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R E Waugh

Publications and source records attributed to R E Waugh.

At least 37 records · Page 2Linked to original sources
PubMed1993

Passive mechanical behavior of human neutrophils: power-law fluid.

The mechanical behavior of the neutrophil plays an important role in both the microcirculation and the immune system. Several laboratories in the past have developed mechanical models to describe different aspects of neutrophil deformability. In this study, the passive mechanical properties of normal human neutrophils have been further characterized. The cellular mechanical properties were assessed by single cell micropipette aspiration at fixed aspiration pressures. A numerical simulation was developed to interpret the experiments in terms of cell mechanical properties based on the Newtonian liquid drop model (Yeung and Evans, Biophys. J., 56: 139-149, 1989). The cytoplasmic viscosity was determined as a function of the ratio of the initial cell size to the pipette radius, the cortical tension, aspiration pressure, and the whole cell aspiration time. The cortical tension of passive neutrophils was measured to be about 2.7 x 10(-5) N/m. The apparent viscosity of neutrophil cytoplasm was found to depend on aspiration pressure, and ranged from approximately 500 Pa.s at an aspiration pressure of 98 Pa (1.0 cm H2O) to approximately 50 Pa.s at 882 Pa (9.0 cm H2O) when tested with a 4.0-micron pipette. These data provide the first documentation that the neutrophil cytoplasm exhibits non-Newtonian behavior. To further characterize the non-Newtonian behavior of human neutrophils, a mean shear rate gamma m was estimated based on the numerical simulation. The apparent cytoplasmic viscosity appears to decrease as the mean shear rate increases. The dependence of cytoplasmic viscosity on the mean shear rate can be approximated as a power-law relationship described by mu = mu c(gamma m/gamma c)-b, where mu is the cytoplasmic viscosity, gamma m is the mean shear rate, mu c is the characteristic viscosity at characteristic shear rate gamma c, and b is a material coefficient. When gamma c was set to 1 s-1, the material coefficients for passive neutrophils were determined to be mu c = 130 +/- 23 Pa.s and b = 0.52 +/- 0.09 for normal neutrophils. The power-law approximation has a remarkable ability to reconcile discrepancies among published values of the cytoplasmic viscosity measured using different techniques, even though these values differ by nearly two orders of magnitude. Thus, the power-law fluid model is a promising candidate for describing the passive mechanical behavior of human neutrophils in large deformation. It can also account for some discrepancies between cellular behavior in single-cell micromechanical experiments and predictions based on the assumption that the cytoplasm is a simple Newtonian fluid.

Biomechanical Phenomena↗
PubMed1993

Bending rigidity of SOPC membranes containing cholesterol.

Bilayer membranes in the fluid state exhibit a large resistance to changes in surface area, negligible resistance to surface shear deformation, and a small but finite resistance to bending. The presence of cholesterol in the membrane is known to increase its resistance to area dilation. In this report, a new method for measuring bilayer membrane bending stiffness has been used to investigate the effect of cholesterol on the bending rigidity of SOPC (1,stearoyl-2,oleoyl-phosphatidylcholine) membranes. The curvature elasticity (kc) for membranes saturated with cholesterol was measured to be 3.3 x 10(-19) J, approximately 3-fold larger than that the modulus for cholesterol-free SOPC membrane. These findings are consistent with previous measurements of bending stiffness based on thermal fluctuations, which showed a similar approximately 3-fold increase in the modulus with cholesterol addition (Evans and Rawicz, 1990, Phys. Rev. Lett. 64:2094) and provide further substantiation of the important contribution that cholesterol makes to membrane cohesion and stability.

Cholesterol↗
PubMed1992

Rheologic properties of senescent erythrocytes: loss of surface area and volume with red blood cell age.

The rheologic properties of senescent erythrocytes have been examined using two models of red blood cell (RBC) aging. In the rabbit, aged erythrocytes were isolated after biotinylation, in vivo aging, and subsequent recovery on an avidin support. Aged RBCs from the mouse were obtained using the Ganzoni hypertransfusion model that suppresses erythropoiesis for prolonged periods of time allowing preexisting cells to age in vivo. In both cases, the aged erythrocytes were found by ektacytometry to have decreased deformability due to diminished surface area and cellular dehydration. The aged rabbit erythrocytes were further characterized by micropipette methods that documented an average surface area decrease of 10.5% and a volume decrease of 8.4% for the cells that were 50 days old. Because both the surface area and volume decreased with cell age, there was little change in surface-to-volume ratio (sphericity) during aging. The aged cells were found to have normal membrane elasticity. In addition, human RBCs were fractionated over Stractan density gradients and the most dense cells were found to have rheologic properties similar to those reported for the aged RBCs from rabbits and mice, although the absolute magnitude of the changes in surface area and volume were considerably greater for the human cells. Thus, stringent density fractionation protocols that result in isolation of the most dense 1% of cells can produce a population of human cells with rheologic properties similar to senescent cells obtained in other species. The data indicate that progressive loss of cell area and cell dehydration are characteristic features of cell aging.

Animals↗
PubMed1992

Role of lamellar membrane structure in tether formation from bilayer vesicles.

A theoretical analysis is presented of the formation of membrane tethers from micropipette-aspirated phospholipid vesicles. In particular, it is taken into account that the phospholipid membrane is composed of two layers which are in contact but unconnected. The elastic energy of the bilayer is taken to be the sum of contributions from area expansivity, relative expansivity of the two monolayers, and bending. The vesicle is aspirated into a pipette and a constant point force is applied at the opposite side in the direction away from the pipette. The shape of the vesicle in approximated as a cylindrical projection into the pipette with a hemispherical cap, a spherical section, and a cylindrical tether with a hemispherical cap. The dimensions of the different regions of the vesicle are obtained by minimizing its elastic energy subject to the condition that the volume of the vesicle is fixed. The range of values for the parameters of the system is determined at which the existence of a tether is possible. Stability analysis is performed showing which of these configurations are stable. The importance of the relative expansion and compression of the constituent monolayers is established by recognizing that local bending energy by itself does not stabilize the vesicle geometry, and that in the limit as the relative expansivity modulus becomes infinitely large, a tether cannot be formed. Predictions are made for the functional relationships among experimentally observable quantities. In a companion report, the results of this analysis are applied to experimental measurements of tether formation, and used to calculate values for the membrane material coefficients.

Biophysical Phenomena↗
PubMed1992

Local and nonlocal curvature elasticity in bilayer membranes by tether formation from lecithin vesicles.

Bilayer membranes exhibit an elastic resistance to changes in curvature. This resistance depends both on the intrinsic stiffness of the constituent monolayers and on the curvature-induced expansion or compression of the monolayers relative to each other. The monolayers are constrained by hydrophobic forces to remain in contact, but they are capable of independent lateral redistribution to minimize the relative expansion or compression of each leaflet. Therefore, the magnitude of the expansion and compression of the monolayers relative to each other depends on the integral of the curvature over the entire membrane capsule. The coefficient characterizing the membrane stiffness resulting from relative expansion is the nonlocal bending modulus kr. Both the intrinsic (local) bending modulus (kc) and the nonlocal bending modulus (kr) can be measured by the formation of thin cylindrical membrane strands (tethers) from giant phospholipid vesicles. Previously, we reported measurements of kc based on measurements of tether radius as a function of force (Song and Waugh, 1991, J. Biomech. Engr. 112:233). Further analysis has revealed that the contribution from the nonlocal bending stiffness can be detected by measuring the change in the aspiration pressure required to establish equilibrium with increasing tether length. Using this approach, we obtain a mean value for the nonlocal bending modulus kr of approximately 4.1 x 10(-19)J. The range of values is broad (1.1-10.1 x 10(-19)J) and could reflect contributions other than simple mechanical equilibrium. Inclusion of the nonlocal bending stiffness in the calculation of kc results in a value for that modulus of approximately 1.20 +/- 0.17 x 10(-19)J, in close agreement with values obtained by other methods.

Biophysical Phenomena↗
PubMed1991

Reticulocyte rigidity and passage through endothelial-like pores.

The importance of cell rigidity in regulating the release of reticulocytes from the bone marrow has been investigated in a model system. Reticulocytes were obtained from phlebotomized rabbits and separated from whole blood by discontinuous density gradient centrifugation. The mechanical properties of the cells were tested. Using single-cell micromechanical techniques, the membrane elastic rigidity and the viscoelastic response of reticulocyte and mature cell populations were measured. The reticulocyte membranes were more rigid than the mature membranes, but the reticulocyte properties were heterogeneous, and some cells exhibited behavior indistinguishable from the mature cells. The mean time constant for viscoelastic recovery was the same for reticulocytes as for mature cells, but the variability within the reticulocyte population was greater. The possible influence of this increased rigidity on cell egress from the bone marrow was tested using an in vitro model of the thin endothelial pores found within the marrow. A silicon wafer approximately 0.1 microns in thickness and containing a small (1.2-microns diameter) pore in its center was cemented over the tip of a large (15.0-microns ID) micropipette. The passage of cells through the pore was observed as a function of the pressure across the pore. Consistent with the difference in mechanical properties, the reticulocytes required greater pressures (as great as 4.0 mm Hg compared with less than 1.0 mm Hg) and took longer to traverse the pore. These measurements support the postulate that deformability is important in the regulation of the release of cells from bone marrow.

Animals↗
PubMed1990

Bilayer membrane bending stiffness by tether formation from mixed PC-PS lipid vesicles.

Recently, a new approach to measure the bending stiffness (curvature elastic modulus) of lipid bilayer membrane was developed (Biophys. J., Vol. 55; pp. 509-517, 1989). The method involves the formation of cylindrical membrane strands (tethers) from bilayer vesicles. The bending stiffness (B) can be calculated from measurements of the tether radius (Rt) as a function of the axial force (f) on the tether: B = f.Rt/2 pi. In the present report, we apply this method to determine the bending stiffness of bilayer membranes composed of mixtures of SOPC (1-stearoyl-2-oleoyl phosphatidyl choline) and POPS (1-palmitoyl-2-oleoyl phosphatidyl serine). Three different mixtures were tested: pure SOPC, SOPC plus 2 percent (mol/mol) POPS, and SOPC plus 16 percent POPS. The bending stiffness determined for these three different lipid mixtures were not significantly different (1.6-1.8 x 10(-12) ergs). Because POPS carries a net negative charge, these results indicate that changes in the density of the membrane surface charge have no effect on the intrinsic rigidity of the membrane. The values we obtain are consistent with published values for the bending stiffness of other membranes determined by different methods. Measurements of the aspiration pressure, tether radius and the tether force were used to verify a theoretical relationship among these quantities at equilibrium. The ratio of the theoretical force to the measured force was 1.12 +/- 0.17.

Biomechanical Phenomena↗
PubMed1989

Determination of bilayer membrane bending stiffness by tether formation from giant, thin-walled vesicles.

The curvature elastic modulus (bending stiffness) of stearoyloleoyl phosphatidylcholine (SOPC) bilayer membrane is determined from membrane tether formation experiments. R. E. Waugh and R. M. Hochmuth 1987. Biophys. J. 52:391-400) have shown that the radius of a bilayer cylinder (tether) is inversely related to the force supported along its axis. The coefficient that relates the axial force on the tether to the tether radius is the membrane bending stiffness. Thus, the bending stiffness can be calculated directly from measurements of the tether radius as a function of force. Giant (10-50-microns diam) thin-walled vesicles were aspirated into a micropipette and a tether was pulled out of the surface by gravitational forces on small glass beads that had adhered to the vesicle surface. Because the vesicle keeps constant surface area and volume, formation of the tether requires displacement of material from the projection of the vesicle in the pipette. Tethers can be made to grow longer or shorter or to maintain equilibrium by adjusting the aspiration pressure in the micropipette at constant tether force. The ratio of the change in the length of the tether to the change in the projection length is proportional to the ratio of the pipette radius to the tether radius. Thus, knowing the density and diameter of the glass beads and measuring the displacement of the projection as a function of tether length, independent determinations of the force on the tether and the tether radius were obtained. The bending stiffness for an SOPC bilayer obtained from these data is approximately 2.0 x 10(-12) dyn cm, for tether radii in the range of 20-100 nm. An equilibrium relationship between pressure and tether force is derived which closely matches experimental observation.

Elasticity↗
PubMed1989

Flexural rigidity of marginal bands isolated from erythrocytes of the newt.

The marginal band is a bundle of microtubules residing at the periphery of nucleated erythrocytes of nonmammalian vertebrates and some invertebrates. Marginal bands from erythrocytes of the newt (Notopthalmus viridescens) were isolated from the cells as intact structures by treatment with detergent and either mild protease or high salt. Isolated bands were subjected to mechanical testing by stretching the band between a glass microhook and a calibrated glass fiber. The deflection of the fiber provided a measure of the force on the band. The flexural rigidity of the band was determined from measurements of the band deformation as a function of applied force. Bands isolated with either of two proteases (pepsin or elastase) or with high salt exhibited elastic behavior with a flexural rigidity of approximately 9.0 X 10(-12) dyn.cm2. Treatment of bands with chymopapain caused an increase in band rigidity and inelastic behavior. Estimates of the contribution of the band to cellular rigidity are made based on the measurements of the structural properties of the isolated band. The band provides the cell with a large resistance to indentations at the rim and to large extensions, while maintaining a high degree of flexibility in small extensions or flexure.

Animals↗
PubMed1988

Reductions of erythrocyte membrane viscoelastic coefficients reflect spectrin deficiencies in hereditary spherocytosis.

Hereditary spherocytosis is a common hemolytic anemia associated with deficiencies in spectrin, the principal structural protein of the erythrocyte membrane-skeleton. We have examined 20 different individuals from 10 spherocytosis kindreds and 2 elliptocytosis kindreds to determine the effects of different levels of spectrin deficiency on the viscoelastic properties of the erythrocyte membrane. Micropipettes were used to perform single-cell micromechanical measurements of approximately 1,000 individual cells to determine the membrane elastic shear modulus, the apparent membrane bending stiffness, and whole cell recovery time constant for the different cell populations. The membrane viscosity was calculated by the product of the shear modulus and the recovery time constant. Results show correlation between the fractional reduction in shear modulus and the fractional reduction in spectrin content (determined by spectrin radioimmunoassay) and spectrin density (determined by the ratios of spectrin to band 3 on electrophoresis gels) suggesting that membrane shear elasticity is directly proportional to the surface density of spectrin on the membrane (P less than 0.001). The apparent membrane bending stiffness is also reduced in proportion to the density of spectrin (P less than 0.001). The membrane viscosity is reduced relative to control (P less than 0.001), but the nature of the relationship between spectrin density and membrane viscosity is less clearly defined. These studies document striking relationships between partial deficiencies of erythrocyte spectrin and specific viscoelastic properties of the mutant membranes.

Elasticity↗
PubMed1987

Effects of intracellular Ca2+ and proteolytic digestion of the membrane skeleton on the mechanical properties of the red blood cell membrane.

Intracellular Ca2+ at concentrations ranging from 0 to 10 mumol/l increases the shear modulus of surface elasticity (mu) and the surface viscosity (eta) of human red blood cells by 20% and 70%, respectively. K+ selective channels in the red cell membrane become activated by Ca2+. The activation still occurs to the same extent when the membrane skeleton is degraded by incorporation of trypsin into resealed red cell ghosts, suggesting that the channel activation is not controlled by the proteins of the membrane skeleton and is independent of mu and eta. Incorporation of trypsin at concentrations ranging from 0 to 100 ng/ml into red cell ghosts leads to a graded digestion of spectrin, a cleavage of the band 3 protein and a release of the binding proteins ankyrin and band 4.1. These alterations are accompanied by an increase of the lateral mobility of the band 3 protein which, at 40 ng/ml trypsin, reaches a plateau value where the rate of lateral diffusion is enhanced by about two orders of magnitude above the rate measured in controls without trypsin. Proteolytic digestion by 10-20 ng/ml trypsin leads to a degradation of more than 40% of the spectrin and increases the rate of lateral diffusion to about 20-70% of the value observed at the plateau. Nevertheless, mu and eta remain virtually unaltered. However, the stability of the membrane is decreased to the point where a slight mechanical extension, or the shear produced by centrifugation results in disintegration and vesiculation, precluding measurements of eta and mu in ghosts treated with higher concentrations of trypsin. These findings indicate that alterations of the structural integrity of the membrane skeleton exert drastically different effects on mu and eta on the one hand and on the stability of the membrane on the other.

Calcium↗
PubMed1987

Mechanical equilibrium of thick, hollow, liquid membrane cylinders.

The mechanical equilibrium of bilayer membrane cylinders is analyzed. The analysis is motivated by the observation that mechanically formed membrane strands (tethers) can support significant axial loads and that the tether radius varies inversely with the axial force. Previously, thin shell theory has been used to analyze the tether formation process, but this approach is inadequate for describing and predicting the equilibrium state of the tether itself. In the present work the membrane is modeled as two adjacent, thick, anisotropic liquid shells. The analysis predicts an inverse relationship between axial force and tether radius, which is consistent with experimental observation. The area expansivity modulus and bending stiffness of the tether membrane are calculated using previously measured values of tether radii. These calculated values are consistent with values of membrane properties measured previously. Application of the analysis to precise measurements of the relationship between tether radius and axial force will provide a novel method for determining the mechanical properties of biomembrane.

Kinetics↗
PubMed1987

Effects of inherited membrane abnormalities on the viscoelastic properties of erythrocyte membrane.

Several workers have identified molecular abnormalities associated with inherited blood disorders. The present work examines how these alterations in molecular structure affect the viscoelastic properties of the red blood cell membrane. Changes in the membrane shear modulus, the membrane viscosity, and the apparent membrane bending stiffness were observed in cells of eight patients having a variety of disorders: Two had reductions in the number of high-affinity ankyrin binding sites, two had abnormalities associated with the protein band 4.1, and six were known to be deficient in spectrin. The data suggest that the membrane shear modulus is proportional to the density of spectrin on the membrane and support the view that spectrin is primarily responsible for membrane shear elasticity. Although membranes having abnormalities associated with the function of ankyrin or band 4.1 exhibited reduced elasticity, the degree of mechanical dysfunction was quantitatively inconsistent with the extent of the molecular abnormality. This indicates that these skeletal components do not play a primary role in determining membrane shear elasticity. The membrane viscosity was reduced in seven of the eight patients studied. The reduction in viscosity was usually greater than the reduction in shear modulus, but the degree of reduction in viscosity was variable and did not correlate well with the degree of molecular abnormality.

Ankyrins↗
PubMed1986

Measurement of the extensional and flexural rigidities of a subcellular structure: marginal bands isolated from erythrocytes of the newt.

The elastic properties of marginal band, a microtubular structure isolated from the newt (notophthalmus viridescens) have been measured. Bands were isolated using Triton X-100 and pepsin at pH 6.8 according to the method of Cohen (1978). Isolated bands were manipulated with a glass microhook in a buffer-filled chamber under the microscope. Single bands were stretched between the hook and a thin glass fiber. The fiber was calibrated so that the force on the band could be calculated from the displacement of the fiber. The data pairs of force versus band deflection were analyzed according to the theoretical work of Libai and Simmonds (1983) to obtain the flexural and extensional rigidities of the band. Band dimensions calculated from the data were consistent with microscopically determined values. The average flexural rigidity of the bands (EI) was 5.3 X 10(-13) dyn X cm2 and the average extensional rigidity (EA) was 0.017 dyn. Compared to the cell membrane, the marginal band is nearly inextensible and has a much greater resistance to bending, indicating that the band makes an important contribution to the deformability of the circulating cell.

Animals↗
PubMed1986

Effects of 2,3-diphosphoglycerate on the mechanical properties of erythrocyte membrane.

Investigation by Schindler et al and Sheetz and Casaly have indicated that high (approximately 10 mmol/L) concentrations of 2,3-diphosphoglycerate (2,3-DPG) have a destabilizing effect on erythrocyte membrane and the membrane skeleton. We have investigated changes in the membrane mechanical properties that occur at elevated 2,3-DPG levels in both intact cells and ghosts. The membrane shear modulus, viscoelastic recovery time constant, critical force, "plastic" viscosity, and material relaxation time constant were measured by standard micropipette and flow channel techniques. Intact cells showed no change in properties at physiologic ionic strength and 2,3-DPG concentrations of about 20 mmol/L, except for an increase in membrane viscosity resulting from an increased cellular hemoglobin concentration that occurs when the 2,3-DPG concentration is elevated. At ionic strengths 20% below physiologic and 2,3-DPG concentrations of approximately 20 mmol/L, decreases in membrane shear modulus and membrane viscosity were observed. In ghosts, no changes in these properties were observed at a 2,3-DPG concentration of 10 mmol/L and ionic strengths as low as 25% below physiologic, but a decrease in the force required to form tethers (critical force) was observed at physiologic ionic strength. The decrease in membrane shear modulus and viscosity of intact cells and the reduced critical force in ghosts are consistent with the results of other investigators. However, the difference in the effects of 2,3-DPG on ghosts and intact cells indicates that the effects of 2,3-DPG depend strongly on the conditions of the experiment. It appears unlikely that 2,3-DPG affects erythrocyte membrane material properties under physiologic conditions.

2,3-Diphosphoglycerate↗
PubMed1986

An in vitro model of erythroid egress in bone marrow.

An in vitro system has been developed that mimics the passage of erythrocytes from the bone marrow to the circulation. Bone marrow egress and its proper regulation are vital physiologic processes. However, because of the inaccessibility of the marrow, it is difficult to evaluate the various factors important in controlling these processes or even to define the precise mechanism by which egress occurs. The in vitro system has been designed to evaluate the importance of different physical parameters in regulating egress. It consists of a thin silicon wafer (thickness approximately equal to 1.0 micron) cemented over the tip of a large (15.0 micron ID) micropipette. The wafer contains a single circular pore. Cells were observed under the microscope as they passed through the pore under controlled pressures. The rate and duration of passage were obtained from videorecordings of the experiment. The measured passage times agreed well with the predictions of a simple analytical model of a cell passing through a thin aperture. The experimental results confirm the conclusion reached from the analysis that the pressures needed to drive a cell through the pore are well within the physiologic range, and the time needed to complete egress is typically less than 1.0 seconds. These results support the hypothesis that erythrocyte egress may be driven by a hydrostatic pressure difference across the pore.

Bone Marrow↗