaction of peripheral and central nervous system cells with two distinct cell-binding domains of fibronectin

Mechanisms of cell interaction with fibronectin have been studied with proteolytic fibronectin fragments that have well-defined ligand binding properties. Results of a previous study (Rogers, S. L., J. B. McCarthy, S. L. Palm, L. T. Furcht, and P. C. Letourneau, 1985, J. Neurosci., 5:369-378) demonstrated that (a) central (CNS) and peripheral (PNS) nervous system neurons adhere to, and extend neurites on a 33-kD carboxyl terminal fibronectin fragment that also binds heparin, and (b) neurons from the PNS, but not the CNS, have stable interactions with a 75-kD cell-binding fragment and with intact fibronectin. In the present study domain-specific reagents were used in inhibition assays to further differentiate cell surface interactions with the two fibronectin domains, and to define the significance of these domains to cell interactions with the intact fibronectin molecule. These reagents are (a) a soluble synthetic tetrapeptide Arg-Gly-Asp-Ser (RGDS; Pierschbacher, M. D., and E. Ruoslahti, 1984, Nature (Lond.), 309:30-33) representing a cell-binding determinant in the 75-kD fragment, and (b) an antibody raised against the 33-kD fragment that binds specifically to that fragment. Initial cell attachment to, and neurite extension upon, fibronectin and the two different fragments was evaluated in the presence and absence of the two reagents. Attachment of both PNS and CNS cells to intact fibronectin was reduced in the presence of RGDS, the former more so than the latter. In contrast, the antibody to the 33-kD fragment did not affect attachment of PNS cells to fibronectin, but significantly decreased attachment of CNS cells to the molecule. RGDS inhibited attachment of CNS cells to the molecule. RGDS inhibited attachment of both cell types to the 75-kD fragment to a greater degree than it did attachment to the intact molecule. Cell interaction with the 33-kD fragment was not affected by RGDS. Reduction of neurite lengths (determined after 24 h of culture) by the domain-specific reagents paralleled the reduction in initial adhesion to each substratum. Therefore, it appears that (a) both PNS and CNS cells have receptors for each cell-binding domain of fibronectin, (b) the receptor(s) for the two domains are distinct, with attachment to the 33-kD fragment being independent of RGDS, and (c) the relative importance of each domain to cell interaction with intact fibronectin is different for CNS and PNS cells.     

Exogenous polyamines enhance copper tolerance of Nymphoides peltatum

The protective effects of polyamines (PAs) against copper (Cu) toxicity were investigated in the leaves of Nymphoides peltatum. Cu treatment increased the putrescine (Put) level and lowered spermidine (Spd) and spermine (Spm) levels, thereby reducing the (Spd+Spm)/Put ratio in leaves. Exogenous application of Spd or Spm markedly reversed these Cu-induced effects for all three PAs and partially restored the (Spd+Spm)/Put ratio in leaves. It also significantly enhanced the level of proline, retarded the loss of soluble protein, decreased the rate of O2*- generation and H2O2 content, and prevented Cu-induced lipid peroxidation. Furthermore, exogenous Spd and Spm reduced the accumulation of Cu and effectively maintained the balance of nutrient elements in plant leaves under Cu stress. These results suggest that exogenous application of Spd or Spm can enhance the tolerance of N. peltatum to Cu by increasing the levels of endogenous Spd and Spm as well as the (Spd+Spm)/Put ratio.     

Characterization of the interaction between fibronectin andTreponema pallidum

The interaction betweenTreponema pallidum and rabbit plasma fibronectin was characterized. Fibronectin was isolated from rabbit plasma and radioiodinated by the lactoperoxidase method. Fibronectin bound to the surface ofT. pallidum, reaching saturation at approximately 54 g/ml. The association affinity constant was 2.85×10⁷ M ^–1, much lower than that ofStaphylococcus aureus (5.6×10⁹ M ^–1) Fibronectin binding plateaued within 15 min at 20° and 37°C, with some reelution at 37°C by 30 min. Little fibronectin, bound toT. pallidum at 4°C. The greatest amount of fibronectin was bound at the lowest pH tested (pH 6.0); the poorest binding was at pH 7.5. Approximately 90% of the binding was reversible in the presence of excess unlabeled fibronectin. The data indicate a more dynamic and weaker interaction betweenT. pallidum and fibronectin than that seen withS. aureus.