Fluctuations of the red blood cell membrane: relation to mechanical properties and lack of ATP dependence

We have analyzed the fluctuations of the red blood cell membrane in both the temporal ((ω(s⁻¹)) and spatial (q(m⁻¹)) frequency domains. The cells were examined over a range of osmolarities leading to cell volumes from 50% to 170% of that in the isotonic state. The fluctuations of the isotonic cell showed an ∼q⁻³-dependence, indicative of a motion dominated by bending, with an inferred bending modulus of ∼9 × 10⁻¹⁹J. When the cells were osmotically swollen to just below the point of lysis (166% of physiological volume), a q⁻¹-dependence of the fluctuations supervened, implying that the motion was now dominated by membrane tension; estimated as ∼1.3 × 10⁻⁴ nm⁻¹. When, on the other hand, the cells were osmotically dehydrated, the fluctuation amplitude progressively decreased. This was caused by a rise in internal viscosity, as shown by measurements on resealed ghosts containing a reduced hemoglobin concentration, which displayed no such effect. We examined, in addition, cells depleted of ATP, before the onset of echinocytosis, and could observe no change in fluctuation amplitude. We conclude that the membrane fluctuations of the red cell are governed by bending modulus, membrane tension, and cytosolic viscosity, with little or no dependence on the presence or absence of ATP.     

Interaction between bending and tension forces in bilayer membranes.

A theoretical analysis is presented of the bending mechanics of a membrane consisting of two tightly-coupled leaflets, each of which shears and bends readily but strongly resists area changes. Structures of this type have been proposed to model biological membranes such as red blood cell membrane. It is shown that when such a membrane is bent, anisotropic components of resultant membrane tension (shear stresses) are induced, even when the tension in each leaflet is isotropic. The induced shear stresses increase as the square of the membrane curvature, and become significant for moderate curvatures (when the radius of curvature is much larger than the distance between the leaflets). This effect has implications for the analysis of shape and deformation of freely suspended and flowing red blood cells.     

Theoretical model and experimental study of red blood cell (RBC) deformation in microchannels

The motion and deformation of red blood cells (RBCs) flowing in a microchannel were studied using a theoretical model and a novel automated rheoscope. The theoretical model was developed to predict the cells deformation under shear as a function of the cells geometry and mechanical properties. Fluid dynamics and membrane mechanics are incorporated, calculating the traction and deformation in an iterative manner. The model was utilized to evaluate the effect of different biophysical parameters, such as: inner cell viscosity, membrane shear modulus and surface to volume ratio on deformation measurements. The experimental system enables the measurement of individual RBCs velocity and their deformation at defined planes within the microchannel. Good agreement was observed between the simulation results, the rheoscope measurements and published ektacytometry results. The theoretical model results imply that such deformability measuring techniques are weakly influenced by changes in the inner viscosity of the cell or the ambient fluid viscosity. However, these measurements are highly sensitive to RBC shear modulus. The shear modulus, estimated by the model and the rheoscope measurements, falls between the values obtained by micropipette aspiration and laser trapping. The study demonstrates the integration of a theoretical model with a microfabricated device in order to achieve a better understanding of RBC mechanics and their measurement using microfluidic shear assays. The system and the model have the potential of serving as quantitative clinical tools for diagnosing deformability disorders in RBCs.     

Red cell mechanics: what changes are needed to adversely affect in vivo circulation

The ability of red cells to deform is essential to allow their circulation. However the degree of rheological abnormality which can be tolerated before flow is impaired is not so clear. Red cell rheology has been characterised in a number of physiological, pathological and genetic conditions, and some inferences can be drawn. In vivo aging causes a small loss of cell deformability attributable to increased membrane and internal viscosity; volume and surface area are also lost. These changes cannot be sufficient to cause cellular removal, since the cells sampled had continued to circulate. In sickle cell disease, the oxygenated blood contains dense cells that are more severely abnormal than dense, aged cells from normal individuals. Melanesian ovalocytes have comparable rigidity to dense SS cells, but this condition has no marked circulatory pathology. Thus circulatory problems in SS disease probably stem from deoxygenation-induced sickling which causes extreme loss of deformability, rather than from the abnormal cells in oxygenated blood. In falciparum malaria, immature parasites cause appreciable loss of deformability but continue to circulate. Maturation of the parasites causes much greater rheological changes, including attachment to vascular endothelium, and the cells cease to circulate. In summary, quite marked changes in cell mechanics can occur without loss of ability to circulate. It thus seems that slight rheological alterations reported in some clinical studies are unlikely to cause appreciable flow disruption.     

Effect of hydroperoxides on red blood cell membrane mechanical properties

We investigate the effect of oxidative stress on red blood cell membrane mechanical properties in vitro using detailed analysis of the membrane thermal fluctuation spectrum. Two different oxidants, the cytosol-soluble hydrogen peroxide and the membrane-soluble cumene hydroperoxide, are used, and their effects on the membrane bending elastic modulus, surface tension, strength of confinement due to the membrane skeleton, and 2D shear elastic modulus are measured. We find that both oxidants alter significantly the membrane elastic properties, but their effects differ qualitatively and quantitatively. While hydrogen peroxide mainly affects the elasticity of the membrane protein skeleton (increasing the membrane shear modulus), cumene hydroperoxide has an impact on both membrane skeleton and lipid bilayer mechanical properties, as can be seen from the increased values of the shear and bending elastic moduli. The biologically important implication of these results is that the effects of oxidative stress on the biophysical properties, and hence the physiological functions, of the cell membrane depend on the nature of the oxidative agent. Thermal fluctuation spectroscopy provides a means of characterizing these different effects, potentially in a clinical milieu.     

Effect of sickling on dimyristoylphosphatidylcholine-induced vesiculation in sickle red blood cells

To study the effect of sickling on dimyristoylphosphatidylcholine (DMPC)-induced vesiculation, sickle (SS) red blood cells were incubated with sonicated suspensions of DMPC under either room air or nitrogen. Like normal red cells, when sickle cells were incubated with DMPC under oxygenated conditions, incorporation of DMPC into the erythrocyte membrane occurred, followed by echinocytic shape transformation and subsequent release of membrane vesicles. On the other hand, when SS cells were induced to sickle by deoxygenation, DMPC-induced vesiculation of these cells was dramatically reduced. However, upon reoxygenation, release of vesicles from these sickle erythrocytes occurred immediately. When SS cells were incubated under hypertonic (500 mosM) and deoxygenated conditions (where hemoglobin polymerization occurs but red cells do not show the typical sickle morphology), a similar decrease in the extent of vesiculation was observed. Experiments with radiolabelled lipid vesicles indicated that incorporation of DMPC into erythrocyte membranes occurred in all cases and therefore was not the limiting factor in the reduction of vesiculation in deoxygenated SS cells. Taken together, these results indicate that cellular viscosity and membrane rigidity, both of which are influenced by hemoglobin polymerization, are two important factors in process of vesicle release from sickle erythrocytes.     

Effect of intracellular magnesium and oxygen tension on K+-Cl- cotransport in normal and sickle human red cells.

In red cells from normal individuals (HbA cells), the K+-Cl- cotransporter (KCC) is inactivated by low O2 tension whilst in those from sickle cell patients (HbS cells), it remains fully active. Changes in free intracellular [Mg2+] have been proposed as a mechanism. In HbA cells, KCC activity was stimulated by Mg2+ depletion and inhibited by Mg2+ loading but the effect of O2 was independent of Mg2+. At all [Mg2+]is, the transporter was stimulated in oxygenated cells, minimally active in deoxygenated ones. By contrast, the stimulatory effects of O2 was abolished by inhibitors of protein (de)phosphorylation. HbS cells had elevated KCC activity, which was of similar magnitude in oxygenated and deoxygenated cells, regardless of Mg2+ clamping. In deoxygenated cells, the antisickling agent dimethyl adipimidate inhibited sickling, Psickle and KCC. Results indicate a role for protein phosphorylation in O2 dependence of KCC, with different activities of the relevant enzymes in HbA and HbS cells, probably dependent on Hb.     

Local membrane curvature affects spontaneous membrane fluctuation characteristics

Mechanical fluctuations on erythrocyte cell membranes wee measured by phase-contrast optics at the cell centre and cel rim. Intensity changes were digitized by a linear charge-coupled device array and both frequency spectra and autocorrelation functions were calculated to detect fluctuation characteristics at these areas. Validation was performed with glutaraldehydetreated cells. The influence of viscosity and membrane elasticity changes was evaluated by testing cells in solution of different osmolarities (239-392 mosmol I(-1)), and cells at different stages of diamide treatment (0.5-5.0 mmol I(-1)). The calculated membrane bending modulus of 1.4 E-19 J is in accordance with other findings. Despite an increase of endoplasmic viscosity, no homogeneous attenuation of the fluctuation amplitudes was observed, but a frequency shift was observed. Spectrin linkage caused by diamide has no effect on membrane fluctuations at the cell centre but it influences fluctuations at the cell rim, which can be explained by the higher membrane curvature at the cell rim compared with the lower, or even negative, membrane curvature at the cell centre.     

Mechanics of curved plasma membrane vesicles: resting shapes, membrane curvature, and in-plane shear elasticity

Highly curved cell membrane structures, such as plasmalemmal vesicles (caveolae) and clathrin-coated pits, facilitate many cell functions, including the clustering of membrane receptors and transport of specific extracellular macromolecules by endothelial cells. These structures are subject to large mechanical deformations when the plasma membrane is stretched and subject to a change of its curvature. To enhance our understanding of plasmalemmal vesicles we need to improve the understanding of the mechanics in regions of high membrane curvatures. We examine here, theoretically, the shapes of plasmalemmal vesicles assuming that they consist of three membrane domains: an inner domain with high curvature, an outer domain with moderate curvature, and an outermost flat domain, all in the unstressed state. We assume the membrane properties are the same in these domains with membrane bending elasticity as well as in-plane shear elasticity. Special emphasis is placed on the effects of membrane curvature and in-plane shear elasticity on the mechanics of vesicle during unfolding by application of membrane tension. The vesicle shapes were computed by minimization of bending and in-plane shear strain energy. Mechanically stable vesicles were identified with characteristic membrane necks. Upon stretch of the membrane, the vesicle necks disappeared relatively abruptly leading to membrane shapes that consist of curved indentations. While the resting shape of vesicles is predominantly affected by the membrane spontaneous curvatures, the membrane shear elasticity (for a range of values recorded in the red cell membrane) makes a significant contribution as the vesicle is subject to stretch and unfolding. The membrane tension required to unfold the vesicle is sensitive with respect to its shape, especially as the vesicle becomes fully unfolded and approaches a relative flat shape.     

Deoxygenation affects fluorescence photobleaching recovery measurements of red cell membrane protein lateral mobility.

We have used the fluorescence photobleaching recovery technique to study the dependence on oxygen tension of the lateral mobility of fluorescently labeled band 3, the phospholipid analogue fluorescein phosphatidylethanolamine, and glycophorins in normal red blood cell membranes. Band 3 protein and sialic acid moieties on glycophorins were labeled specifically with eosin maleimide and fluorescein thiosemicarbazide, respectively. The band 3 diffusion rate increased from 1.7 x 10(-11) cm2 s-1 to 6.0 x 10(-11) cm2 s-1 as oxygen tension was decreased from 156 to 2 torr, and a further increase to 17 x 10(-11) cm2 s-1 occurred as oxygen tension was decreased from 2 to 0 torr. The fractional mobility of band 3 decreased from 58 to 32% as oxygen tension was decreased from 156 to 0 torr. The phospholipid diffusion coefficient remained constant as oxygen tension was decreased from 156 to 20 torr, but increased from 2.3 x 10(-9) cm2 s-1 to 7.1 x 10(-9) cm2 s-1 as oxygen tension was decreased from 20 to 0 torr. Neither the diffusion coefficient nor the fractional mobility of glycophorins changed significantly at low oxygen tension. Under non-bleaching excitation conditions, intensities of fluorescence emission were identical for oxygenated and deoxygenated eosin-labeled RBCs. Deoxygenated eosin-labeled RBCs required 160-fold greater laser intensities than did oxygenated RBCs to achieve comparable extents of photobleaching, however. Oxygen seems to act as a facilitator of fluorophore photobleaching and may thereby protect the fluorescently labeled red cell membrane from photodamage.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cholesterol Depletion by MβCD Enhances Cell Membrane Tension and Its Variations-Reducing Integrity

Cholesterol depletion by methyl-β-cyclodextrin (MβCD) remodels the plasma membrane’s mechanics in cells and its interactions with the underlying cytoskeleton, whereas in red blood cells, it is also known to cause lysis. Currently it’s unclear if MβCD alters membrane tension or only enhances membrane-cytoskeleton interactions—and how this relates to cell lysis. We map membrane height fluctuations in single cells and observe that MβCD reduces temporal fluctuations robustly but flattens spatial membrane undulations only slightly. Utilizing models explicitly incorporating membrane confinement besides other viscoelastic factors, we estimate membrane mechanical parameters from the fluctuations’ frequency spectrum. This helps us conclude that MβCD enhances membrane tension and does so even on ATP-depleted cell membranes where this occurs despite reduction in confinement. Additionally, on cholesterol depletion, cell membranes display higher intracellular heterogeneity in the amplitude of spatial undulations and membrane tension. MβCD also has a strong impact on the cell membrane’s tenacity to mechanical stress, making cells strongly prone to rupture on hypo-osmotic shock with larger rupture diameters—an effect not hindered by actomyosin perturbations. Our study thus demonstrates that cholesterol depletion increases membrane tension and its variability, making cells prone to rupture independent of the cytoskeletal state of the cell.     

Nitric oxide scavenging by red blood cells as a function of hematocrit and oxygenation

The reaction rate between nitric oxide and intraerythrocytic hemoglobin plays a major role in nitric oxide bioavailability and modulates homeostatic vascular function. It has previously been demonstrated that the encapsulation of hemoglobin in red blood cells restricts its ability to scavenge nitric oxide. This effect has been attributed to either factors intrinsic to the red blood cell such as a physical membrane barrier or factors external to the red blood cell such as the formation of an unstirred layer around the cell. We have performed measurements of the uptake rate of nitric oxide by red blood cells under oxygenated and deoxygenated conditions at different hematocrit percentages. Our studies include stopped-flow measurements where both the unstirred layer and physical barrier potentially participate, as well as competition experiments where the potential contribution of the unstirred layer is limited. We find that deoxygenated erythrocytes scavenge nitric oxide faster than oxygenated cells and that the rate of nitric oxide scavenging for oxygenated red blood cells increases as the hematocrit is raised from 15% to 50%. Our results 1) confirm the critical biological phenomenon that hemoglobin compartmentalization within the erythrocyte reduces reaction rates with nitric oxide, 2) show that extra-erythocytic diffusional barriers mediate most of this effect, and 3) provide novel evidence that an oxygen-dependent intrinsic property of the red blood cell contributes to this barrier activity, albeit to a lesser extent. These observations may have important physiological implications within the microvasculature and for pathophysiological disruption of nitric oxide homeostasis in diseases.     

Minimum energy analysis of membrane deformation applied to pipet aspiration and surface adhesion of red blood cells.

An experimental procedure is demonstrated which can be used to determine the interfacial free energy density for red cell membrane adhesion and membrane elastic properties. The experiment involves micropipet aspiration of a flaccid red blood cell and manipulation of the cell proximal to a surface where adhesion occurs. A minimum free energy method is developed to model the equilibrium contour of unsupported membrane regions and to evaluate the partial derivatives of the total free energy, which correspond to the micropipet suction force and the interfacial free energy density of adhesion. It is shown that the bending elasticity of the red cell membrane does not contribute significantly to the pressure required to aspirate a flaccid red cell. Based on experimental evidence, the upper bound for the bending or curvature elastic modulus of the red cell membranes is 10-12 ergs (dyn-cm). Analysis of the adhesion experiment shows that interfacial free energy densities for red cell adhesion can be measured from a lower limit of 10-4 ergs/cm2 to an upper limit established by the membrane tension for lysis of 5-10 ergs/cm2.     

Nitric oxide red blood cell membrane permeability at high and low oxygen tension.

Red blood cell (RBC) encapsulated hemoglobin in the blood scavenges nitric oxide (NO) much more slowly than cell-free hemoglobin would. Part of this reduced NO scavenging has been attributed to an intrinsic membrane barrier to diffusion of NO through the RBC membrane. Published values for the permeability of RBCs to NO vary over several orders of magnitude. Recently, the rate that RBCs scavenge NO has been shown to depend on the hematocrit (percentage volume of RBCs) and oxygen tension. The difference in rate constants was hypothesized to be due to oxygen modulation of the RBC membrane permeability, but also could have been due to the difference in bimolecular rate constants for the reaction of NO and oxygenated vs deoxygenated hemoglobin. Here, we model NO scavenging by RBCs under previously published experimental conditions. A finite-element based computer program model is constrained by published values for the reaction rates of NO with oxygenated and deoxygenated hemoglobin as well as RBC NO scavenging rates. We find that the permeability of RBCs to NO under oxygenated conditions is between 4400 and 5100 microm s(-1) while the permeability under deoxygenated conditions is greater than 64,000 microm s(-1). The permeability changes by a factor of 10 or more upon oxygenation of anoxic RBCs. These results may have important implications with respect to NO import or export in physiology.     

Inhibition of adrenergic proton extrusion in rainbow trout red cells by nitrite-induced methaemoglobinaemia

Summary The effects of nitrite-induced methaemoglobinaemia on adrenergic proton extrusion from rainbow trout red blood cells were studied using the pH-stat method. In control conditions adrenergic proton extrusion was completely inhibited by amiloride and was greater in deoxygenated than in oxygenated erythrocytes. Nitrite-induced methaemoglobinaemia was associated with a pronounced reduction in the catecholamine-stimulated proton efflux from both deoxygenated and oxygenated erythrocytes. In deoxygenated erythrocytes the initial proton efflux upon catecholamine stimulation decreased by 60–70%, while the percentage of methaemoglobin in the red cells increased from the control level of 1–3% to 20%. In oxygenated erythrocytes the decrease was 30% at the same methaemoglobin percentage range. It is suggested that the pronounced influence of nitriteinduced methaemoglobinaemia on adrenergic proton efflux results from an inhibition of the red cell sodium/proton exchanger by the R-like haemoglobin conformations.Abbreviations DIDS 4,4-diisothiocyanostilbene-2,2-disulfonic acid - DMO 5,5-dimethyloxazolidine-2,4-dione - RBC red blood cell     

Spectrin, human erythrocyte shapes, and mechanochemical properties.

Physical studies of human erythrocyte spectrin indicate that isolated spectrin dimers and tetramers in solution are worm-like coils with a persistence length of approximately 20 nm. This finding, the known polyelectrolytic nature of spectrin, and other structural information about spectrin and the membrane skeleton molecular organization have lead us to the hypothesis that the human erythrocyte membrane skeleton constitutes a two-dimensional ionic gel (swollen ionic elastomer). This concept is incorporated in what we refer to as the protein gel-lipid bilayer membrane model. The model accounts quantitatively for red elastic shear modulus and the maximum elastic extension ratio reported for the human erythrocytes membrane. Gel theory further predicts that depending on the environmental conditions, the membrane skeleton modulus of area compression may be small or large relative to the membrane elastic shear modulus. Our analyses show that the ratio between these two parameters affects both the geometry and the stability of the favored cell shapes and that the higher the membrane skeleton compressibility the smaller the values of the gel tension needed to induce cell shape transformations. The main virtue of the protein gel-lipid bilayer membrane model is that it offers a novel theoretical and molecular basis for the various mechanical properties of the membrane skeleton such as the membrane skeleton modulus of area compression and osmotic tension, and the effects of these properties on local membrane skeleton density, cell shape, and shape transformations.     

Renormalization of the tension and area expansion modulus in fluid membranes.

Renormalization of the membrane tension and elastic area expansion modulus by thermally induced bending fluctuations is treated in terms of the formalism of Brochard, De Gennes, and Pfeuty (J. de Phys. (France). 37:1099-1104, 1976). The dependence of the renormalized tension on the bare membrane tension parallels the dependence on the fractional area extension of giant vesicles found experimentally by Evans and Rawicz (Physiol. Rev. Lett. 64:2094-2097, 1990), and suggests conditions for molecular dynamics simulations with membrane patches of limited size that might best represent the properties of macroscopic vesicles.     

Atrial natriuretic peptide: direct effects on human red blood cell dynamics.

The ability to deform is an important feature of red blood cells (RBCs) for performing their function of oxygen delivery. Little is known about the hormonal regulation of RBC deformability. Here we report that human atrial natriuretic peptide (ANP) acts directly on human RBCs leading to the elevation of local bending fluctuations of the cell membrane. These changes are accompanied by an increase in the filterability of RBCs. These ANP effects were mimicked by cyclic GMP analogues, suggesting modulation of local membrane bending fluctuations and RBC filterability via a cyclic GMP-dependent pathway. The effect of ANP on the mechanical properties of RBCs suggests that ANP may increase the passage red blood cells through capillaries resulting in an improved oxygen delivery to the tissues.     

基于图像分析技术的红细胞膜力学特性多参数动态测定的研究

探索以图像分析技术,在无扰、在位、实时的情况下,对单个活态红细胞的多个力学参量：弯曲模量KC、剪切模量μ及切向与弯曲模量之比ε等进行非侵入性连续动态测定的新方法。以该技术对红细胞在不同外部条件（温度、氧分压、渗透压）下的力学参量进行动态监测,不但揭示出有关变量条件对细胞各个力学参量的影响。还证明了本技术适于对细胞的各种生理和病理过程进行连续监测。     

Measuring optical and mechanical properties of a living cell with defocusing microscopy

Defocusing microscopy (DM) is a recently developed technique that allows quantitative analysis of membrane surface dynamics of living cells using a simple bright-field optical microscope. According to DM, the contrast of defocused images is proportional to cell surface curvature. Although, until now, this technique was used mainly to determine size and amount of membrane shape fluctuations, such as ruffles and small random membrane fluctuations, in macrophages, its applications on cell biology extend beyond that. We show how DM can be used to measure optical and mechanical properties of a living macrophage, such as cell refractive index n, membrane bending modulus K(c), and effective cell viscosity eta for membrane-actin meshwork relaxation. Experimental data collected from defocused images of bone marrow-derived macrophages were used to evaluate these parameters. The obtained values, averaged over several different macrophages, are n = (1.384 +/- 0.015), K(c) approximately 3.2 x 10(-19) J, and eta approximately 459 Pa.s. We also estimate the amplitude of the small fluctuations to be of the order of 3 nm, which is around the step size of a polymerizing actin filament.