Correlation between the oscillatory and adhesion activities in erythroid cells

The attachment kinetics of erythroid cells, such as human erythrocytes, their saponin ghosts, and erythroleukemic cells K562 to a glass surface has been studied in the presence of substances inhibiting spontaneous fluctuations of cell membranes. It has been shown that wheat germ agglutinin (WGA) slows down the attachment kinetics of K562 cells, as is the case in intact erythrocytes. Concanavalin A (Con A), which inhibits the attachment of erythrocytes to glass does not affect the adhesion of K562 cells to glass due to the absence of band 3 proteins in the membranes of K562 cells. Both lectins slow down the adhesion rate of saponin ghosts of human erythrocytes, as it takes place in intact erythrocytes. Suramin and the anionic dye ANS bind specifically to the actin protofilaments of the erythrocyte skeleton and also inhibit cell adhesion to glass. At the same time, these substances do not affect the oscillatory and adhesion activities of intact erythrocytes due to the impermeability of erythrocyte membranes for these drugs. The results obtained allow the conclusion that inhibition of erythrocyte adhesion by lectins is due to lectin binding to different constituents of the erythrocyte membrane--sialic acid moieties of glycophorin in the case of WGA and band 3 proteins in the case of Con A. The most probable mechanism of erythrocyte and K562 cell attachment to glass is the formation of the so-called local contacts between cells and the glass surface. It is also suggested that the cell surface oscillations facilitate the formation of cell contacts.     

Influence of flow velocity and cell concentration on dynamic adhesion of erythrocytes to glass surface

Comparative studies were carried out on dynamic adhesion of 51Cr-labelled erythrocytes to the surface of glass beads in the presence of serum in the medium (50 microng of protein/ml) and in protein-free medium. The influence of cell concentration (within the range 4 X 10(5) to 8 X 10(6)/ml) and of cellular flow velocity (within the range 1.5-0.4 cm/min) on the value of adhesion was investigated. It was found that when serum was present in the medium, the decisive influence on erythrocyte adhesion was exerted by the velocity with which the cells pass though the glass bead layer. Cell concentration under these conditions has only a very slight effect. When the medium does not contain serum, erythrocyte adhesion to the bead layer seems to depend on both cell concentration and flow velocity. Preliminary data were obtained concerning the release of 51Cr from the bead layer after erythrocyte adhesion.     

Rapid local oscillations of the surface of the human erythrocyte

The transverse displacements of the human erythrocyte surface with amplitude 300-400 nm in the frequency range 0.2-30 Hz are recorded on the minimal area erythrocyte rim (approximately 0.5 X 0.5 microns). These local oscillations of the surface are diminished at hypoosmotic erythrocyte swelling, on addition of substances which increase the membrane rigidity (0.01% glutaraldehyde, 0.5 mM 4-hydroxymercuribenzoate, cell membrane stain--0.002% Heliogen Blue) and on discocyte--echinocyte transformation due to addition of 1-2 mM 2,4-dinitrophenol. The amplitude of transverse displacements is reduced by 1.7-2 times on erythrocytes of patients with inherent microspherocytosis. These erythrocytes have inherent defects in spectrin. It is suggested that spectrin is important for rapid local oscillations of the human erythrocyte surface.     

Adhesion of human erythrocytes to glass: The nature of the interaction and the effect of serum and plasma

Studies of normal human erythrocyte adhesiveness to glass have demonstrated quantitative and qualitative differences between cell-glass and cell-cell contact interaction. Identifiable charged groups on the red cell surface were of minor importance in the adhesive process and it is postulated that direct cell-glass contact attraction involves nonpolar regions of the red cell surface. However, erythrocyte adhesion to glass in the presence of serum and plasma is affected by electrostatic forces. At least two critical factors are present in serum and plasma: a heat-stable factor(s) diminishing adhesion and a heat-labile factor(s) promoting cell-glass interaction. A postulate is presented concerning the role of soluble fibrin monomer complexes in promoting cell adhesion to glass.     

Fibrinogen inhibits red cell adhesion to glass

Studies of human erythrocyte adhesion to glass have demonstrated consistently greater adhesion with serum-containing media than with a comparable concentration of plasma. This serum-plasma difference is explained by the adhesion-inhibiting property of plasma fibrinogen. The fibrinogen effect is probably mediated through its firm binding to glass, since no adsorption onto the red cell surface could be demonstrated. The ability of more red cells to adhere to a foreign surface after plasma coagulation (the formation of serum from plasma) may be significant in the red cell surface interactions necessary for the formation of a fibrin-red cell thrombus.     

Coherent oscillations of the molecular state of protein in live cells

The paper describes results of experimental studies of changes in the refractive index of the live cell (human erythrocyte) substance. The measurements were carried out using automated interference microscope. Oscillations of the refractive index were detected with the amplitude 5-10% and period 1 x 10(3)-3 x 10(3). In other words, oscillations with a near one hour period is equal to 10-45 min. It is supposed that these oscillations may result from coherent changes in the state of protein molecules.     

The growth and adhesion to glass of algal (Chlorella) cells: the effects of iron and other mineral nutrients

Summary A convenient quantitative method for measuring the adhesion of cells to a surface is described. The method is based on the readiness of algal cells to adhere to a clean glass surface. A trace element mixture was found to decrease cell adhesion but it also decreased the maximum specific growth rate of a Chlorella-type green alga. Cell adhesion was also decreased by reducing the magnesium ion concentration of the medium. The biomass yield from iron was 1.51×10³ g dry biomass/g iron consumed.     

The measurement of Bacillus mycoides spore adhesion using atomic force microscopy,simple counting methods,and a spinning disk technique

An atomic force microscope has been used to study the adhesion of Bacillus mycoides spores to a hydrophilic glass surface and a hydrophobic-coated glass surface. AFM images of spores attached to the hydrophobic-coated mica surface allowed the measurement of spore dimensions in an aqueous environment without desiccation. The spore exosporium was observed to be flexible and to promote the adhesion of the spore by increasing the area of spore contact with the surface. Results from counting procedures using light microscopy matched the density of spores observed on the hydrophobic-coated glass surface with AFM. However, no spores were observed on the hydrophilic glass surface with AFM, a consequence of the weaker adhesion of the spores at this surface. AFM was also used to quantify directly the interactions of B. mycoides spores at the two surfaces in an aqueous environment. The measurements used "spore probes" constructed by immobilizing a single spore at the apex of a tipless AFM cantilever. The data showed that stretching and sequential bond breaking occurred as the spores were retracted from the hydrophilic glass surface. The greatest spore adhesion was measured at the hydrophobic-coated glass surface. An attractive force on the spores was measured as the spores approached the hydrophobic-coated surface. At the hydrophilic glass surface, only repulsive forces were measured during the approach of the spores. The AFM force measurements were in qualitative agreement with the results of a hydrodynamic shear adhesion assay that used a spinning disk technique. Quantitatively, AFM measurements of adhesive force were up to 4 x 10(3) times larger than the estimates made using the spinning disk data. This is a consequence of the different types of forces applied to the spore in the different adhesion assays. AFM has provided some unique insights into the interactions of spores with surfaces. No other instrument can make such direct measurements for single microbiological cells.     

Determination of binding strength and kinetics of binding initiation. A model study made on the adhesive properties of P388D1 macrophage-like cells

The adhesive properties of the mouse P388D1 macrophage-like line were explored. Cells were deposited in glass capillary tubes, and the kinetics of adhesion and spreading were studied. Binding involved the cell metabolism since it was decreased by cold, azide, or a divalent cation chelator. Glass-adherent cells were subjected to calibrated laminar shear flows with a highly viscous dextran solution. A tangential force of about 5 X 10(-3) dyn/cell was required to achieve substantial detachment. The duration of application of the shearing force strongly influenced cell-substrate separation when this was varied from 1-10 s. Further, this treatment resulted in marked cell deformation, with the appearance of an elongated shape. Hence, cell-substrate separation is a progressive process, and binding strength is expected to be influenced by cell deformability. The minimum time required for adhesion was also investigated by making cells adhere under flow conditions. The maximum flow rate compatible with adhesion was about 1000-fold lower than that required to detach glass-bound cells. A simple model was devised to provide a quantitative interpretation for the experimental results of kinetic studies. It is concluded that cell-to-glass adhesion required a cell-substrate contact longer than a few seconds. This first step of adhesion was rapidly followed by a large (about 1000-fold) increase of adhesion strength. It is therefore emphasized that adhesion is heavily dependent on the duration of cell-to-cell encounter, as well as the force used to remove so-called unbound cells.     

Interaction of neoplastic cells with glass surface under flow conditions

The adhesion to glass of L 1210 cells flowing in transparent parallel plate microchannel was studied by a cinematographic method. Most cells settle on the surface when their velocity immediately preceding attachment does not exceed approx. 100 μm/sec, the greatest adhesion rate accompanying relatively small velocities. The arrest of cells on the glass surface is either permanent or temporary and in a certain range of fluid velocities numerous cells are arrested several times consecutively for brief periods. Two types of surface attachment may be distinguished: cells are either totally immobilized on the surface (firm adhesion) or are able to perform under the influence of the fluid impulses some movements around the attachment site (loose adhesion). When the adherent cells are subjected to the shearing force of rapidly flowing fluid, they detach from the surface, the tearing away being frequently preceded by an accelerating gliding movement. The influence of hydrodynamic forces on the cell-surface interaction and adhesion processes is discussed, as well as some problems concerning possible mechanisms of the cell binding to the surface under dynamic conditions.     

Use of cell contour analysis to evaluate the affinity between macrophages and glutaraldehyde-treated erythrocytes.

Recently, several authors evaluated the affinity between lipid bilayers or erythrocyte membranes by analyzing the deformation of cells or vesicles they brought into close contact using micromanipulators. In the present report, we extend this approach in a study of the adhesive properties of rough nucleated cells. Rat peritoneal macrophages were made to bind human red cells modified with glutaraldehyde or glutaraldehyde and polylysine. Conjugates were examined with electron microscopy, and photomicrographs were digitized for quantification of cell surface roughness in and out of adhesion areas. Also, macrophages were subjected to micropipette aspiration to find a relationship between apparent surface tension and area increase. Assuming that this increase was a direct consequence of a smoothing of the cell surface on the submicrometer scale, the actual affinity between macrophages and erythrocytes was estimated. The obtained values ranged between 8.4 X 10(-5) and 18.2 X 10(-5) J/m2. It is concluded that cell surface roughness may be an important parameter of cell adhesion and perhaps deformation. This is made amenable to experimental study by the present approach.     

Locomotion of granulocytes on an inclined plane

The paper presents a quantitative study of the trajectories of rat granulocytes (PMNs) migrating on a glass surface inclined at various angles, i.e. under the action of gravitational force component parallel to the plane. The action of the force of the order of 5 X 10(-13) N (component parallel to the plane inclined at 80 degrees) accompanied by the decrease of a gravitational component perpendicular to the surface does not disrupt the adhesion contact of migrating PMNs with the serum coated glass surface. Under the action of the external force parallel to the surface, the PMNs exhibit a tendency to migrate in the direction of the force vector and the angles between elementary segments (steps) of cell trajectories are smaller in comparison with migration on a horizontal plane (0 degrees inclination). It has been found that the mean velocity of motion of PMNs locomoting on a steep slope (70 degrees and 80 degrees) is greater in comparison with the migration velocity on a horizontal surface. The increase of velocity concerns not only cells migrating in the downward direction, but also those which move upwards. Possible mechanisms of the influence of external force on direction and rate of migration of granulocytes are discussed, namely modification of adhesion force, stimulation of cell motile activity, individual variability of cell adhesive and migration properties, shortening of transient locomotory adhesions.     

The role of exopolymers in the attachment of sphingomonas paucimobilis

The importance of exopolymers in the adhesion of Sphingomonas paucimobilis was established by studying the attachment to glass of three mutants with defective gellan production. The attachment assays were performed in either phosphate buffered saline (controls) or in the exopolymeric solutions produced by the mutants. The exopolymer was found to have surface active properties, changing the glass surface from hydrophilic to hydrophobic, making adhesion thermodynamically favourable. Only the cells that had a substantial polymeric layer surrounding their walls were able to significantly colonise glass coated with the exopolymer. It is hypothesised that the exopolymer bound to the glass and the exopolymer present at the surface of the bacteria bound together, overcoming the energy barrier created by the negative charge of both surfaces. It is concluded that the exopolymer from S. paucimobilis has a dual role in the process of adhesion by both coating the surface thereby strengthening adhesion and by enhancing adhesion through the establishment of polymeric bridges.     

Kinetics of granulocyte phagocytosis: rate limited by cytoplasmic viscosity and constrained by cell size

Micromanipulation of yeast particles and blood granulocytes has been used to study the kinetics of single phagocytosis events. The ingestion process was quantitated by observation of sequential adhesion and encapsulation times. Both adherence and encapsulation times were found to increase greatly as the temperature was reduced below 37 degrees C; calcium in solution facilitated adhesion of the particle to the phagocyte but not encapsulation; both adhesion and encapsulation processes required a minimum level of plasma components (presumably complement). The general nature of these observations were confirmatory of previous studies, but this study is unique in that the specific time course of single particle ingestion was quantitated. It was immediately apparent that the phagocytosis process was 100% efficient above the threshold concentrations required for plasma and temperature, but variations in times from cell to cell indicated heterogeneity in the population. The total time for ingestion varied from as low as 2 sec/particle at 37 degrees C to above several min/particle below 15 degrees C. Encapsulation times for particles were normalized by estimates of particle surface areas to establish a specific time/unit area of particle surface: from 0.5 sec/10(-8) cm2 at 37 degrees C to greater than 8 sec/10(-8) cm2 at 15 degrees C. The temperature dependence of the encapsulation time correlated well with the temperature dependence of the "apparent" viscosity for granulocytes measured by micropipet aspiration. As such, the kinetic properties observed in these phagocytosis tests are consistent with a model that both assembly of the contractile system and the displacement of the surface by active contraction in phagocytosis are limited by viscous dissipation in the cell.(ABSTRACT TRUNCATED AT 250 WORDS)     

Spontaneous and chemoattractant-induced oscillations of cytosolic free calcium in single adherent human neutrophils

Studies with fluorescent Ca2+ indicators in large populations of neutrophils in suspension reveal a stable base line followed by a rapid agonist-induced elevation of cytosolic free calcium, [Ca2+]i, concomitant with other parameters of cellular activation. To study the role of adhesion in cell activation, we monitored [Ca2+]i in single neutrophils adhered to albumin-coated or fibronectin-coated glass coverslips before and after stimulation with the chemotactic peptide N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP). Human neutrophils loaded with 2 microM fura 2/AM were allowed to adhere to coverslips for 15-20 min at 37 degrees C. [Ca2+]i was monitored with a dual excitation microfluorimeter with a time resolution of 200 ms. Statistical analysis was performed using an algorithm allowing to detect significant [Ca2+]i peaks. 54% of the cells showed spontaneous [Ca2+]i oscillations. The amplitude of these [Ca2+]i peaks averaged 77 +/- 10 nM above basal levels (mean value of 110 +/- 20 nM), and their mean duration was 28 +/- 5 s; periods of [Ca2+]i bursts could last up to 15 min. In "silent" cells exhibiting a stable [Ca2+]i base line without spontaneous oscillations, low concentrations of fMLP (10(-10)-10(-9) M) could induce sustained [Ca2+]i oscillations. By contrast, higher agonist concentrations (10(-6) M) induced a single [Ca2+]i transient followed by a stable base line. 47% of the cells showing spontaneous [Ca2+]i oscillations did not respond to fMLP. Spontaneous [Ca2+]i oscillations depended on the continuous presence of extracellular Ca2+. Therefore: (i) spontaneous oscillations of [Ca2+]i occur in neutrophils adherent to various substrata; (ii) these oscillations do not preclude and can be dissociated from the response to fMLP; (iii) neutrophil functions might be controlled by [Ca2+]i oscillations rather than by sustained alterations of [Ca2+]i.     

Affinity of red blood cell membrane for particle surfaces measured by the extent of particle encapsulation.

An experimental technique and a simple analysis are presented that can be used to quantitate the affinity of red blood cell membrane for surfaces of small beads or microsomal particles up to 3 micrometers Diam. The technique is demonstrated with an example of dextran-mediated adhesion of small spherical red cell fragments to normal red blood cells. Cells and particles are positioned for contact by manipulation with glass micropipets. The mechanical equilibrium of the adhesive contact is represented by the variational expression that the decrease in interfacial free energy due to a virtual increase in contact area is balanced by the increase in elastic energy of the membrane due to virtual deformation. The surface affinity is the reduction in free energy per unit area of the interface associated with the formation of adhesive contact. From numerical computations of equilibrium configurations, the surface affinity is derived as a function of the fractional extent of particle encapsulation. The range of surface affinities for which the results are applicable is increased over previous techniques to several times the value of the elastic shear modulus. It is shown that bending rigidity of the membrane has little effect on the analytical results for particles 1--3 micrometers Diam and that results are essentially the same for both cup- and disk-shaped red cells. A simple analytical model is shown to give a good approximation for surface affinity (normalized by the elastic shear modulus) as a function of the fractional extent of particle encapsulation. The model predicts that a particle would be almost completely vacuolized for surface affinities greater than or equal to 10 times the elastic shear modulus. Based on an elastic shear modulus of 6.6 x 10(-3) dyn/cm, the range for the red cell-particle surface affinity as measured by this technique is from approximately 7 x 10(-4) to 7 x 10(-2) erg/cm2. Also, an approximate relation is derived for the level of surface affinity necessary to produce particle vacuolization by a phospholipid bilayer surface which possesses bending rigidity and a fixed tension.     

The physico-chemical characterization of casein-modified surfaces and their influence on the adhesion of spores from a Geobacillus species

To gain a better understanding of the factors influencing spore adhesion in dairy manufacturing plants, casein-modified glass surfaces were prepared and characterized and their effect on the adhesion kinetics of spores from a Geobacillus sp., isolated from a dairy manufacturing plant (DMP) was assessed using a flow chamber. Surfaces were produced by initially silanizing glass using (3-glycidyloxypropyl) trimethoxysilane (GPS) or (3-aminopropyl) triethoxysilane to form epoxy-functionalized (G-GPS) or amino-functionalized glass (G-NH(2)) substrata. Casein was grafted to the G-GPS directly by its primary amino groups (G-GPS-casein) or to G-NH(2) by employing glutaraldehyde as a linking agent (G-NH(2)-glutar-casein). The surfaces were characterised using streaming potential measurements, contact angle goniometry, infrared spectroscopy and scanning electron microscopy. The attachment rate of spores suspended in 0.1?M KCl at pH 6.8, was highest on the positively charged (+14 mV) G-NH(2) surface (333 spores cm(-2) s(-1)) compared to the negatively charged glass (-22 mV), G-GPS (-20 mV) or G-GPS-casein (-21 mV) surfaces (162, 17 or 6 spores cm(-2) s(-1) respectively). Whilst there was a clear decrease in attachment rate to negatively charged casein-modified surfaces compared to the positively charged amine surface, there was no clear relationship between surface hydrophobicity and spore attachment rate.     

Saturated C21-C33 hydrocarbons are involved in the self-regulation of Pseudomonas fluorescens adhesion to a glass surface

One of the two putative groups of antiadhesions was identified in Pseudomonas fluorescens by the method of gas chromatography-mass spectrometry. A mixture of high-molecular unbranched hydrocarbons (HC) with a chain length from 21 to 33 carbon atoms reduced cell adhesion to a glass surface. These HC accumulated in the culture liquid to a total concentration of 10-15 micrograms/l; the concentrations of individual HC ranged from 0.1 to 3.0 micrograms/l. After the addition of individual HC to the bacterial culture, the number of cells attached to the glass surface decreased. This decrease in cell adhesion was due to the enhanced aggregation of the bacterial cells, which promoted mechanical (hydrodynamic) cell detachment from the surface.     

Spontaneous and forced oscillations of cell membrane of normal human erythrocytes: absence of resonance frequencies in a range of 0.03-500 Hz

The spectra of natural oscillations of human erythrocyte cell membranes were studied experimentally and theoretically. The measurements were carried out at room temperature for both single normal cells and erythrocyte rouleaux in a range of 0.03-500 Hz. The spectra were measured at a resolution better than 1% using two techniques: registration of spontaneous membrane oscillations induced by thermal agitation in the surrounding medium and registration of the amplitude-frequency characteristics of the forced oscillations of erythrocyte elongation induced by a high-frequency electric field with the amplitude harmonic modulation. The spectra measured by both techniques had no resonance frequencies and decreased monotonically with the frequency increase. These results are confirmed by the theory developed for the extracellular excitation mechanisms of membrane oscillations. The spectra of active oscillatory biomechanical processes were measured for comparison. These processes are ciliary beating of human bronchial epithelium and ciliary beating and artificial periodic contractions of the cytoplasm of the ciliate Spirostomum ambiguum. The quality of the resonance lines of the order of 10-20 registered may serve as estimates for the line width in search of the resonance oscillations in erythrocytes induced by active cell processes.     

Measurement of charge transfer during bacterial adhesion to an indium tin oxide surface in a parallel plate flow chamber.

An experimental method is described for the measurement of charge transfer during bacterial adhesion in situ to a transparent, semiconducting indium tin oxide (ITO) coated glass plate in a parallel plate flow chamber. Bacterial adhesion is measured simultaneously with either the electric potential or the capacitance of the surface. Initial bacterial adhesion was accompanied by a change in electric potential of the surface with no measurable change in capacitance. Consequently, it can be assumed that the change in electric potential of the surface is due to charge transfer between bacteria and the surface, and it can be calculated that, on average, a charge of about 10(-14) C per bacterium is exchanged during initial adhesion, which corresponds to only several percent of the total surface charge of a bacterium. Charge transfer could either be to or from the bacterial cell surface, dependent on the bacterial strain involved and the ionic strength used.