Role of glucosyltransferase B in interactions of Candida albicans with Streptococcus mutans and with an experimental pellicle on hydroxyapatite surfaces.

Candida albicans and mutans streptococci are frequently detected in dental plaque biofilms from toddlers afflicted with early childhood caries. Glucosyltransferases (Gtfs) secreted by Streptococcus mutans bind to saliva-coated apatite (sHA) and to bacterial surfaces, synthesizing exopolymers in situ, which promote cell clustering and adherence to tooth enamel. We investigated the potential role Gtfs may play in mediating the interactions between C. albicans SC5314 and S. mutans UA159, both with each other and with the sHA surface. GtfB adhered effectively to the C. albicans yeast cell surface in an enzymatically active form, as determined by scintillation spectroscopy and fluorescence imaging. The glucans formed on the yeast cell surface were more susceptible to dextranase than those synthesized in solution or on sHA and bacterial cell surfaces (P < 0.05), indicating an elevated α-1,6-linked glucose content. Fluorescence imaging revealed that larger numbers of S. mutans cells bound to C. albicans cells with glucans present on their surface than to yeast cells without surface glucans (uncoated). The glucans formed in situ also enhanced C. albicans interactions with sHA, as determined by a novel single-cell micromechanical method. Furthermore, the presence of glucan-coated yeast cells significantly increased the accumulation of S. mutans on the sHA surface (versus S. mutans incubated alone or mixed with uncoated C. albicans; P < 0.05). These data reveal a novel cross-kingdom interaction that is mediated by bacterial GtfB, which readily attaches to the yeast cell surface. Surface-bound GtfB promotes the formation of a glucan-rich matrix in situ and may enhance the accumulation of S. mutans on the tooth enamel surface, thereby modulating the development of virulent biofilms.

Image correlation microscopy for uniform illumination.

Image cross-correlation microscopy is a technique that quantifies the motion of fluorescent features in an image by measuring the temporal autocorrelation function decay in a time-lapse image sequence. Image cross-correlation microscopy has traditionally employed laser-scanning microscopes because the technique emerged as an extension of laser-based fluorescence correlation spectroscopy. In this work, we show that image correlation can also be used to measure fluorescence dynamics in uniform illumination or wide-field imaging systems and we call our new approach uniform illumination image correlation microscopy. Wide-field microscopy is not only a simpler, less expensive imaging modality, but it offers the capability of greater temporal resolution over laser-scanning systems. In traditional laser-scanning image cross-correlation microscopy, lateral mobility is calculated from the temporal de-correlation of an image, where the characteristic length is the illuminating laser beam width. In wide-field microscopy, the diffusion length is defined by the feature size using the spatial autocorrelation function. Correlation function decay in time occurs as an object diffuses from its original position. We show that theoretical and simulated comparisons between Gaussian and uniform features indicate the temporal autocorrelation function depends strongly on particle size and not particle shape. In this report, we establish the relationships between the spatial autocorrelation function feature size, temporal autocorrelation function characteristic time and the diffusion coefficient for uniform illumination image correlation microscopy using analytical, Monte Carlo and experimental validation with particle tracking algorithms. Additionally, we demonstrate uniform illumination image correlation microscopy analysis of adhesion molecule domain aggregation and diffusion on the surface of human neutrophils.

Rheological analysis and measurement of neutrophil indentation.

Aspects of neutrophil mechanical behavior relevant to the formation of adhesive contacts were assessed by measuring the dependence of the contact area between the cell and a spherical substrate under controlled loading. Micropipettes were used to bring neutrophils into contact with spherical beads under known forces, and the corresponding contact area was measured over time. The neutrophil was modeled as a viscous liquid drop with a constant cortical tension. Both the equilibrium state and the dynamics of the approach to equilibrium were examined. The equilibrium contact area increased monotonically with force in a manner consistent with a cell cortical tension of 16-24 pN/microm. The dynamic response matched predictions based on a model of the cell as a growing drop using published values for the effective viscosity of the cell. The contact pressure between the cell and substrate at equilibrium is predicted to depend on the curvature of the contacting substrate, but to be independent of the impingement force. The approach to equilibrium was rapid, such that the time-averaged stress for a two-second impingement was within 20% of the equilibrium value. These results have implications for the role of mechanical force in the formation of adhesive contacts.

Neutrophil adhesive contact dependence on impingement force.

Neutrophil capture and recruitment from the circulation requires the formation of specific receptor/ligand bonds under hydrodynamic forces. In the present study we examine bond formation between beta2-integrins on neutrophils and immobilized ICAM-1 while using micropipettes to control the force of contact between the cell and substrate. Magnesium was used to induce the high affinity conformation of the integrins, and bond formation was assessed by measuring the probability of adhesion during repeated contacts. Increasing the impingement force caused an increase in the contact area and led to a proportional increase in adhesion probability (from approximately 20 to 50%) over the range of forces tested (50-350 pN). In addition, different-sized beads were used to change the force per unit area in the contact zone (contact stress). We conclude that for a given contact stress, the rate of bond formation increases linearly with contact area, but that increasing contact stress results in higher intrinsic rates of bond formation.

Elastic properties of the red blood cell membrane that determine echinocyte deformability.

The natural biconcave shape of red blood cells (RBC) may be altered by injury or environmental conditions into a spiculated form (echinocyte). An analysis is presented of the effect of such a transformation on the resistance of RBC to entry into capillary sized cylindrical tubes. The analysis accounts for the elasticity of the membrane skeleton in dilation and shear, and the local and nonlocal resistance of the bilayer to bending, the latter corresponding to different area strains in the two leaflets of the bilayer. The shape transformation is assumed to be driven by the equilibrium area difference (delta A(0), the difference between the equilibrium areas of the bilayer leaflets), which also affects the energy of deformation. The cell shape is approximated by a parametric model. Shape parameters, skeleton shear deformation, and the skeleton density of deformed membrane relative to the skeleton density of undeformed membrane are obtained by minimization of the corresponding thermodynamic potential. Experimentally, delta A(0) is modified and the corresponding discocyte-echinocyte shape transition obtained by high-pressure aspiration into a narrow pipette, and the deformability of the resulting echinocyte is examined by whole cell aspiration into a larger pipette. We conclude that the deformability of the echinocyte can be accounted for by the mechanical behavior of the normal RBC membrane, where the equilibrium area difference delta A(0) is modified.

Fractional occurrence of defects in membranes and mechanically driven interleaflet phospholipid transport.

The picture of biological membranes as uniform, homogeneous bileaflet structures has been revised in recent times due to the growing recognition that these structures can undergo significant fluctuations both in local curvature and in thickness. In particular, evidence has been obtained that a temporary, localized disordering of the lipid bilayer structure (defects) may serve as a principal pathway for movement of lipid molecules from one leaflet of the membrane to the other. How frequently these defects occur and how long they remain open are important unresolved questions. In this report, we calculate the rate of molecular transport through a transient defect in the membrane and compare this result to measurements of the net transbilayer flux of lipid molecules measured in an experiment in which the lipid flux is driven by differences between the mechanical stress in the two leaflets of the membrane bilayer. Based on this comparison, we estimate the frequency of defect occurrence in the membrane. The occurrence of defects is rare: the probability of finding a defect in 1.0 microm2 of a lecithin membrane is estimated to be approximately 6.0x10(-6). Based on this fractional occurrence of defects, the free energy of defect formation is estimated to be approximately 1.0x10(-19) J. The calculations provide support for a model in which interleaflet transport in membranes is accelerated by mechanically driven lipid flow.

Membrane instability in late-stage erythropoiesis.

During maturation of the red blood cell (RBC) from the nucleated normoblast stage to the mature biconcave discocyte, both the structure and mechanical properties of the cell undergo radical changes. The development of the mechanical stability of the membrane reflects underlying changes in the organization of membrane-associated cytoskeletal proteins, and so provides an assessment of the time course of the development of membrane structural organization. Membrane stability in maturing erythrocytes was assessed by measuring forces required to form thin, tubular, lipid strands (tethers) from the surfaces of mononuclear cells obtained from fresh human marrow samples, marrow reticulocytes, circulating reticulocytes, and mature erythrocytes. Cells were biotinylated and manipulated with a micropipette to form an adhesive contact with a glass microcantilever, which gave a measure of the tethering force. The cell was withdrawn at controlled velocity and aspiration pressure to form a tether from the cell surface. The mean force required to form tethers from marrow reticulocytes and normoblasts was 27 +/- 9 pN, compared to 54 +/- 14 pN for mature cells. The energy of dissociation of the bilayer from the underlying skeleton increases 4-fold between the marrow reticulocyte stage and the mature cell, demonstrating that the mechanical stability of the membrane is not completely established until the very last stages of RBC maturation.

A microcantilever device to assess the effect of force on the lifetime of selectin-carbohydrate bonds.

A microcantilever technique was used to apply force to receptor-ligand molecules involved in leukocyte rolling on blood vessel walls. E-selectin was adsorbed onto 3-microm-diameter, 4-mm-long glass fibers, and the selectin ligand, sialyl Lewis(x), was coupled to latex microspheres. After binding, the microsphere and bound fiber were retracted using a computerized loading protocol that combines hydrodynamic and Hookean forces on the fiber to produce a range of force loading rates (force/time), r(f). From the distribution of forces at failure, the average force was determined and plotted as a function of ln r(f). The slope and intercept of the plot yield the unstressed reverse reaction rate, k(r)(o), and a parameter that describes the force dependence of reverse reaction rates, r(o). The ligand was titrated so adhesion occurred in approximately 30% of tests, implying that >80% of adhesive events involve single bonds. Monte Carlo simulations show that this level of multiple bonding has little effect on parameter estimation. The estimates are r(o) = 0.048 and 0.016 nm and k(r)(o) = 0.72 and 2.2 s(-1) for loading rates in the ranges 200-1000 and 1000-5000 pN s(-1), respectively. Levenberg-Marquardt fitting across all values of r(f) gives r(o) = 0.034 nm and k(r)(o) = 0.82 s(-1). The values of these parameters are in the range required for rolling, as suggested by adhesive dynamics simulations.

Adaptation and survival of surface-deprived red blood cells in mice.

The consequences of lost membrane area for long-term erythrocyte survival in the circulation were investigated. Mouse red blood cells were treated with lysophosphatidylcholine to reduce membrane area, labeled fluorescently, reinfused into recipient mice, and then sampled periodically for 35 days. The circulating fraction of the modified cells decreased on an approximately exponential time course, with time constants ranging from 2 to 14 days. The ratio of volume to surface area of the surviving cells, measured using micropipettes, decreased rapidly over the first 5 days after infusion to within 5% of normal. This occurred by both preferential removal of the most spherical cells and modification of others, possibly due to membrane stress developed during transient trapping of cells in the microvasculature. After 5 days, the cell area decreased with time in the circulation, but the ratio of volume to surface area remained essentially constant. These results demonstrate that the ratio of cell volume to surface area is a major determinant of the ability of erythrocytes to circulate properly.

Theoretical analysis of the effect of the transbilayer movement of phospholipid molecules on the dynamic behavior of a microtube pulled out of an aspirated vesicle.

Observations over extended times of a lipid microtube (tether) formed from a lecithin vesicle have shown that under constant external loads the tether exhibits a continuous slow growth. It is considered that this growth is a consequence of the net transbilayer movement of phospholipid molecules in a direction which relieves the membrane strain resulting from the elastic deformation of the vesicle. The elastic deformation mode responsible for this effect is identified as the relative expansion of the two membrane layers reflecting the non-local contribution to membrane bending. An equation for the consequent rate of transbilayer movement of phospholipid molecules is derived. The dynamic behavior of the system is modeled by including frictional contributions due to interlayer slip and Stokes drag on the glass bead used to form the tether. The general numerical solution reveals a complex dependence of the tether growth rate on the system parameters and a continuous increase in the rate of tether growth at long times. Closed form expressions approximating the system behavior are derived and the conditions under which they can be applied are specified. Modeling the mechanically-driven lipid transport as a simple, stochastic, thermal process, allows the rate of lipid translocation to be related to the equilibrium transbilayer exchange rate of phospholipid molecules. Consideration of experimental results shows that the time constant for mechanically-driven translocation is shorter than the time for diffusion-driven translocation by approximately two orders of magnitude, indicating that lipid translocation is not a simple diffusive process.

Passive mechanical behavior of human neutrophils: effects of colchicine and paclitaxel.

The role of microtubules in determining the mechanical rigidity of neutrophils was assessed. Neutrophils were treated with colchicine to disrupt microtubules, or with paclitaxel to promote formation of microtubules. Paclitaxel caused an increase in the number of microtubules in the cells as assessed by immunofluorescence, but it had no effect on the presence or organization of actin filaments or on cellular mechanical properties. Colchicine at concentrations <1.0 microM caused disruption of microtubular structures, but had little effect on either F-actin or on cellular mechanical properties. Higher concentrations of colchicine disrupted microtubular structure, but also caused increased actin polymerization and increases in cell rigidity. Treatment with 10 microM colchicine increased F-actin content by 17%, the characteristic cellular viscosity by 30%, the dependence of viscosity on shear rate by 10%, and the cortical tension by 18%. At 100 microM colchicine the corresponding increases were F-actin, 25%; characteristic viscosity, 50%; dependence of viscosity on shear rate, 20%; and cortical tension, 21%. These results indicate that microtubules have little influence on the mechanical properties of neutrophils, and that increases in cellular rigidity caused by high concentrations of colchicine are due to a secondary effect that triggers actin polymerization. This study supports the conclusion that actin filaments are the primary structural determinants of neutrophil mechanical properties.

Energy of dissociation of lipid bilayer from the membrane skeleton of red blood cells.

The association between the lipid bilayer and the membrane skeleton is important to cell function. In red blood cells, defects in this association can lead to various forms of hemolytic anemia. Although proteins involved in this association have been well characterized biochemically, the physical strength of this association is only beginning to be studied. Formation of a small cylindrical strand of membrane material (tether) from the membrane involves separation of the lipid bilayer from the membrane skeleton. By measuring the force required to form a tether, and knowing the contribution to the force due to the deformation of a lipid bilayer, it is possible to calculate the additional contribution to the work of tether formation due to the separation of membrane skeleton from the lipid bilayer. In the present study, we measured the tethering force during tether formation using a microcantilever (a thin, flexible glass fiber) as a force transducer. Numerical calculations of the red cell contour were performed to examine how the shape of the contour affects the calculation of tether radius, and subsequently separation work per unit area W(sk) and bending stiffness k(c). At high aspiration pressure and small external force, the red cell contour can be accurately modeled as a sphere, but at low aspiration pressure and large external force, the contour deviates from a sphere and may affect the calculation. Based on an energy balance and numerical calculations of the cell contour, values of the membrane bending stiffness k(c) = 2.0 x 10(-19) Nm and the separation work per unit area W(sk) = 0.06 mJ/m2 were obtained.

Surface area and volume changes during maturation of reticulocytes in the circulation of the baboon.

Changes in the surface area and volume of reticulocytes were measured in vivo during late stage maturation. Baboons were treated with erythropoietin to produce mild reticulocytosis. Reticulocyte-rich cohorts of cells were obtained from whole blood by density gradient centrifugation. The cohorts were labeled with biotin, reinfused into the animal, and recovered from whole blood samples by panning on avidin supports. Changes in the surface area, volume, and membrane deformability were measured using micropipettes during the 2 to 6 weeks subsequent to reinfusion. For the entire cohort, the membrane area decreased by 10% to 15% and the cell volume decreased by approximately 8.5%, mostly within 24 hours after reinfusion. Estimates of the cellular dimensions of the reticulocyte subpopulation within this cohort indicated larger reductions in the mean cell area (12% to 30%) and mean cell volume (approximately 15%) of the reticulocytes themselves. Two weeks after reinfusion, the distribution of cell size for the cohort was indistinguishable from that of whole blood. There was evidence of slightly elevated membrane shear rigidity in some reticulocytes before reinfusion, but this slight increase disappeared within 24 hours after reinfusion. These are the first direct measurements of changes in the membrane physical properties of an identifiable cohort of reticulocytes as they mature in vivo.

Effects of lost surface area on red blood cells and red blood cell survival in mice.

The effects of removing area from mouse red blood cells on the fate of the cells after reinfusion were investigated. When cells were made nearly spherical (by reducing cell area by approximately 35%) and then reinfused into the animal, most were cleared from the circulation within 1-2 h, although approximately 20% of the cells survived for 4 h or longer. When only 20% of the area was removed (leaving a 15% excess), more than 90% of the cells continued to circulate for 4 h. After reinfusion, the mean surface area of the surviving cells remained constant (73-75 microns2), but the mean volume decreased, from 56.6 +/- 2.1 to 19.1 +/- 1.5 microns3 (+/- SD of 5 replicates) over 4 h. These changes did not occur in cells suspended in plasma but not reinfused into the animal. Thus a loss of surface area results in a decrease in cell volume, as if to maintain a requisite degree of deformability. The results support the hypothesis that the increase in cell density associated with increasing cell age may be a consequence of surface area loss.

Accelerated interleaflet transport of phosphatidylcholine molecules in membranes under deformation.

Biological membranes are lamellar structures composed of two leaflets capable of supporting different mechanical stresses. Stress differences between leaflets were generated during micromechanical experiments in which long thin tubes of lipid (tethers) were formed from the surfaces of giant phospholipid vesicles. A recent dynamic analysis of this experiment predicts the relaxation of local differences in leaflet stress by lateral slip between the leaflets. Differential stress may also relax by interleaflet transport of lipid molecules ("flip-flop"). In this report, we extend the former analysis to include interleaflet lipid transport. We show that transmembrane lipid flux will evidence itself as a linear increase in tether length with time after a step reduction in membrane tension. Multiple measurements were performed on 24 different vesicles composed of stearoyl-oleoyl-phosphatidylcholine plus 3% dinitrophenol-linked di-oleoyl-phosphatidylethanolamine. These tethers all exhibited a linear phase of growth with a mean value of the rate of interlayer permeation, cp = 0.009 s-1. This corresponds to a half-time of approximately 8 min for mechanically driven interleaflet transport. This value is found to be consistent with longer times obtained for chemically driven transport if the lipids cross the membrane via transient, localized defects in the bilayer.

A piconewton force transducer and its application to measurement of the bending stiffness of phospholipid membranes.

The bending stiffness of a phospholipid bilayer (Kc) was measured by forming thin bilayer cylinders (tethers) from giant phospholipid vesicles. Based on the balance of forces, the tether force was expected to be proportional to the square root of the membrane tension, with a constant of proportionality containing Kc. The membrane tension was controlled via the aspiration pressure in a micropipette used to hold the vesicle. The force on the tether was generated by an electromagnet acting on a paramagnetic bead attached to the vesicle surface. The magnitude of the force was determined from measurements of the magnet current, which was adjusted to maintain the position of the bead. Measurements were performed on vesicles composed of stearoyl-oleoyl-phosphatidylcholine plus 5% (by mole) biotinylated phosphatidylethanolamine to mediate adhesion to streptavidin-coated beads. From each vesicle, tethers were formed repeatedly at different values of the membrane tension. The expected relationship between membrane tension and tether force was observed. The mean value of Kc for 10 different vesicles was 1.17 x 10(-19) J (SD = 0.08 x 10(-19) J). The precision of these data demonstrates the reliability of this approach, which avoids uncertainties of interpretation and measurement that may be associated with other methods for determining Kc.

Cell cycle-dependence of HL-60 cell deformability.

In this study, the role of cytoskeleton in HL-60 deformability during the cell cycle was investigated. G1, S, and G2/M cell fractions were separated by centrifugal elutriation. Cell deformability was evaluated by pipette aspiration. Tested at the same aspiration pressures, S cells were found to be less deformable than G1 cells. Moreover, HL-60 cells exhibited power-law fluid behavior: mu = mu c(gamma m/ gamma c)-b, where mu is cytoplasmic viscosity, gamma m is mean shear rate, mu c is the characteristic viscosity at the characteristic shear rate gamma c, and b is a material constant. At a given shear rate, S cells (mu c = 276 +/- 14 Pa.s, b = 0.51 +/- 0.03) were more viscous than G1 cells (mu c = 197 +/- 25, b = 0.53 +/- 0.02). To evaluate the relative importance of different cytoskeletal components in these cell cycle-dependent properties, HL-60 cells were treated with 30 microM dihydrocytochalasin B (DHB) to disrupt F-actin or 100 microM colchicine to collapse microtubules. DHB dramatically softened both G1 and S cells, which reduced the material constants mu c by approximately 65% and b by 20-30%. Colchicine had a limited effect on G1 cells but significantly reduced mu c of S cells (approximately 25%). Thus, F-actin plays the predominate role in determining cell mechanical properties, but disruption of microtubules may also influence the behavior of proliferating cells in a cell cycle-dependent fashion.

Elastic energy of curvature-driven bump formation on red blood cell membrane.

Model calculations were performed to explore quantitative aspects of the discocyte-echinocyte shape transformation in red blood cells. The shape transformation was assumed to be driven by changes in the preferred curvature of the membrane bilayer and opposed by the elastic shear rigidity of the membrane skeleton. The energy required for echinocyte bump formation was calculated for a range of bump shapes for different preferred curvatures. Energy minima corresponding to nonzero bump heights were found when the stress-free area difference between the membrane leaflets or the spontaneous curvature of the membrane became sufficiently large, but the calculations predict that the membrane can tolerate significant differences in the resting areas of the inner and outer leaflets or significant spontaneous curvature without visible changes in shape. Thus, if the cell is near the threshold for bump formation, the calculations predict that small changes in membrane properties would produce large changes in cellular geometry. These results provide a rational framework for interpreting observations of shape transformations in red cells and for understanding the mechanism by which small changes in membrane elastic properties might lead to significant changes in geometry.

Remodeling the shape of the skeleton in the intact red cell.

The role of the membrane skeleton in determining the shape of the human red cell was probed by weakening it in situ with urea, a membrane-permeable perturbant of spectrin. Urea by itself did not alter the biconcave disk shape of the red cell; however, above threshold conditions (1.5 M, 37 degrees C, 10 min), it caused an 18% reduction in the membrane elastic shear modulus. It also potentiated the spiculation of cells by lysophosphatidylcholine. These findings suggest that the contour of the resting cell is not normally dependent on the elasticity of or tension in the membrane skeleton. Rather, the elasticity of the skeleton stabilizes membranes against deformation. Urea treatment also caused the projections induced both by micropipette aspiration and by lysophosphatidylcholine to become irreversible. Furthermore, urea converted the axisymmetric conical spicules induced by lysophosphatidylcholine into irregular, curved and knobby spicules; i.e., echinocytosis became acanthocytosis. Unlike controls, the ghosts and membrane skeletons obtained from urea-generated acanthocytes were imprinted with spicules. These data suggest that perturbing interprotein associations with urea in situ allowed the skeleton to evolve plastically to accommodate the contours imposed upon it by the overlying membrane.

Changes in HL-60 cell deformability during differentiation induced by DMSO.

We have investigated changes in cellular deformability during promyelocytic leukemic HL-60 cell maturation. HL-60 cells were induced to mature with 1.25% dimethyl sulfoxide. Cellular deformability was evaluated by single-cell micropipette aspiration at one day, four days and seven days after induction. HL-60 cells were found to decrease in size and increase in deformability with maturation. When tested under the same aspiration pressures (0.5-1.3 kPa), cytoplasmic viscosity was found to vary from 210 to 85 Pa.s for cells prior to induction; it varied from 85 to 40 Pa.s for cells seven days after induction. Further, cytoplasmic viscosity exhibits power-law dependence on shear rate, mu = mu c (gamma m/gamma c)-b, where mu is cytoplasmic viscosity, gamma m is mean shear rate during cell entry, mu c is the characteristic viscosity at the characteristic shear rate, gamma c, and b is a material coefficient. Cells of all maturities showed similar dependence on shear rate (b approximately 0.5), but the characteristic viscosity decreased with maturation except for Day 1. When gamma c was set to 1 s-1, mu c = 236 +/- 5 Pa.s for cells prior to induction, mu c = 239 +/- 7, 209 +/- 7 and 175 +/- 14 Pa.s for cells on Days 1, 4 and 7 of induction, respectively.