Colloidal iron binding to the surface of HeLa cells,spreading in monolayer culture

Summary We have used the colloidal iron (CI) binding technique, adapted for transmission electron microscopy, for semiquantitative evaluation of the negative charge density at the surface of HeLa cells in monolayer culture. The surface area increases when HeLa cells spread on the substrate. This increase brings about a decrease in the thickness of the CI rim, indicating a decrease in negative surface charge density. This phenomenon implicates lowering of the electrostatic repulsion, and explains the formation of intercellular contacts at the level of spread parts of the cell. Because of lack of penetration, CI particles are absent in regions of close apposition between cells and between cells and substrates. Absence of CI binding in broader intercellular or cell-substrate spaces was explained through masking of the anionic groups.Supported by a Grant from the Algemene Spaar- en Lijfrentekas Cancer FundThe authors acknowledge the technical assistance of B. Buysse, O. Claeys and J. Roels van Kerckvoorde     

Evidence for long-range electrostatic repulsion between HeLa cells

Agglutination curves obtained on addition of low molecular weight poly-l-lysines (mol. wt 4 000–23 000) to HeLa cells, show a deviation from linearity at low polymer concentration. This probably indicates the existence of a ζ-potential which has to be lowered before agglutination can take place. Experiments with dilysine support the assumption that cell surface charge is lowered on addition of low concentrations of short chain poly-l-lysines.Long poly-l-lysine molecules (mol. wts 70 000; 100 000) yield linear agglutination curves already at the lowest polymer concentrations. This might indicate that these polymers are able to bridge the original repulsion gap between HeLa cells.After removal of peripheral sialic acid by neuraminidase, linear agglutination curves are obtained with all poly-l-lysines irrespective of their chain lengths. This is interpreted as evidence for involvement of sialic acid residues in the charge organization responsible for electrostatic repulsion.The magnitude of the presumed repulsion effect is shown to vary with the cell density at the time the HeLa cells were harvested from the culture. The largest repulsion effect is obtained with cells from density inhibited cultures which also have the lowest tendency for mutual adhesion. With fast growing cells from low density cultures linear agglutination curves are obtained with short chain poly-l-lysines. This is interpreted as evidence for a strong diminishment or absence of long-range electrostatic repulsion between such cells.     

Modulation of WGA binding sites on marrow sinus endothelium in state of stimulated erythropoiesis: a possible mechanism regulating the rate of cell egress

To study the regulation of cellular and molecular traffic across the marrow-blood barrier, rat marrow endothelial surface was incubated with ferritin-conjugated concanavalin A, wheat germ agglutinin (WGA), recinus communis agglutinin I, and phytohemagglutinin. Normal animals were compared with those after erythropoietic stimulation (phenylhydrazine-induced hemolysis, phlebotomy). A selective and significant reduction in the density of WGA receptors, but not other lectins was noted congruent to the degree of reticulocytosis. Neuraminidase treatment also reduced WGA binding sites and the surface negative charge as detected by polycationic ferritin (PCF). Thus, the reduction in WGA binding sites, may reflect a decrease in the density of membrane sialic acid, rendering the endothelial surface charge less negative and providing an electrostatic attraction for the negatively charged surface of reticulocytes. The findings may also be explained by an increase in the frequency of WGA-excluding fenestrae in the endothelium. These areas, lacking sialic acid, may provide unstable areas in the membrane suitable for the passage of cells and molecules in both directions. It is concluded that, by modulating the density of sialic acid residues, the endothelium may regulate the traffic of cells and molecules across the marrow-blood barrier.     

A simple electrostatic model can explain the effect of pH upon the force-pCa relation of skinned frog skeletal muscle fibers.

The relative force-pCa relation of skinned frog skeletal muscle fibers is shifted along the pCa axis by changes in pH. This shift has been interpreted as arising from competition between H+ and Ca2+ for a binding site on troponin. Unfortunately, binding studies have been unable to confirm such competition. Alternatively, however, the data fit a model where H+ influences the degree of dissociation of ionizable groups on the surface of the thin filaments, thus altering the electrostatic potential surrounding the filaments. Alterations in the potential will, in turn, change the concentration of Ca2+ near the troponin binding sites in accordance with the Boltzmann relation. A simple model, based upon the Gouy-Chapman relation between surface potential and charge density, provides a quantitative explanation for the shift of the relative force-pCa curve with pH, given a reasonable estimate of the surface charge density on the thin filament. A best fit is obtained when the ionizable groups giving rise to the potential have a log proton ionization constant (pKa) of 6.1, similar to that for the imidazole group on histidine, and when the density of these groups is near that estimated from amino acid analysis of thin filament proteins and from filament geometry. In preliminary experiments, reaction of skinned frog fibers with diethylpyrocarbonate (DEP) at pH 6 shifted the force-pCa curve toward lower Ca2+. This would be expected in the model since DEP at pH 6 is reported to specifically react with histidine imidazole groups and to irreversibly decrease their pKa, which would increase the net negative charge of the filaments.     

Histochemical study of the distribution of sialic acid on the surface of HeLa cells in culture

Summary The distribution of sialic acid on the surface of HeLa cells is studied using Hale's staining technique. Treatment of the cells with neuraminidase before staining, indicates that the staining technique is specific for the demonstration of sialic acid.HeLa cell monolayers, grown in Leighton tubes, are treated with a solution of E.D.T.A. During separation and rounding up, cells are fixed in calcium-formalin and stained. We found a gradual increase of the Hale's positivity during treatment with E.D.T.A.HeLa cells from suspension cultures are grown in Rose-chambers. They are fixed and stained after various periods of incubation. We found a decrease of Hale's positivy during spreading out of cells and monolayer formation.These findings are discussed in terms of surface charge density and formation of stable cell contacts.The authors thank Mr. C. Dragonetti for technical assistance.     

Cell density-dependent regulation of cell surface expression of two types of human tumor necrosis factor receptors and its effect on cellular response

Tumor necrosis factor (TNF) is a multipotential cytokine known to regulate the growth of a wide variety of normal and tumor cells. It has been shown that the density of cells in culture can modulate the growth regulatory activities of TNF, the mechanism of which, however, is not understood. In this report, we investigated the effect of cell density on the expression of TNF receptors. The receptors were examined on epithelial cells (e.g., HeLa), which primarily express the p60 form, and on myeloid cells (e.g., HL-60) known to express mainly the p80 form. We observed that binding of TNF to both cell lines decreased with increase in cell density. Scatchard analysis of binding on HeLa and HL-60 cells revealed a 4- to 5-fold reduction in the number of TNF receptors without any significant change in receptor affinity in both cell types at high density. The decrease in TNF receptor numbers at high cell density was also observed in several other epithelial and myeloid cell lines. The downmodulation at high cell density was unique to TNF receptors, since minimum change in other cell surface proteins was observed as revealed by fluorescent activated cell sorter analysis. Neutralization of binding with antibodies specific to each type of the receptors revealed that both the p60 and p80 forms of the TNF receptor were equally downmodulated. A decrease in leucine incorporation into proteins was observed with increase in cell density, suggesting a reduction in protein synthesis. Since inhibition of protein synthesis by cycloheximide also leads to a decrease in TNF receptors, it is possible that the density-dependent reduction in TNF receptor number is due to an overall decrease in protein synthesis. The density-dependent decrease in TNF receptors was accompanied by a decrease in intracellular reduced glutathione levels. A reduction in the number of receptors on TNF sensitive tumor cells induced by cell-density correlated with increase in resistance to the cytokine.     

Contribution of interfacial tension changes during cellular interaction to the energy balance

The energetics of cell-cell and cell-substrate interactions has been analyzed in terms of the lyophobic colloid stability theory adapted to biological conditions. Some important differences that exist between lyophobic particles and living cells are recognized and taken into account. The protein-aceous coat exterior to the lipid cell membrane (glycocalyx) is treated as a very thick Stern layer which has a constant electric capacitance. The cell itself is viewed as a fluid droplet due to the semi-fluid state of the cell membrane, and its outer boundary is assumed to have a constant electric charge density. When particles with constant surface charge density interact, their surface potential increases. Then the potential at the lipid-protein interface will also increase, hence the interfacial tension should decrease. The magnitude of the interfacial tension change at the lipid-protein interface occurring during the interaction of cells has been calculated for various thicknesses of the glycocalyx. This term, obtained for cells with a relatively thin proteinaceous coat, was found to dominate the energy balance, making the total energy of interaction negative.     

Interaction of polynucleotides with cultured mammalian cells : II. Cell surface charge density and RNA uptake

This report describes the uptake of high molecular weight RNA by Ehrlich ascites tumor cells treated with enzymes and polycations which reduce cell net negative surface charge density. Enzyme treatment had little effect on RNA uptake, but treatment with poly- -lysine resulted in increased binding and uptake of RNA. Present data indicate that decreased cell surface charge, increased availability of positive surface sites, and cell death, all contribute to increased RNA uptake. The individual contributions of these factors has been partially resolved. A possible mechanism for polyanion uptake by cells is proposed.     

The Surface Charge of Isolated Toad Bladder Epithelial Cells : Mobility, effect of pH and divalent ions

The surface charge of epithelial cells isolated from the toad bladder has been determined by the microscope method of cell electrophoresis. The cells possess a net negative charge, and a net surface charge density of 3.6 x 10⁴ electronic charges per square micron at pH 7.3. Estimates of net surface charge over the alkaline pH range indicate (a) that an average distance of the order of 40 A separates the negatively charged groups, and (b) that amino as well as acid groups are present at the electrophoretic surface of shear. A significant increase in mobility following cyanate treatment of the cells suggests that a large proportion of the amino groups are the ε-amino groups of lysine. In view of the known effects of calcium and other divalent ions on cell permeability and cell adhesion, the extent of binding of calcium and magnesium to the cell surface was determined by the electrophoretic technique. Mobility was significantly decreased in the presence of calcium or magnesium, indicating that these ions are bound by surface groups. When the pH was lowered from 7.3 to 5.2, calcium binding was markedly decreased, an observation consistent with competition between calcium and hydrogen ions for a common receptor site.     

Electrostatic effects and the dynamics of enzyme reactions at the surface of plant cells. 2. The role of pectin methyl esterase in the modulation of electrostatic effects in soybean cell walls

The pectin methyl esterase from soybean cell walls has been isolated and purified to homogeneity. It is a protein with a relative molecular mass close to 33 000. The enzyme is maximally active at a pH close to 8 and its pH dependence may be explained by a classical Dixon model, where the two interconvertible enzyme ionization states coexist. The outflux of protons from cell walls, upon raising the ionic strength, may be taken as an indirect estimate of the fixed charge density. If the cell-wall fragments are pre-incubated at pH values between 5 and 9, the outflux of protons rises with the pH of pre-incubation. This implies, as postulated from the theory developed in the preceding paper, that alkaline pH favours the activity of pectin methyl esterase and that this enzyme effectively generates the fixed negative charges of the cell wall. Therefore the pectin methyl esterase reaction builds up the Donnan potential, delta psi, at the cell surface. The cell-wall charge density, estimated from the proton outflux, as well as from the titration of methyl groups on the cell wall, reaches a maximum between the third and the fourth day of growth. While the cell-wall volume increases and reaches a plateau, the fixed charge density increases at first and then declines. This is understandable if one assumes that the building up of a high charge density is a co-operative phenomenon and that the local pH inside the wall rises during cell growth. When both the cell-wall volume and the charge density increase together, this suggests that the local pH inside the wall lies within the critical pH range associated with the steep response of the system. When the cell-wall volume increases together with a decrease of the fixed charge density, the local pH should have dropped below this critical pH range. Under these conditions the pectin methyl esterase remains inactive, or poorly active. As the number of fixed negative charges increases, calcium becomes tightly bound to cell walls. This binding is so tight that the net charge density is minimum when the calcium concentration is maximum. The experimental results, presented above, offer experimental support to two important ideas discussed in the preceding paper, namely that pectin methyl esterase reaction builds up the Donnan potential at the cell surface, and that this response may be co-operative with respect to pH.     

Changes in the cell surface of human diploid fibroblasts during cellular aging.

The electrophoretic mobility of 13 human diploid cell strains, TIG-1, TIG-2, TIG-3, TIG-7, WI-38, IMR-90, MRC-5, MRC-9, TIG-1H, TIG-1L, TIG-2M, TIG-2B, and TIG-3S, which were established from different tissues of human embryos, was studied at different passages. The net negative surface charge of the cells was characteristic for each cell strain and decreased significantly during the in vitro aging of the cells. The decrease in the net negative charge of the cells correlated well with the decrease in cell density throughout the life span of the cells. A strict linear correlation between the electrophoretic mobility and the number of cells harvested at each passage was obtained for all the human diploid cell strains. Moreover, almost the same linear regression coefficient of the cells was obtained among these cell strains. Therefore, the net negative surface charge of human diploid cell strains could serve as a cell surface marker for in vitro cellular aging.     

The use of cationized ferritin to measure cell surface charge of mouse bone marrow cells by flow cytometry

Summary We have prepared fluorescein isothiocyanate (FITC) conjugates of cationised ferritin (CF) and have investigated the usefulness of this CF-FITC to measure the negative cell surface charge of mouse bone marrow cells by flow cytometry. CF-FITC conjugates of low fluorochrome to protein ratios (F/P ratio) gave insufficient fluorescence and/or formed large aggregates when stored. CF-FITC conjugates of high F/P ratios (above 25) bound specifically to bone marrow cells, giving sufficient fluorescence, the intensity of which differed for the different cell types. When stored at –20° C the CF-FITC was stable and could be used over prolonged periods. CF-FITC could be used to selectively enrich for pluripotent stem cells (CFU-S) and granulocyte/macrophage progenitors (CFU-C) by fluorescence activated cell sorting (FACS), although the CF-FITC binding to CFU-S and CFU-C was unexpectedly low. No correlation between CF-FITC fluorescence, cell size and electrophoretic mobility (EMP) was observed of bone marrow cells fractionated by free flow electrophoresis. Neuraminidase treatment to remove negatively charged sialic acid groups from the cell surface resulted in an increased binding of CF-FITC, although the EPM was decreased. The biotin conjugate of CF bound to bone marrow cells and could be visualised by avidin-FITC. The relative fluorescence intensity for the individual cell types showed a good correlation with the cell surface charge as determined by the EPM of the different cell types.The mechanism of binding CF-FITC to the cell surface was not by electrostatic interaction of the negative cell surface and positively charged CF because CF-FITC of F/P ratios of above 20 was negatively charged. This has been shown by theoretical calculations and determination of the pI of CF-FITC by iso-electric focussing. Binding of CF-FITC to the cell surfaces was probably caused by hydrophobic interaction between bound fluorescein molecules and lipid domains in the cell surface membrane aided by some ionic interaction. CF-biotin is still positively charged and is probably bound through electrostatic interactions with negatively charged cell surface groups. The indirect detection of bound CF-biotin with avidin-FITC of high F/P ratio results in a high fluorescence signal, which is a measure of the negative cell surface charge density, in the FACS.In honour of Prof. P. van Duijn     

Heparin interferes with translocation of Yop proteins into HeLa cells and binds to LcrG, a regulatory component of the Yersinia Yop apparatus

Yersiniae are equipped with the Yop virulon, an apparatus that allows extracellular bacteria to deliver toxic Yop proteins inside the host cell cytosol in order to sabotage the communication networks of the host cell or even to cause cell death. LcrG is a component of the Yop virulon involved in the regulation of secretion of the Yops. In this paper, we show that LcrG can bind HeLa cells, and we analyse the role of proteoglycans in this phenomenon. Treatment of the HeLa cells with heparinase I, but not chondroitinase ABC, led to inhibition of binding. Competition assays indicated that heparin and dextran sulphate strongly inhibited binding, but that other glycosaminoglycans did not. This demonstrated that binding of HeLa cells to purified LcrG is caused by heparan sulphate proteoglycans. LcrG could bind directly to heparin-agarose beads and, in agreement with these results, analysis of the protein sequence of Yersinia enterocolitica LcrG revealed heparin-binding motifs. In vitro production and secretion by Y . enterocolitica of the Yops was unaffected by the addition of heparin. However, the addition of exogenous heparin decreased the level of YopE–Cya translocation into HeLa cells. A similar decrease was seen with dextran sulphate, whereas the other glycosaminoglycans tested had no significant effect. Translocation was also decreased by treatment of HeLa cells with heparinitase, but not with chondroitinase. Thus, heparan sulphate proteoglycans have an important role to play in translocation. The interaction between LcrG and heparan sulphate anchored at the surface of HeLa cells could be a signal triggering deployment of the Yop translocation machinery. This is the first report of a eukaryotic receptor interacting with the type III secretion and associated translocation machinery of Yersinia or of other bacteria.     

Cell surface charge alterations occurring during dimethylsulfoxide-induced erythrodifferentiation of Friend leukemia cells

Dimethylsulfoxide-induced erythrodifferentiation of Friend leukemia cells caused a decrease in net negative cell surface charge which began two days after exposure to the polar solvent and continued throughout the maturation process. Neuraminidase treatment caused a marked reduction in mobility of both untreated and dimethylsulfoxide-treated cells suggesting that sialic acid residues are the major anionogenic moieties of the surface membrane of Friend cells. A decrease in the content of total glycosidically bound sialic acid in dimethylsulfoxide-treated cells also occurred. The findings provide evidence to support an association between erythrodifferentiation of Friend cells and net negative surface charge dependent upon sialic acid residues.     

Adsorption of gamma-aminobutyric acid to phosphatidylserine membranes.

The interaction of the negatively-charged phosphatidylserine (PS) and gamma-Aminobutyric acid (GABA) is examined in black lipid membranes (BLM) and inverse micelles. GABA does not permeate through PS membranes and, in concentrations of 10(-5)-10(-4) M, it reduces the negative potential at the membrane-aqueous solution interface. The effect is owing to the adsorption of the GABA cationic species and the consequent decrease of the negative surface charge density of the membrane. When the intrinsic pH of the membrane-solution interface is considered, the Gouy-Chapman-Stern theory describes the GABA screening effect and makes it possible to calculate the GABA-PS binding constant. This value is compared with that obtained measuring the partition of 14C-GABA between an organic phase containing PS and the aqueous solution. The results presented strongly suggest that the electrostatic force plays a major role in GABA-PS interaction.     

A fluorescence spectroscopy study on the interactions of the TAT-PTD peptide with model lipid membranes

Protein-transduction domains (PTDs) have been shown to translocate into and through the living cells in a rapid manner by an as yet unknown mechanism. Regardless of the mechanism of translocation, the first necessary step must be binding of the PTD peptide to the surface of the lipid membrane. We used fluorescence spectroscopy to study the interaction between PTD of the HIV-1 Tat protein (TAT-PTD; residues 47-60 of Tat, fluorescently labeled with tryptophan) and the lipid bilayer labeled with various fluorescence membrane probes. The TAT-PTD tryptophan exhibited a decrease in fluorescence intensity and an increase in anisotropy upon interaction with lipid bilayers. The fluorescence changes were linearly proportional to the density of negative charge in the membrane. Kinetic analysis of the interaction showed two apparent dissociation constants. The value of one dissociation constant (Kd1 = 2.6 +/- 0.6 microM), which accounted for 24% of the interaction, was found to be independent of the negative charge density, suggesting its nonelectrostatic nature. The value of the second dissociation constant (Kd2), which accounted for 76% of the interaction, decreased linearly from 610 +/- 150 to 130 +/- 30 microM with an increase in negative charge density from 0 to 25 mol %, suggesting this interaction is electrostatic in nature. Even though the binding was predominantly electrostatic, it could not be reversed by high salt, indicating the presence of a second, irreversible, step in the interaction with lipid. When TAT-PTD was bound to lipid vesicles labeled with 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH), fluorescence resonance energy transfer between the tryptophan and the probe occurred at a distance of 3.4 nm. No change in fluorescence anisotropy of either TMA-DPH or DPH was observed upon the interaction with TAT-PTD, indicating no significant disruption or perturbation of the lipid bilayer by the peptide. TAT-PTD did not cause dissipation of membrane potential (165 mV, negative inside). Inclusion of 3% pyrene-labeled phosphatidylglycerol (pyrene-PG) in the membrane revealed that TAT-PTD preferentially bound to the membrane in the liquid state. We conclude that membrane fluidity is an important physicochemical parameter, which may regulate binding of TAT-PTD to the membrane.     

The effect of polycations on cell membrane stability and transport processes

Summary The interaction of poly-l-lysines of different molecular weights (PL) with Ehrlich ascites tumor cells was studied experimentally with respect to cell surface binding, cell electrophoresis, cytotoxicity and membrane permeability. Although they decrease the net negative charge of Ehrlich ascites cells similarly at low PL concentrations, low molecular weight PL was less cytotoxic and less damaging to the potassium transport mechanism than was high molecular weight PL. At certain PL concentrations, membrane damage was reversible on reincubation in PL-free media. The amount of bound polylysine as determined with fluorescent labeled polylysine was compared by electrophoresis to the amount of polylysine expressed on the electrokinetic surface. The results indicated that only a small fraction of polylysine bound to Ehrlich ascites tumor cells was electrokinetically detectable. The adsorption of polylysine to Ehrlich ascites tumor cells was not describable by the usual adsorption isotherms. It is suggested that the same number of monomeric lysine units of high and low molecular weight PL are adsorbed at the cell electrokinetic surface, but cytotoxicity is dependent on molecular weight. Although the negative charge of human red blood cells could be reversed at low PL concentrations, no such effect could be observed for ELD (a subline of Ehrlich ascites carcinoma) cells even at high PL concentrations. The relationship of PL binding to the stimulation of macromolecular uptake is discussed.     

The role of surface charge on the accelerating action of heparin on the antithrombin III-inhibited activity of alpha-thrombin

We have compared surface charge and the surface charge density on the polyanions heparin and potassium polyvinyl sulfate (KPVS), as well as on hydrolyzed heparin and KPVS, with their accelerating effect on the inhibitory action of antithrombin III on thrombin. Polyelectrolyte titration of thrombin with KPVS or heparin at pH 7.4 clearly indicates an electrostatic interaction. In contrast, at the same pH no electrostatic interaction is observed between polyanions and antithrombin III. KPVS accelerates the inhibitory action of antithrombin III to the same extent as heparin on the basis of charge equivalence. Heparin and KPVS with a mean distance between two charged centers of less than 0.75 and 0.95 nm, respectively, accelerate strongly whereas hydrolysates with lower charge densities are far less active. The following observations are indicated. Intramolecular neutralization of oppositely charged residues occurs within thrombin, antithrombin III, and partially hydrolyzed heparin. Heparin acts on the antithrombin III-thrombin reaction through cooperative electrostatic binding to thrombin and nonelectrostatic interaction with antithrombin III. This indicates a quasi-catalytic action of the polyelectrolyte. Hydrolysis of only a few N-sulfate residues within the heparin molecule decreases the linear surface charge density to such an extent that the accelerating action is drastically reduced. The loss of accelerating capacity agrees with the sudden loss of counterion condensation due to the decrease of the linear surface charge density beyond limits postulated by Manning in a theory of polyelectrolytes.