Dynamics of oscillating erythrocyte doublets after electrofusion

Erythrocytes were electrofused with multiple rectangular voltage pulses to show an oscillatory movement, divided into swell phases and pump events. During each swell phase, which lasted from 0.5 s to more than 180 s, the fused cells' (doublets') volume increased by colloid osmotic swelling, and the membrane area was expanded until rupture. Membrane rupture initiated the pump event, where the doublets' volume and membrane area decreased with an almost exponential time course and time constants between 2 ms and 8 ms. Simultaneously, a portion of cytosolic hemoglobin solution was ejected into extracellular space ("jet"). Pump event time constants and swell phase durations decreased with rising chamber temperature, indicating that both parts of the oscillatory movements were determined by physical properties of membrane and liquids. Relative volume change developments express a gradual loss of membrane elasticity during the oscillation, decreasing the elastic forces stored in the membrane. Evidence is given that the first rupture causes a weakening of the membrane at the rupture site. Heat treatment up to 45 degrees C had a negligible effect on swell times, pump time constants, and relative volume changes. A heat treatment of 50 degrees C prevented oscillatory movements. The rupture location accorded with theories of potential induced membrane electropermeabilization.     

Membrane perturbations of erythrocyte ghosts by spectrin release

The cytoskeleton plays an important role in the stability and function of the membrane. Spectrin release from erythrocyte ghosts makes the membrane more fragile. However, the detail of membrane fragility has remained unclear. In the present study, the effects of incubation temperatures and polyamines on the membrane structure of ghosts under hypotonic conditions have been examined. Upon exposure of ghosts to a hypotonic buffer at 0-37 degrees C, reduction of ghost volume, spectrin release and decrease of band 3-cytoskeleton interactions were clearly observed above 30 degrees C. However, such changes were completely inhibited by spermine and spermidine. Interestingly, conformational changes of spectrin induced at 37 degrees C or 49 degrees C were not suppressed by both polyamines. Flow cytometry of fluorescein isothiocyanate-labelled ghosts exposed to 37 degrees C demonstrated the two peaks corresponding to ghosts with normal spectrin content and decreased one. Taken together, these results indicate that the degree of spectrin release from the membrane under hypotonic conditions is not same in all ghosts, and that polyamines inhibit the spectrin release followed by changes in the membrane structure, but not conformational changes of spectrin.     

Regulation of the glycophorin C-protein 4.1 membrane-to-skeleton bridge and evaluation of its contribution to erythrocyte membrane stability

The band 3-ankyrin-spectrin bridge and the glycophorin C-protein 4.1-spectrin/actin bridge constitute the two major tethers between the erythrocyte membrane and its spectrin skeleton. Although a structural requirement for the band 3-ankyrin bridge is well established, the contribution of the glycophorin C-protein 4.1 bridge to red cell function remains to be defined. In order to explore this latter bridge further, we have identified and/or characterized five stimuli that sever the linkage in intact erythrocytes and have examined the impact of this rupture on membrane mechanical properties. We report here that elevation of cytosolic 2,3-bisphosphoglycerate, an increase in intracellular Ca(2+), removal of cell O(2), a decrease in intracellular pH, and activation of erythrocyte protein kinase C all promote dissociation of protein 4.1 from glycophorin C, leading to reduced retention of glycophorin C in detergent-extracted spectrin/actin skeletons. Significantly, where mechanical studies could be performed, we also observe that rupture of the membrane-to-skeleton bridge has little or no impact on the mechanical properties of the cell, as assayed by ektacytometry and nickel mesh filtration. We, therefore, suggest that, although regulation of the glycophorin C-protein 4.1-spectrin/actin bridge likely occurs physiologically, the role of the tether and the associated regulatory changes remain to be established.     

Changes in passive electric parameters of human erythrocyte membrane during hyperthermia: role of spectrin phosphorylation.

In prefixed by 1 mmol/l OsO4 human erythrocytes, the discocyte shape was preserved upon heating to temperatures which include the denaturation temperature of the main peripheral protein spectrin. Nevertheless, the suspension of fixed cells displayed threshold decrease in its capacitance and resistance at the temperature range where spectrin denaturates. The same changes were established using intact cells and their resealed ghosts. For packed cells (ghosts), the capacitance and resistance decreased about 17% (31%) and 30% (19%). These data indicate a decrease in the beta dispersion of erythrocyte membrane associated, according to a previous study (Ivanov 1997), with the heat denaturation of spectrin at 49.5 degrees C. The amplitude of the 49.5 degrees C decrease in beta dispersion was reversibly reduced in intact erythrocytes and white ghosts following reversible decrease in the phosphorylation of their membrane proteins. It was fully eliminated in ghosts following their resealing with alkaline phosphatase (0.1 mg/ml) which dephosphorylated membrane proteins. These findings are discussed in relation to similar changes found in normal and tumour tissues and cells during hyperthermia.     

Effects of hyperthermia on spectrin expression patterns of murine lymphocytes

In this study the influence of whole-body hyperthermia on the distribution of spectrin in murine lymphocytes isolated from various lymphoid tissues is examined. Lymphocytes normally vary in terms of the pattern of spectrin distribution within the cell. In certain populations of lymphocytes, spectrin is distributed into a dense submembranous aggregate that can be easily identified by immunofluorescence microscopy. In these lymphocytes, little or no spectrin is seen at the plasma membrane region in the rest of the cell. Other lymphocytes have no such cytoplasmic aggregates, and the protein is seen at the region of the plasma membrane. Following whole-body hyperthermia (40.5 degrees C for 90 min) there is a 100% increase in cells exhibiting polar spectrin aggregates in the spleen, while lymphocytes from the thymus show no alteration in the number of cells showing such aggregates. The increase in the percentage of splenic cells that express aggregated spectrin is a result of increases occurring in both T- and B-cell subsets. This increase gradually returns to control levels by 48 h post-heating. During recovery to control levels this phenomenon is resistant to additional changes when a second heat treatment is applied. The effects described above are not observed when the experiments are performed in vitro; therefore, it is likely that the in vivo heat-induced alteration in the splenic lymphocyte population reflects the physiological response of lymphocytes to stimuli during a natural fever. The role that spectrin may play in the modulation of lymphocyte membrane properties is discussed.     

Transient dichroism studies of spectrin rotational diffusion in solution and bound to erythrocyte membranes.

Spectrin was purified from human erythrocytes and labeled with the triplet probe eosin-5-maleimide. Rotational diffusion of spectrin was investigated by observing transient dichroism following flash excitation of the probe. Measurements were performed at 4 degrees C in solutions of varying viscosity and with spectrin rebound to spectrin/actin-depleted erythrocyte membranes. In solution, complex anisotropy decays were observed which could not be satisfactorily fitted by the equations for a rod-shaped molecule of appropriate dimensions. When spectrin was rebound to the erythrocyte membrane, a decay in the anisotropy was still present but was markedly less sensitive to solution viscosity and flatter at longer times. In order to overcome the objection that the cytoskeleton is only partially reconstituted when spectrin is rebound, a method was developed for labeling spectrin with eosin-5-maleimide in situ. Anisotropy decays for these labeled membranes exhibited features similar to those obtained for spectrin labeled in solution and subsequently rebound. Taken together, the results provide good evidence for segmental motion of spectrin when incorporated into the erythrocyte cytoskeleton. Upon increasing the temperature, the initial anisotropy ro for both rebound and in situ labeled spectrin decreases, and above 30 degrees C the measured anisotropies are small. Thus, at physiological temperature the probe is almost completely randomized by motions with correlation times less than 10 microseconds.     

Suppression of high-pressure-induced hemolysis of human erythrocytes by preincubation at 49 degrees C.

When human erythrocytes were preincubated at 37-52 degrees C under atmospheric pressure before exposure to a pressure of 200 MPa at 37 degrees C, the value of hemolysis was constant (about 43%) up to 45 degrees C but became minimal at 49 degrees C. The results from anti-spectrin antibody-entrapped red ghosts, spectrin-free vesicles, and N-(1-pyrenyl)iodoacetamide-labeled ghosts suggest that the denaturation of spectrin is associated with such behavior of hemolysis at 49 degrees C. The vesicles released at 200 MPa by 49 degrees C-preincubated erythrocytes were smaller than those released by the treatment at 49 degrees C or 200 MPa alone. The size of vesicles released at 200 MPa was independent of preincubation temperature up to 45 degrees C, and the vesicles released from 49 degrees C-preincubated erythrocytes became smaller with increasing pressure up to 200 MPa. Thus, hemolysis and vesiculation under high pressure are greatly affected by the conformation of spectrin before compression. Since spectrin remains intact up to 45 degrees C, the compression of erythrocytes at 200 MPa induces structural changes of spectrin followed by the release of large vesicles and hemolysis. On the other hand, in erythrocytes that are undergoing vesiculation due to spectrin denaturation at 49 degrees C, compression produces smaller vesicles, so that the hemolysis is suppressed.     

Rotational dynamics of erythrocyte spectrin

The rotational diffusion of erythrocyte spectrin has been measured using time-resolved phosphorescence anisotropy. The anisotropy of the spectrin dimer decays to zero with a time constant of 3 microseconds at 21 degrees C. The results are compared with the correlation times predicted for the anisotropy decay of an equivalent sphere and rigid rod. The data indicate that the ribbon-like spectrin molecule possesses considerable torsional and segmental flexibility. These motions are restricted, but not abolished, when spectrin is reconstituted into cross-linked cytoskeletal protein networks, or bound to spectrin-actin depleted erythrocyte membrane vesicles.     

Native ultrastructure of the red cell cytoskeleton by cryo-electron tomography

Erythrocytes possess a spectrin-based cytoskeleton that provides elasticity and mechanical stability necessary to survive the shear forces within the microvasculature. The architecture of this membrane skeleton and the nature of its intermolecular contacts determine the mechanical properties of the skeleton and confer the characteristic biconcave shape of red cells. We have used cryo-electron tomography to evaluate the three-dimensional topology in intact, unexpanded membrane skeletons from mouse erythrocytes frozen in physiological buffer. The tomograms reveal a complex network of spectrin filaments converging at actin-based nodes and a gradual decrease in both the density and the thickness of the network from the center to the edge of the cell. The average contour length of spectrin filaments connecting junctional complexes is 46 ± 15 nm, indicating that the spectrin heterotetramer in the native membrane skeleton is a fraction of its fully extended length (∼190 nm). Higher-order oligomers of spectrin were prevalent, with hexamers and octamers seen between virtually every junctional complex in the network. Based on comparisons with expanded skeletons, we propose that the oligomeric state of spectrin is in a dynamic equilibrium that facilitates remodeling of the network as the cell changes shape in response to shear stress.     

Spectrin involvement in a 40 degrees C structural transition of the red blood cell membrane

Proteins involved in a structural transition detected in red blood cell membranes at 40 degrees C by spin labeling methods have been investigated. Antibodies specific for spectrin, band 3, and protein 4.1 have been used as specific probes to modify membrane thermotropic properties. Spectrin seems to be involved in a 40 degrees C transition detected in ghosts by both a stearic acid spin label (16-doxyl stearic) and a sulfhydryl-specific maleimide analogue spin label. Circular dichroism and maleimide spin labeling studies of purified spectrin show a slow unfolding of the protein structure starting at 25-30 degrees C and a massive transition with an onset temperature of 48 and 40 degrees C, respectively. This thermotropic behavior of spectrin could be the process that modifies membrane physicochemical properties above 40 degrees C that are detected by the stearic acid spin label. The transition detected by the stearic acid spin label was modified both by antispectrin antibodies and anti-4.1 protein antibodies, but not by antibodies specific for the cytoplasmic domain of band 3. These results suggest an involvement of protein 4.1 in regulating spectrin unfolding at the membrane level. A selective inhibition of the transition detected by the maleimide spin label has been obtained with a monoclonal antispectrin antibody at 1:1 molar ratio. The involvement in this transition of a localized spectrin domain(s) containing few exposed sulfhydryl groups is proposed.     

Spectrin promotes the association of F-actin with the cytoplasmic surface of the human erythrocyte membrane

We studied the binding of actin to the erythrocyte membrane by a novel application of falling ball viscometry. Our approach is based on the notion that if membranes have multiple binding sites for F-actin they will be able to cross-link and increase the viscosity of actin. Spectrin- and actin-depleted inside-out vesicles reconstituted with purified spectrin dimer or tetramer induce large increases in the viscosity of actin. Comparable concentrations of spectrin alone, inside-out vesicles alone, inside-out vesicles plus heat-denatured spectrin dimmer or tetramer induce large increases in the viscosity of actin. Comparable concentrations of spectrin alone, inside-out vesicles alone, inside-out plus heat denatured spectrin, ghosts, or ghosts plus spectrin have no effect on the viscosity of actin. Centrifugation experiments show that the amount of actin bound to the inside-out vesicles is enhanced in the presence of spectrin. The interactions detected by low-shear viscometry reflect actin interaction with membrane- bound spectrin because (a) prior removal of band 4.1 and ankyrin (band 2.1, the high- affinity membrane attachment site for spectrin) reduces both spectrin binding to the inside-out vesicles and their capacity to stimulate increase in viscosity of actin in the presence of spectrin + actin are inhibited by the addition of the water-soluble 72,000- dalton fragment of ankyrin, which is known to inhibit spectrin reassociation to the membrane. The increases in viscosity of actin induced by inside-out vesicles reconstituted with purified spectrin dimer or tetramer are not observed when samples are incubated at 0 degrees C. This temperature dependence may be related to the temperature-dependent associations we observe in solution studies with purified proteins: addition of ankyrin inhibits actin cross-linking by spectrin tetramer plus band 4.1 at 0 degrees C, and enhances it at 32 degrees C. We conclude (a) that falling ball viscometry can be used to assay actin binding to membranes and (b) that spectrin is involved in attaching actin filaments or oligomers to the cytoplasmic surface of the erythrocyte membrane.     

The role of human red blood cell membrane skeletal proteins in repetitive membrane strain and rupture

The influence of the membrane skeleton on cell membrane deformability, elasticity, and rupture after repetitive cycles of membrane strain, release and rupture was investigated. Human red blood cells were electrofused to doublets, which showed the post-fusion oscillation-movement. Geometrical developments of heat-treated cells were measured and compared to control cells. Alterations of cluster length and fusion zone diameter during repetitive colloidosmotic swelling period grow with heat treatment, and the number of precedent swell phases has minor influence on these values. Irrespective of the treatment, the geometrical doublet configuration at which a membrane rupture is initiated has an almost constant roundness index of 0.89. Increasing heat treatment temperature was shown to affect both deformability and elasticity of the membrane, such that doublets start each swell phase of the oscillation cycle from decreased roundness values. Evidence is given that there is a difference in the mechanical properties between the membrane at the fusion zone and the membrane of native red blood cells.     

The role of human red blood cell membrane skeletal proteins in repetitive membrane strain and rupture

The influence of the membrane skeleton on cell membrane deformability, elasticity, and rupture after repetitive cycles of membrane strain, release and rupture was investigated. Human red blood cells were electrofused to doublets, which showed the post-fusion oscillation-movement. Geometrical developments of heat-treated cells were measured and compared to control cells. Alterations of cluster length and fusion zone diameter during repetitive colloidosmotic swelling period grow with heat treatment, and the number of precedent swell phases has minor influence on these values. Irrespective of the treatment, the geometrical doublet configuration at which a membrane rupture is initiated has an almost constant roundness index of 0.89. Increasing heat treatment temperature was shown to affect both deformability and elasticity of the membrane, such that doublets start each swell phase of the oscillation cycle from decreased roundness values. Evidence is given that there is a difference in the mechanical properties between the membrane at the fusion zone and the membrane of native red blood cells.     

Direct involvement of spectrin thiols in maintaining erythrocyte membrane thermal stability and spectrin dimer self-association

Human erythrocytes vesiculate upon exposure to temperatures of 49 degrees C and above. Pretreatment of the cells with the thiol-alkylating agent N-ethylmaleimide (NEM) lowers the temperature needed to produce the same effect. Concomitant with the cells' heat susceptibility, skeletal mechanical instability and an increase in spectrin dissociation have been reported (Smith and Palek (1983) Blood 62, 1190). In the present study, similar results were achieved by preincubation of the cells with diamide, which could be reversed by reduction with dithiothreitol. Another oxidative agent, sodium tetrathionate, could only induce the temperature susceptibility, with little effect on spectrin dissociation. Incubation of spectrin solutions with NEM or diamide caused decreased association of spectrin dimers and increased dissociation of spectrin tetramers. Estimation of membrane and spectrin thiols in the treated cells showed that NEM was effective while blocking less than 20% of the thiols. Diamide and tetrathionate blocked more than 50% of the thiols, but were less effective than NEM. It is suggested that some very defined population of thiols is essential for spectrin self-association and for membrane thermal stability. They are more available to NEM than to diamide and less so to tetrathionate. Other thiols participate in maintaining the membrane thermal stability only.     

Hemoglobin enhances the self-association of spectrin heterodimers in human erythrocytes

Spectrin in isolated erythrocyte membranes is known to undergo tetramer to dimer transformation upon hypotonic incubation at 37 degrees C. In the present study, we detect no such transformation in intact erythrocytes in which hypotonicity is achieved by valinomycin treatment followed by hypotonic swelling. The inhibition of spectrin tetramer to dimer transformation is attributable to intracellular hemoglobin, since the addition of hemoglobin to isolated membranes or spectrin extracts blocks a similar spectrin transformation. However, the inhibitory effect is not limited to hemoglobin; other proteins including heme-containing proteins and basic proteins such as cytochrome c, ribonuclease, and albumin are also effective. The magnitude of their effect is proportional to the increased pI value of these proteins. We conclude that the stabilizing effect of these proteins on spectrin tetramers under hypotonic conditions is partly due to their non-ideality, which excludes water from spectrin and thus increases the effective concentration of spectrin, and to their electrostatic interactions with spectrin. In addition, promotion of spectrin self-association by hemoglobin under hypotonic conditions increases the stability of membrane skeletons against mechanical shearing. More importantly, the hemoglobin effect on spectrin self-association is demonstrable at physiological hemoglobin concentration, pH, and osmolarity, suggesting that in intact red cells the spectrin dimer-dimer association, as well as the membrane skeletal structure, is strengthened by intracellular hemoglobin.     

Tetracaine modifies the fragmentation mode of heated human erythrocytes and can induce heated cell fusion

It is known that human erythrocytes in saline fragment by development of an unstable surface wave on the cell rim when cells are heated through the denaturation temperature of the structural protein, spectrin. Here the influence of tetracaine on the fragmentation process has been recorded and analysed by video microscopy of cells heated in rectangular glass microcapillaries. The number of waves per cell rim decreases with increasing tetracaine concentration until, at 0.5 mM tetracaine, wave growth on the cell rim is suppressed on most cells and the cells internalize membrane at the cell dimple. The rate constant for the change in the number of waves per cell with increasing tetracaine concentration is 9.6 mM^?1 at a heating rate of 0.5 K/s. 50% of heated cells internalize membrane at 0.14 mM tetracaine. When cells are heated rapidly in suspension in test tubes the presence of tetracaine reduces the temperature for 50% haemolysis from 66°C for washed control cells to 60.5°C for cells in 2 mM tetracaine. Cells heated in microcapillaries in tetracaine concentrations of 3 mM and higher begin to swell before the spectrin denaturation temperature is reached. Cell fusion was observed at and above the spectrin denaturation temperature in cells heated in 3 and 4 mM tetracaine. It was also noted that the morphology of erythrocytes maintained in 3.6 mM tetracaine for times up to 30 min at 37°C or 20°C was strongly dependent on temperature and time.     

Modifying effect of concanavalin A and diamide on erythrocyte sensitivity to cold shock]

The temperature (0 degrees C and 37 degrees C) and the medium tonicity (0.15-1.20 M NaCl) were shown to affect erythrocyte agglutination by concanavalin A. Treatment of cells with lectin caused no significant decrease in the erythrocyte hemolysis upon cooling. Diamide, unlike concanavalin A used at concentrations above 2.0 M decreases the cell sensitivity to the cold shock. The changes in the erythrocyte susceptibility to cooling within the temperature range of 37-0 degrees C correlate with changes in the electrophoretic spectrum of membrane proteins. The progressive decrease in the spectrin bands intensity with a simultaneous formation of high molecular weight protein aggregates not included in the gel composition was observed after diamide treatment. The diamide effect depends on the medium tonicity, at which the treatment was performed, being especially well pronounced in hypertonic media with 0.8-1.2 M NaCl concentrations, the maximal spectrin aggregation being observed under these conditions. It is suggested that the main factor of the mechanism underlying the erythrocyte hypertonic cold shock is the increase in the association of peripheral cytoskeleton proteins with plasma membrane in osmotically dehydrated cells which limits the ability of lipids to adapt during cooling and results in the stabilization of defects in the membrane structure at low temperatures. Diamide eliminates these unfavourable changes eventually resulting in the dissociation of peripheral proteins from the cytoplasmic surface of the membrane on the protein aggregation.     

Rapid local oscillations of the surface of the human erythrocyte

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

Adhesively-tensed cell membranes: lysis kinetics and atomic force microscopy probing

Membrane tension underlies a range of cell physiological processes. Strong adhesion of the simple red cell is used as a simple model of a spread cell with a finite membrane tension-a state which proves useful for studies of both membrane rupture kinetics and atomic force microscopy (AFM) probing of native structure. In agreement with theories of strong adhesion, the cell takes the form of a spherical cap on a substrate densely coated with poly-L-lysine. The spreading-induced tension, sigma, in the membrane is approximately 1 mN/m, which leads to rupture over many minutes; and sigma is estimated from comparable rupture times in separate micropipette aspiration experiments. Under the sharpened tip of an AFM probe, nano-Newton impingement forces (10-30 nN) are needed to penetrate the tensed erythrocyte membrane, and these forces increase exponentially with tip velocity ( approximately nm/ms). We use the results to clarify how tapping-mode AFM imaging works at high enough tip velocities to avoid rupturing the membrane while progressively compressing it to a approximately 20-nm steric core of lipid and protein. We also demonstrate novel, reproducible AFM imaging of tension-supported membranes in physiological buffer, and we describe a stable, distended network consistent with the spectrin cytoskeleton. Additionally, slow retraction of the AFM tip from the tensed membrane yields tether-extended, multipeak sawtooth patterns of average force approximately 200 pN. In sum we show how adhesive tensioning of the red cell can be used to gain novel insights into native membrane dynamics and structure.     

Pyruvate kinase-deficiency anemia: membrane approach

Low ionic strength extraction (37 degrees C, 30 min) of ghosts from PK-deficient erythrocytes provided crude spectrin extract. No significant differences in the extract composition compared to normal donors were observed. The reticulocyte-dependent spectrin extractability was found among the subjects with PK-deficiency anemia. Likewise ATP-depletion affects spectrin extractability and also leads to the adsorption of cytoplasmic protein MW 50,000 to the reticulocyte membrane. The measurement of membrane fluidity using the fluorescence probe DPH did not reveal significant alterations in the moiety of integral membrane constituents.