Mechanical Properties of the Red Cell Membrane: I. Membrane Stiffness and Intracellular Pressure

The technique of Mitchison and Swann (1954) was modified for determining the resistance to deformation, or “stiffness,” of the red cell membrane and the pressure gradient across the cell wall. It requires a measure of the pressure needed to suck a portion of the cell into a micropipette. Stiffness of hypertonically crenated cells was less than that of biconcave discs or hypotonically swollen cells. Crenated cells showed zero pressure gradient and a stiffness, probably due to pure bending, equivalent to 0.007 ± 0.001 (SE) dynes/cm. Normal and swollen cells showed a pressure gradient of 2.3 ± 0.8 (SE) mm H₂O and a stiffness, due to bending and tension in the membrane, equivalent to 0.019 ± 0.002 (SE) dynes/cm. No difference in stiffness was found between the rim and the biconcavity of the cell or between biconcave discs and hypotonically swollen cells. Micromanipulation showed that the membrane can withstand large bending strains but limited tangential strains (stretching). These results have significant implications in any theory explaining the cell shape. For example, the data give no indication that the physical properties of the membrane are different at the rim from those of the biconcavities, and the existence of a positive pressure in the normal cell is established.     

New Membrane Concept Applied to the Analysis of Fluid Shear- and Micropipette-Deformed Red Blood Cells

A two-dimensional elastomer material concept of the red cell membrane is applied to the analysis of fluid shear-deformed, point-attached red cells and micropipette aspiration of red cell disks. The elastic constant (corresponding to the “shear” modulus multiplied by the membrane thickness) is of the order 10^-2 dyn/cm for both cases. Additional experimental observations are in agreement with the membrane model, e.g. teardrop and “tether” formation of the sheared disks, pressure difference vs. aspirated length of the cell for micropipette experiments, etc     

Leukocyte relaxation properties.

Study of the mechanical properties of leukocytes is useful to understand their passage through narrow capillaries and interaction with other cells. Leukocytes are known to be viscoelastic and their properties have been established by micropipette aspiration techniques. Here, the recovery of leukocytes to their normal spherical form is studied after prolonged deformation in a pipette which is large enough to permit complete entry of the leukocyte. The recovery history is characterized by the time history of the major diameter (d1) and minor diameter (d2). When the cell is removed from the pipette, it shows initially a small rapid recoil followed by a slower asymptotic recovery to the spherical shape. In the presence of cell activation and formation of pseudopods, the time history for recovery is prolonged compared with passive cell recovery. If a protopod pre-existed during the holding period, the recovery only begins when the protopod starts to retract.     

Viscoelastic properties of transformed cells: role in tumor cell progression and metastasis formation

The micropipette aspiration technique was used to investigate the deformation properties of a panel of nontransformed and transformed rat fibroblasts derived from the same normal cell line. In this method, a step negative pressure is applied to the cell via a micropipette and the aspiration distance into the pipette as a function of time is determined using video techniques. A standard solid viscoelastic model was then used to analyze the viscoelastic properties of the cell. From these results, it is concluded that a direct correlation exists between an increase in deformability and progression of the transformed phenotype from a nontumorigenic cell line into a tumorigenic, metastatic cell line.     

Membrane viscoelasticity.

In this paper, we develop a theory for viscoelastic behavior of large membrane deformations and apply the analysis to the relaxation of projections produced by small micropipette aspiration of red cell discocytes. We show that this relaxation is dominated by the membrane viscosity and that the cytoplasmic and extracellular fluid flow have negligible influence on the relaxation time and can be neglected. From preliminary data, we estimate the total membrane "viscosity" when the membrane material behaves in an elastic solid manner. The total membrane viscosity is calculated to be 10(-3) dyn-s/cm, which is a surface viscosity that is about three orders of magnitude greater than the surface viscosity of lipid membrane components (as determined by "fluidity" measurements). It is apparent that the lipid bilayer contributes little to the fluid dynamic behavior of the whole plasma membrane and that a structural matrix dominates the viscous dissipation. However, we show that viscous flow in the membrane is not responsible for the temporal dependence of the isotropic membrane tension required to produce lysis and that the previous estimates of Rand, Katchalsky, et al., for "viscosity" are six to eight orders of magnitude too large.     

Application of the micropipette technique to the measurement of cultured porcine aortic endothelial cell viscoelastic properties

The viscoelastic deformation of porcine aortic endothelial cells grown under static culture conditions was measured using the micropipette technique. Experiments were conducted both for control cells (mechanically or trypsin detached from the substrate) and for cells in which cytoskeletal elements were disrupted by cytochalasin B or colchicine. The time course of the aspirated length into the pipette was measured after applying a stepwise increase in aspiration pressure. To analyze the data, a standard linear viscoelastic half-space model of the endothelial cell was used. The aspirated length was expressed as an exponential function of time. The actin microfilaments were found to be the major cytoskeletal component determining the viscoelastic response of endothelial cells grown in static culture.     

Adhesivity and rigidity of erythrocyte membrane in relation to wheat germ agglutinin binding

Binding of the plant lectin wheat germ agglutinin (WGA) to erythrocyte membranes causes membrane rigidification. One of our objectives has been to directly measure the effects of WGA binding on membrane rigidity and to relate rigidification to the kinetics and levels of WGA binding. Our other objective has been to measure the strength of adhesion and mechanics of cell separation for erythrocytes bound together by WGA. The erythrocyte membrane rigidity was measured on single cells by micropipette aspiration. The slope of the suction pressure-length data for entry into the pipette provided the measure of the membrane extensional modulus. Data were collected for cells equilibrated with WGA solutions in the range of concentrations of 0.01- 10 micrograms/ml. Erythrocyte-erythrocyte adherence properties were studied by micropipette separation of two-cell aggregates. First, a "test" cell was selected from a WGA solution by aspiration into a small micropipette, then transferred to a separate chamber that contained erythrocytes in WGA-free buffer. Here, a second cell was aspirated with another pipette and maneuvered into close proximity of the test cell surface, and adhesive contact was produced. The flaccid cell was separated from the test cell surface in steps at which the force of attachment was derived from the pipette suction pressure and cell geometry. In addition, we measured the time-dependent binding and release of fluorescently labeled WGA to single erythrocytes with a laser microfluorometry system. The results showed that the stiffening of the erythrocyte membrane and binding of fluorescently labeled WGA to the membrane surface followed the same concentration and time dependencies. The threshold concentration for membrane stiffening was at approximately 0.1 microgram/ml where the time course to reach equilibrium was close to 1 h. The maximal stiffening (almost 30-fold over the normal membrane elastic modulus) occurred in concentrations greater than 2 micrograms/ml where the time to reach equilibrium took less than 1 min. The WGA binding also altered the normal elastic membrane behavior into an inelastic, plastic-like response which indicated that mechanical extension of the membrane caused an increase in cross-linking within the surface plane. Similar to the stiffening effect, we observed that the membrane adhesivity of cells equilibrated with WGA solutions greatly increased with concentration greater than 0.1 microgram/ml.(ABSTRACT TRUNCATED AT 400 WORDS)     

The structure of a model membrane in relation to the viscoelastic properties of the red cell membrane

The molecular arrangement within a lamellar structure composed of human erythrocyte lipids is determined. The 45 A thick lipid layer, in water, is filled in the interior with a liquid-like configuration of the hydrocarbon chains of phospholipid molecules and is covered on both sides by their hydrophilic polar groups. Cholesterol is located so that part of its steroid nucleus is between the polar groups of the phospholipid molecules while the rest of the molecule extends into the inner hydrocarbon layer. This lipid leaflet would be expected to have the mechanical properties of a purely liquid surface, as other authors have shown for the "black" lipid membranes. Data are presented which demonstrate that the intact erythrocyte membrane is a tough viscoelastic substance with a Young''s modulus of 10⁶–10⁸ dynes/cm² and a viscosity of 10⁷–10¹⁰ poises. The parameters and the kinetics of membrane breakdown are incompatible with the model system of pure lipid. Caution must be exercised in applying various data on the model systems to intact membranes.     

A New Turgor/Membrane Potential Probe Simultaneously Measures Turgor and Electrical Membrane Potential

Abstract: A new combined turgor/membrane potential probe (T-EP probe) monitored cell turgor and membrane potential simultaneously in single giant cells. The new probe consisted of a silicone oil-filled micropipette (oil-microelectrode), which conducted electric current. Measurements of turgor and hydraulic conductivity were performed as with the conventional cell pressure probe besides the membrane potential. In internodal cells of Chara corallina, steady state turgor (0.5-0.7 MPa) and resting potentials (-200 to ?220 mV) in APW, and hydraulic conductivity (0.07 to 0.21 × 10～⁵ m s^?1 MPa^?1) were measured with the new probe, and cells exhibited healthy cytoplasmic streaming for at least 24 h during measurements. When internodal cells of Chara corallina were treated with 30, 20, 10, and 5 mM KCI, turgor responded immediately to all concentrations, and the osmotic changes in the medium were measured. Action potentials, which brought the membrane potential to a steady depolarization that measured the concentration difference of K⁺ in the medium, were induced in a concentration — dependent delay and occurred only 30, 20, and 10 mM of KCl. When the solution was changed back to APW, the repolarization of membrane potential consisted of a quick and a following slow phase. During the quick phase, which took place immediately and lasted 1 to 3 min, the plasma membrane remained activated. The membrane was gradually deactivated in the slow phase, and entirely deactivated when the membrane potential recovered to the resting potential in APW. Although the activated plasma membrane was permeable to K⁺, no major ion channels were activated on the tonoplast, and therefore, internodal cells of Chara corallina did not regulate turgor when osmotic potential changed in the surrounding medium.     

Red blood cell deformability and the prostaglandins

The effects of Prostaglandin E₁ and Prostaglandin E₂ on the deformability of the human red blood cell have been studied using the glass micropipette method. In the range of concentrations 10^?13 to 10^?5 M, neither PGE₁ nor PGE₂ alters the red cell deformability.     

Optical evaluation of red blood cell geometry using micropipette aspiration.

Although red blood cell (RBC) geometry has been extensively studied by micropipette aspiration, the small size of RBC and pipettes vs. the optical resolution of light microscopy suggests the need to consider potential errors. The present study addressed such difficulties and investigated four specific problems: (1) use of exact equations to calculate RBC membrane area and volume; (2) calibration of the pipette internal diameter (PID); (3) correction for a noncylindrical pipette barrel; (4) diffraction distortion of the RBC image. The observed PID represents a cylinder lens enlargement that can be theoretically derived from the glass/buffer refractive index ratio (1.49/1.33 = 1.12). This enlargement was experimentally confirmed by: (1) studying pipettes bent to allow measurement through the barrel (horizontal) and at the orifice (vertical), with a resulting diameter ratio of 1.12 +/- 0.01; (2) and by replacing the surrounding buffer with immersion oil and hence abolishing the lens phenomenon (ratio = 1.12 +/- 0.02). In addition, use of aspirated oil droplets demonstrated a 3.2 +/- 0.2% error when the PID is focused at a sharp, maximum diameter. The average pipette cone angle was 1.49 +/- 0.09 degrees and varied considerably with pipette pulling procedures; calculated tongue geometry inside the pipette was affected by the noncylindrical pipette barrel. The RBC diffraction error, demonstrated by touching two aspirated cells held by opposing pipettes, was 0.091 +/- 0.002 microns. The PID, cone angle, and diffraction artifacts significantly (p < 0.001) affected calculated RBC geometry (average errors up to 5.4% for area and 9.6% for volume). Two new methods to calculate, rather than directly measure, the PID from images of a single RBC, during either osmotic or pressure manipulation, were evaluated; the osmotic method closely predicted the PID, whereas the pressure method markedly underestimated the PID. Our results thus confirm the need to consider the above-mentioned phenomena when determining RBC geometric parameters via micropipette aspiration.     

Filtration Coefficient of the Axon Membrane As Measured with Hydrostatic and Osmotic Methods

The hydraulic conductivity of the membranes surrounding the giant axon of the squid, Dosidicus gigas, was measured. In some axons the axoplasm was partially removed by suction. Perfusion was then established by insertion of a second pipette. In other axons the axoplasm was left intact and only one pipette was inserted. In both groups hydrostatic pressure was applied by means of a water column in a capillary manometer. Displacement of the meniscus in time gave the rate of fluid flowing across the axon sheath. In both groups osmotic differences across the membrane were established by the addition of a test molecule to the external medium which was seawater. The hydraulic conductivity determined by application of hydrostatic pressure was 10.6 ± 0.8.10^-8 cm/sec cm H₂O in perfused axons and 3.2 ± 0.6.10^-8 cm/sec cm H₂O in intact axons. When the driving force was an osmotic pressure gradient the conductivity was 4.5 ± 0.6 x 10^-10 cm/sec cm H₂O and 4.8 ± 0.9 x 10^-10 cm/sec cm H₂O in perfused and intact axons, respectively. A comparable result was found when the internal solution was made hyperosmotic. The fluid flow was a linear function of the hydrostatic pressure up to 70 cm of water. Glycerol outflux and membrane conductance were increased 1.6 and 1.1 times by the application of hydrostatic pressure. These increments do not give an explanation of the difference between the filtration coefficients. Other possible explanations are suggested and discussed.     

Biophysical correlates of lysophosphatidylcholine- and ethanol-mediated shape transformation and hemolysis of human erythrocytes. Membrane viscoelasticity and NMR measurement

The effects of monopalmitoylphosphatidylcholine (MPPC or lysophosphatidylcholine) and a series of short-chain primary alcohols (ethanol, 1-butanol and 1-hexanol) on cell shape, hemolysis, viscoelastic properties and membrane lipid packing of human red blood cells (RBCs) were studied. For MPPC, the effective membrane concentration to induce the formation of stage 3 echinocytes (8 x 10(6) molecules per cell) was one order of magnitude lower than that needed to induce 50% hemolysis (7 x 10(7) molecules per cell). In contrast, short-chain alcohols induced both shape changes and hemolysis within close concentration range (2.5 x 10(8) to 3.5 x 10(8) molecules per cell). Viscoelastic properties of the RBCs were studied by micropipette aspiration and correlated with shape change. Ethanol-treated RBCs showed a decrease in membrane elastic modulus and an increase in membrane viscosity in the recovery phase at the early stage of shape change. MPPC-treated cells showed the same type of viscoelastic changes, but these were not observed until the formation of stage 2 echinocytes. High-resolution solid-state 13C nuclear magnetic resonance technique was applied to study membrane lipid packing in the ghost membrane by following the chemical shift of hydrocarbon chains. Both MPPC and ethanol caused the 13C-NMR chemical shift to move upfield, indicating that membrane lipids were expanded due to the intercalation of these exogenous molecules. Using data obtained from model compounds, we convert values of chemical shift into a lipid packing parameter, i.e., number of gauche bonds for fatty acyl hydrocarbon chains. Approximately 10(8) interacting molecules per cell are required to induce a detectable change of lipid packing by both MPPC and ethanol. The results indicate that homolysis occurs at a smaller surface area for MPPC- than ethanol-treated RBCs. Our findings suggest that progressive changes in the molecular packing in the membrane lead eventually to hemolysis, but the mode responsible for shape transformation varies with these amphipaths.     

Influence of preparative procedures on the membrane viscoelasticity of human red cell ghosts

The effects of systematic variations in the preparative procedures on the membrane viscoelastic properties of resealed human red blood cell ghosts have been investigated. Ghosts, prepared by hypotonic lysis at 0 degrees C and resealing at 37 degrees C, were subjected to: measurement of the time constant for extensional recovery (tc); measurement of the membrane shear elastic modulus (mu) via three separate techniques; determination of the membrane viscosity (eta m) via a cone-plate Rheoscope. Membrane viscosity was also determined as eta m = mu X tc. Compared to intact cells, ghosts had shorter tc, regardless of their residual hemoglobin concentration (up to 21.6 g/dl). However, prolonged exposure to hypotonic media did increase their recovery time toward the intact cell value. The shear elastic modulus, as judged by micropipette aspiration of membrane tongues (mu p), was similar for all ghosts and intact cells. This result, taken with the tc data, indicates that ghosts have reduced membrane viscosity. Rheoscopic analysis also showed that eta m was reduced for ghosts, with the degree of reduction (approx. 50%) agreeing well with that estimated by the product mu p X tc. However, flow channel and pipette elongation estimates indicated that the ghost membrane elastic modulus was somewhat elevated compared to intact cells. We conclude that: ghosts have reduced membrane viscosity; ghosts have membrane rigidities close to intact cells, except possibly when the membrane is subjected to very large strains; the reduction in eta m is not directly related to the loss of hemoglobin; prolonged exposure of ghosts to low-ionic strength media increases the membrane viscosity toward its initial cellular level. These data indicate that the mechanical characteristics of ghost membranes can be varied by changing the methods of preparation and thus have potential application to further studies of the structural determinants of red cell membrane viscoelasticity.     

CHANGES IN THICKNESS OF THE RED BLOOD CORPUSCLE MEMBRANE

Measurements of the static capacity per cm.² of membrane for the red corpuscle as changed when the cells are made spherical by the addition of lecithin or rose bengal, show a slight increase of capacity, indicating a thinning of the membrane, although the change is not large enough to make it certain that it is real. Furthermore, the membrane capacity shows a slight decrease when spherical cells are swollen in hypotonic saline, indicating a thickening of the membrane, although the change is hardly outside the experimental error. The fact that there is no increase in capacity lends support to the theory that as the cell swells the membrane does not stretch but new material comes from the interior of the cell to make a new portion of the membrane.     

Membrane effects of imidoesters in hereditary stomatocytosis

The marked increase in cation (Na⁺, K⁺) permeability that results in swollen, cup-shaped red cells in the hereditary stomatocytosis syndrome can be corrected in vitro with a bifunctional crosslinking reagent, dimethyl adipimidate (DMA). ⁴⁵Ca influx in intact RBC, ⁴⁵Ca efflux in red ghosts, and ⁴⁵Ca retention in red ghosts are normal and not influenced by DMA. Endocytosis in resealed red ghosts is strikingly impaired but becomes normal if cells are first treated with 2 mM DMA. Protein kinase mediated phosphorylation of membrane proteins by AT³²P–only 20–40% of normal control values in both short-term (5 min) and more extended (60 min) incubations–is not improved by DMA. After reaction of ¹⁴C-DMA with stomatocytes, radiolabel is found associated with phosphatidyl serine and phosphatidyl ethanolamine and is also widely distributed among membrane proteins. Cation permeability of stomatocytes is corrected at DMA concentrations (1 mM) that result in barely detectable crosslinking of aminophospholipids or proteins, suggesting that either crosslinking of a minor component present in only small quantities or intramolecular (rather than intermolecular) crosslinking is responsible for the permeability effects. DMA, whose maximal crosslinking dimension is 7.3–9 Å, is the most effective bifunctional imidoester of those tested. Shorter (dimethyl malonimidate) or longer (dimethylsuberimidate) reagents are either less effective than DMA or totally without effect.     

Measurement of the Elastic Modulus for Red Cell Membrane Using a Fluid Mechanical Technique

Red cells which adhere to a surface in a parallel plate flow channel are stretched when acted on by a fluid shear stress. Three types of stretching are studied: whole cell stretching, the stretching of a red cell evagination, and tether (long, thin membrane process) stretching. In addition, the stretching of a large scale model cell attached to a surface is studied in a Couette flow channel. The results indicate that the uniaxial stretching of red cell membrane can be described by a linear stress-strain relationship. Simple theories developed from free body diagrams permit the calculation of a value for the modulus of elasticity of cell membrane in each of the three experiments. In all cases the value for the modulus is on the order of 10⁴ dyn/cm² for an assumed membrane thickness of 0.01 μm. It was also observed that red cell tethers steadily increase in length when the fluid shear stress is greater than approximately 1.5 dyn/cm² and tether lengths in excess of 200 μm have been achieved. Tethers appear to possess both fluid and elastic properties.     

Estimating the sensitivity of mechanosensitive ion channels to membrane strain and tension

Bone adapts to its environment by a process in which osteoblasts and osteocytes sense applied mechanical strain. One possible pathway for the detection of strain involves mechanosensitive channels and we sought to determine their sensitivity to membrane strain and tension. We used a combination of experimental and computational modeling techniques to gain new insights into cell mechanics and the regulation of mechanosensitive channels. Using patch-clamp electrophysiology combined with video microscopy, we recorded simultaneously the evolution of membrane extensions into the micropipette, applied pressure, and membrane currents. Nonselective mechanosensitive cation channels with a conductance of 15 pS were observed. Bleb aspiration into the micropipette was simulated using finite element models incorporating the cytoplasm, the actin cortex, the plasma membrane, cellular stiffening in response to strain, and adhesion between the membrane and the micropipette. Using this model, we examine the relative importance of the different cellular components in resisting suction into the pipette and estimate the membrane strains and tensions needed to open mechanosensitive channels. Radial membrane strains of 800% and tensions of 5 10(-4) N.m(-1) were needed to open 50% of mechanosensitive channels. We discuss the relevance of these results in the understanding of cellular reactions to mechanical strain and bone physiology.     

Membrane mediated link between ion transport and metabolism in human red cells

When 10^?6 M oubain is added to human red cells that have been incubated without glucose for two hours, there is a significant shift in the ³¹P nuclear magnetic resonances of both phosphate groups of cellular 2,3-diphosphoglycerate, which is not found in control cells incubated with glucose. This means that an effect induced by ouabain on the outside of the red cell membrane is transmitted through the membrane to alter the environment of an intracellular metabolite. Experiments with glycolytic cycle inhibitors have indicated that the intracellular ligand responsible for the resonance shifts is monophosphoglycerate mutase which requires 2,3-diphosphoglycerate as a cofactor for the reaction it catalyzes. To account for this finding a hypothesis is presented that the (Na⁺ + K⁺)-ATPase in human red cells is linked to monophosphoglycerate mutase through the agency of phosphoglycerate kinase. Evidence is presented for the existence of phosphoglycerate kinase/monophosphoglycerate mutase in solution. It is shown that this complex can interact with the cytoplasmic face of (Na⁺ + K⁺)-ATPase at the outside surface of inside out red cell vesicles, and that this interaction is inhibited when 10^?6 M ouabain is contained within the vesicle. Neither monophosphoglycerate mutase nor phosphoglycerate kinase is significantly bound to the inside surface of the intact human red cell, but glyceraldehyde 3-phosphate dehydrogenase is; it is shown that this enzyme also interacts with the cytoplasmic face of the (Na⁺ + K⁺)-ATPase and that the interaction is inhibited by 10^?6 M ouabain.