Measurement of erythrocyte orientation in flow by spin labeling III--erythrocyte orientation and rheological conditions

The measurements of erythrocyte orientation, obtained through a spin labeling technique, are compared with a phenomenological model. Several rheological conditions are varied: hematocrit, suspending medium viscosity, blood age, artificial reversible aggregation. We found that the onset of orientation is very sensitive to any variation of these conditions, and that its measurement would be a good method to assess erythrocyte deformability. A critical shear rate for the orientation process is then determined and compared to the corresponding parameter obtained from viscosity measurements of identical suspensions. A close qualitative relationship is found between the two sets of values of the critical shear rate.     

Red blood cell orientation in orbit C = 0.

Two modes of behavior of single human red cells in a shear field have been described. It is known that in low viscosity media and at shear rates less than 20 s-1, the cells rotate with a periodically varying angular velocity, in accord with the theory of Jeffery (1922) for oblate spheroids. In media of viscosity greater than approximately 5 mPa s and sufficiently high shear rates, the cells align themselves at a constant angle to the direction of flow with the membrane undergoing tank-tread motion. Also, in low viscosity media, as the shear rate is increased, more and more cells lie in the plane of shear, undergoing spin with their axes of symmetry aligned with the vorticity axis of the shear field in an orbit "C = 0" (Goldsmith and Marlow, 1972). We have explored this latter phenomenon using two experimental methods. First, the erythrocytes were observed in the rheoscope and their diameters measured. Forward light scattering patterns were correlated with the red cell orientation mode. Light flux variations after flow onset or stop were measured, and the characteristic times of erythrocyte orientation and disorientation were assessed. The characteristic time of erythrocyte orientation in Orbit C = 0 is proportional to the inverse of the shear rate. The corresponding coefficient of proportionality depends on the suspending medium viscosity eta o. The disorientation time tau D, after flow has been stopped, is such that the ratio tau D/eta o is independent of the initial applied shear stress. However, tau D is much shorter than one would expect if pure Brownian motion were involved. The proportion of erythrocytes in orbit C = 0 was also measured. It was found that this proportion is a function of both the shear rate and eta o. At low values of eta o, the proportion increases with increasing shear rate and then reaches a plateau. For higher values of eta o (5 to 10 mPa s), the proportion of RBC in orbit C = 0 is a decreasing function of the shear stress. A critical transition between orbit C = 0 and parallel alignment was observed at high values of eta o, when the shear stress is on the order of 1 N/m2. Finally, the effect of altering membrane viscoelastic properties (by heat or diamide treatment) was tested. The proportion of oriented cells is a steep decreasing function of red cell rigidity.     

Microscopic investigation of erythrocyte deformation dynamics

The understanding of erythrocyte deformation under conditions of high shear stress and short exposure time is central to the study of hemorheology and hemolysis within prosthetic blood contacting devices. A combined computational and experimental microscopic study was conducted to investigate the erythrocyte deformation and its relation to transient stress fields. A microfluidic channel system with small channels fabricated using polydimethylsiloxane on the order of 100 mum was designed to generate transient stress fields through which the erythrocytes were forced to flow. The shear stress fields were analyzed by three-dimensional computational fluid dynamics. Microscopic images of deforming erythrocytes were experimentally recorded to obtain the changes in cell morphology over a wide range of fluid dynamic stresses. The erythrocyte elongation index (EI) increased from 0 to 0.54 with increasing shear stress up to 123 Pa. In this shear stress range, erythrocytes behaved like fluid droplets, and deformed and flowed following the surrounding fluid. Cells exposed to shear stress beyond 123 Pa (up to 5170 Pa) did not exhibit additional elongation beyond EI=0.54. Two-stage deformation of erythrocytes in response to shear stress was observed: an initial linear elongation with increasing shear stress and a plateau beyond a critical shear stress.     

Two-component coarse-grained molecular-dynamics model for the human erythrocyte membrane

We present a two-component coarse-grained molecular-dynamics model for simulating the erythrocyte membrane. The proposed model possesses the key feature of combing the lipid bilayer and the erythrocyte cytoskeleton, thus showing both the fluidic behavior of the lipid bilayer and the elastic properties of the erythrocyte cytoskeleton. In this model, three types of coarse-grained particles are introduced to represent clusters of lipid molecules, actin junctions, and band-3 complexes, respectively. The proposed model facilitates simulations that span large length scales (approximately micrometers) and timescales (approximately milliseconds). By tuning the interaction potential parameters, we were able to control the diffusivity and bending rigidity of the membrane model. We studied the membrane under shearing and found that at a low shear strain rate, the developed shear stress was due mainly to the spectrin network, whereas the viscosity of the lipid bilayer contributed to the resulting shear stress at higher strain rates. In addition, we investigated the effects of a reduced spectrin network connectivity on the shear modulus of the membrane.     

Measurement of the erythrocyte orientation in a flow by spin labeling

When a suspension of erythrocytes labeled in their membrane with a fatty acid paramagnetic molecule is allowed to flow in a flat quartz sample cell, the recorded electron paramagnetic spectra change as a function of the orientation of the cell in the magnetic field. This indicates that the red cells are themselves oriented in the flow. Such spectral variations have been reproduced by a numerical simulation procedure, which allowed us to quantify the proportion of oriented red blood cells by measuring the amplitude of some characteristic lines on the experimental spectra. Orientation rates were then measured as a function of various rheological parameters, such as shear rate, hematocrit and viscosity of the suspending medium. The kinetics of the disorientation process was determined by stopping the flow.     

Actin protofilament orientation in deformation of the erythrocyte membrane skeleton

The red cell's spectrin-actin network is known to sustain local states of shear, dilation, and condensation, and yet the short actin filaments are found to maintain membrane-tangent and near-random azimuthal orientations. When calibrated with polarization results for single actin filaments, imaging of micropipette-deformed red cell ghosts has allowed an assessment of actin orientations and possible reorientations in the network. At the hemispherical cap of the aspirated projection, where the network can be dilated severalfold, filaments have the same membrane-tangent orientation as on a relatively unstrained portion of membrane. Likewise, over the length of the network projection pulled into the micropipette, where the network is strongly sheared in axial extension and circumferential contraction, actin maintains its tangent orientation and is only very weakly aligned with network extension. Similar results are found for the integral membrane protein Band 3. Allowing for thermal fluctuations, we deduce a bound for the effective coupling constant, alpha, between network shear and azimuthal orientation of the protofilament. The finding that alpha must be about an order of magnitude or more below its tight-coupling value illustrates how nanostructural kinematics can decouple from more macroscopic responses. Monte Carlo simulations of spectrin-actin networks at approximately 10-nm resolution further support this conclusion and substantiate an image of protofilaments as elements of a high-temperature spin glass.     

Dielectro-deformations of erythrocyte: analysis of the ellipsoidal shear model

Using the theoretical analysis within the framework of the proposed ellipsoidal shear electromechanical model of erythrocyte, the main mechanisms and relationships have been established and studied for the deformations of erythrocytes caused by a spatially homogeneous high-frequency electric field. The main types of the stress-strain curves characteristic of stationary and dynamic deformations caused by the rectangular-pulse and harmonic modulations of the field amplitude have been calculated. The relationship has been established between the parameters of essentially nonlinear stress-strain curves and mechanical, electric, and geometric parameters of erythrocyte. The impossibility of unlimited elongation of erythrocyte by the field, due to the conservation of the cell volume and surface area, has been shown, and the dependence of the maximum possible elongation of the cell on its volume has been calculated. It has been shown that the relaxation time of dynamic deformations of erythrocyte in the presence of an electric field considerably differs from that characteristic of the membrane material and sharply decreases with the increase of the initial elongation of the cell.     

Erythrocyte microtubule assembly in vitro. Determination of the effects of erythrocyte tau, tubulin isoforms, and tubulin oligomers on erythrocyte tubulin assembly, and comparison with brain microtubule assembly

Two tubulin variants, isolated from chicken brain and erythrocytes and known to have different peptide maps and electrophoretic properties, are demonstrated to exhibit different assembly properties in vitro: 1) erythrocyte tubulin assembles with greater efficiency (lower critical concentration, greater elongation rate) but exhibits a lower nucleation rate than brain tubulin, and 2) erythrocyte tubulin readily forms oligomers whose presence significantly retards the rate of elongation, suggesting that tubulin oligomers may also be important for determining the rate of assembly and the length of microtubules in erythrocytes. Erythrocyte tubulin isolated by cycles of in vitro assembly-disassembly is also demonstrated to contain a 67-kDa tau factor that greatly enhances microtubule nucleation but has little effect on elongation rates or critical concentration. Immunofluorescence microscopy with tau antibody indicates that tau is specifically associated with marginal band microtubules, suggesting that it may be important for determining microtubule function in vivo.     

Measurements of viscosity of synthetic erythrocyte suspensions

Measurements were made of the viscosity of suspensions of synthetic erythrocytes composed of hemoglobin solutions encapsulated in liposomes, as a function of shear rate, temperature, suspension concentration, lipid membrane composition, and the viscosity of the suspending medium. It was found that the viscous behavior of the synthetic erythrocyte suspensions was non-Newtonian and nearly the same as that of suspensions of natural erythrocytes prepared similarly, with the major difference being that synthetic erythrocyte suspensions are somewhat more viscous. Suspensions of Fluosol FC-43 prepared similarly were found to be essentially Newtonian fluids, and substantially different and more viscous than either erythrocyte suspension. The higher viscosity of synthetic erythrocyte suspensions probably accounts for the ability of these suspensions to maintain normal systemic vascular resistance in transfusion experiments, in spite of the fact that synthetic erythrocytes are smaller than natural erythrocytes.     

Selective alteration of erythrocyte deformabiliby by SH-reagents: evidence for an involvement of spectrin in membrane shear elasticity

In order to elucidate the molecular basis of membrane shear elasticity, the effect of membrane protein modification by SH-reaents on the deformability of human erythrocytes was studied. Deformability was quuantified by measuring the elongation of erythrocytes subjected to viscometric flow in a transparent cone plate viscometer. Impermeable SH-reagents proved to have no mechanical effect. Many, but not all, permeable SH-reagents markedly decreased the elongation. Among these, bifunctional SH-reagents (e.g. diamide, tetrathionate and N, N' -p-phenylenedimaleimide) able to cross-link membrane SH-groups were more effective than monofunctional SH-reagents (e.g. N-ethylmaleimide and ethacrynic acid). The bifunctional SH-reagents produced a 50% decrease of elongation after modification of less than 5% of the membrane SH-groups. In contrast, for a comparable effect, more than 20% of the SH-groups had to be modified by the monofunctional reagents. The effect of SH-oxidizing agents was fully reversible after treatment with disulfide-reducing agents. All bifunctional SH-reagents induced a dimerization of a small fraction of spectrin. Anaalysis of the distribution of the diamide-induced disulfide bonds among the various membrane protein fractions showed that this agent preferentially acts on the spectrin polypeptides. The results provide direct experimental evidence that the native arrangement of spectrin is essential for the shear resistance of the erythrocyte membrane and that introduction of small numbers of intermolecular cross-links as well as modification within the molecule lead to a rapid loss of this function.     

Dynamics of microtubules from erythrocyte marginal bands.

Microtubules can adjust their length by the mechanism of dynamic instability, that is by switching between phases of growth and shrinkage. Thus far this phenomenon has been studied with microtubules that contain several components, that is, a mixture of tubulin isoforms, with or without a mixture of microtubule-associated proteins (MAPs), which can act as regulators of dynamic instability. Here we concentrate on the influence of the tubulin component. We have studied MAP-free microtubules from the marginal band of avian erythrocytes and compared them with mammalian brain microtubules. The erythrocyte system was selected because it represents a naturally stable aggregate of microtubules; second, the tubulin is largely homogeneous, in contrast to brain tubulin. Qualitatively, erythrocyte microtubules show similar features as brain microtubules, but they were found to be much less dynamic. The critical concentration of elongation, and the rates of association and dissociation of tubulin are all lower than with brain microtubules. Catastrophes are rare, rescues frequent, and shrinkage slow. This means that dynamic instability can be controlled by the tubulin isotype, independently of MAPs. Moreover, the extent of dynamic behavior is highly dependent on buffer conditions. In particular, dynamic instability is strongly enhanced in phosphate buffer, both for erythrocyte marginal band and brain microtubules. The lower stability in phosphate buffer argues against the hypothesis that a cap of tubulin.GDP.Pi subunits stabilizes microtubules. The difference in dynamics between tubulin isotypes and between the two ends of microtubules is preserved in the different buffer systems.     

Stereotypical cell division orientation controls neural rod midline formation in zebrafish

The development of multicellular organisms is dependent on the tight coordination between tissue growth and morphogenesis. The stereotypical orientation of cell divisions has been proposed to be a fundamental mechanism by which proliferating and growing tissues take shape. However, the actual contribution of stereotypical division orientation (SDO) to tissue morphogenesis is unclear. In zebrafish, cell divisions with stereotypical orientation have been implicated in both body-axis elongation and neural rod formation, although there is little direct evidence for a critical function of SDO in either of these processes. Here we show that SDO is required for formation of the neural rod midline during neurulation but dispensable for elongation of the body axis during gastrulation. Our data indicate that SDO during both gastrulation and neurulation is dependent on the noncanonical Wnt receptor Frizzled 7 (Fz7) and that interfering with cell division orientation leads to severe defects in neural rod midline formation but not body-axis elongation. These findings suggest a novel function for Fz7-controlled cell division orientation in neural rod midline formation during neurulation.     

The orientation of eosin-5-maleimide on human erythrocyte band 3 measured by fluorescence polarization microscopy.

The dominant motional mode for membrane proteins is uniaxial rotational diffusion about the membrane normal axis, and investigations of their rotational dynamics can yield insight into both the oligomeric state of the protein and its interactions with other proteins such as the cytoskeleton. However, results from the spectroscopic methods used to study these dynamics are dependent on the orientation of the probe relative to the axis of motion. We have employed polarized fluorescence confocal microscopy to measure the orientation of eosin-5-maleimide covalently reacted with Lys-430 of human erythrocyte band 3. Steady-state polarized fluorescence images showed distinct intensity patterns, which were fit to an orientation distribution of the eosin absorption and emission dipoles relative to the membrane normal axis. This orientation was found to be unchanged by trypsin treatment, which cleaves band 3 between the integral membrane domain and the cytoskeleton-attached domain. this result suggests that phosphorescence anisotropy changes observed after trypsin treatment are due to a rotational constraint change rather than a reorientation of eosin. By coupling time-resolved prompt fluorescence anisotropy with confocal microscopy, we calculated the expected amplitudes of the e-Dt and e-4Dt terms from the uniaxial rotational diffusion model and found that the e-4Dt term should dominate the anisotropy decay. Delayed fluorescence and phosphorescence anisotropy decays of control and trypsin-treated band 3 in ghosts, analyzed as multiple uniaxially rotating populations using the amplitudes predicted by confocal microscopy, were consistent with three motional species with uniaxial correlation times ranging from 7 microseconds to 1.4 ms.     

Selective alteration of erythrocyte deformability by SH-reagents. Evidence for an involvement of spectrin in membrane shear elasticity

In order to elucidate the molecular basis of membrane shear elasticity, the effect of membrane protein modification by SH-reagents on the deformability of human erythrocytes was studied. Deformability was quantified by measuring the elongation of erythrocytes subjected to viscometric flow in a transparent cone-plate viscometer. Impermeable SH-reagents proved to have no mechanical effect. Many, but not all, permeable SH-reagents markedly decreased the elongation. Among these, bifunctional SH-reagents (e.g. diamide, tetrathionate and N,N′-p-phenylenedimaleimide) able to cross-link membrane SH-groups were more effective than monofunctional SH-reagents (e.g. N-ethylmaleimide and ethacrynic acid). The bifunctional SH-reagents produced a 50% decrease of elongation after modification of less than 5% of the membrane SH-groups. In contrast, for a comparable effect, more than 20% of the SH-groups had to be modified by the monofunctional reagents. The effect of SH-oxidizing agents was fully reversible after treatment with disulfide-reducing agents. All bifunctional SH-reagents induced a dimerization of a small fraction of spectrin. Analysis of the distribution of the diamide-induced disulfide bonds among the various membrane protein fractions showed that this agent preferentially acts on the spectrin polypeptides.The results provide direct experimental evidence that the native arrangement of spectrin is essential for the shear resistance of the erythrocyte membrane and that introduction of small numbers of intermolecular cross-links as well as modification within the molecule lead to a rapid loss of this function.     

Effect of pH on the velocity of erythrocyte aggregation

The effect of pH on the velocity of aggregation of human erythrocytes was quantitatively examined with a rheoscope combined with a video-camera, an image analyzer and a computer, in relation to the morphological changes of erythrocytes and their aggregates. (i) With increasing pH of the medium, the velocity of erythrocyte aggregation increased. (ii) The rouleaux formed at high pH were longer in shape and more stable against the increase of shear rate than those formed at low pH. (iii) With increasing pH, the diameter of erythrocyte increased, the (maximum) thickness decreased, and the cell volume decreased. The pH dependency of erythrocyte aggregation may be mainly due to the morphological change of erythrocytes, and partly due to the changes of erythrocyte deformability and of interaction with macromolecules.     

Trunk orientation causes asymmetries in leg function in small bird terrestrial locomotion

In contrast to the upright trunk in humans, trunk orientation in most birds is almost horizontal (pronograde). It is conceivable that the orientation of the heavy trunk strongly influences the dynamics of bipedal terrestrial locomotion. Here, we analyse for the first time the effects of a pronograde trunk orientation on leg function and stability during bipedal locomotion. For this, we first inferred the leg function and trunk control strategy applied by a generalized small bird during terrestrial locomotion by analysing synchronously recorded kinematic (three-dimensional X-ray videography) and kinetic (three-dimensional force measurement) quail locomotion data. Then, by simulating quail gaits using a simplistic bioinspired numerical model which made use of parameters obtained in in vivo experiments with real quail, we show that the observed asymmetric leg function (left-skewed ground reaction force and longer leg at touchdown than at lift-off) is necessary for pronograde steady-state locomotion. In addition, steady-state locomotion becomes stable for specific morphological parameters. For quail-like parameters, the most common stable solution is grounded running, a gait preferred by quail and most of the other small birds. We hypothesize that stability of bipedal locomotion is a functional demand that, depending on trunk orientation and centre of mass location, constrains basic hind limb morphology and function, such as leg length, leg stiffness and leg damping.     

Strain rate does not affect cortical microtubule orientation in the isolated epidermis of sunflower hypocotyls

A hypothesis exists that external and internal factors affect the orientation of cortical microtubules in as much as these lead to changes in cell elongation rate. Factors that stimulate elongation are proposed to lead to transverse microtubule orientation, whereas factors that inhibit elongation lead to longitudinal orientation. The elongation rate is equal to the rate of longitudinal irreversible strain in cell walls. Incubated epidermis peeled from sunflower hypocotyls does not extend unless it is stretched by loading and the pH of the incubation medium is appropriately low. Thus, peels provide a convenient model to investigate the relationship between longitudinal strain rate and cortical microtubule orientation. In the present study, it was found that peeling affects microtubule orientation. Peels were incubated for several hours in Murashige & Skoog medium (both unbuffered and buffered) to attain a steady state of microtubule orientation before loading. The effects of loading and pH on strain rate and orientation of microtubules under the outer epidermal walls were examined in three portions of peels positioned with respect to the cotyledonary node. Appropriate loading caused longitudinal strain of peels at pH 4.5 but not at pH 6.5. However, no clear effect of strain rate on microtubule orientation in the peels was observed. Independent of applied load and pH of the incubation medium, the microtubule orientation remained unchanged, i.e. orientation was mainly oblique. Our results show that strain rate does not affect cortical microtubule orientation in isolated epidermis of the sunflower hypocotyl model system, although orientation could be changed by white light.     

The elongation and orientation of cultured endothelial cells in response to shear stress

Vascular endothelial cells appear to be aligned with the flow in the immediate vicinity of the arterial wall and have a shape which is more ellipsoidal in regions of high shear and more polygonal in regions of low shear stress. In order to study quantitatively the nature of this response, bovine aortic endothelial cells grown on Thermanox plastic coverslips were exposed to shear stress levels of 10, 30, and 85 dynes/cm2 for periods up to 24 hr using a parallel plate flow chamber. A computer-based analysis system was used to quantify the degree of cell elongation with respect to the change in cell angle of orientation and with time. The results show that (i) endothelial cells orient with the flow direction under the influence of shear stress, (ii) the time required for cell alignment with flow direction is somewhat longer than that required for cell elongation, (iii) there is a strong correlation between the degree of alignment and endothelial cell shape, and (iv) endothelial cells become more elongated when exposed to higher shear stresses.     

Appearance of an erythrocyte population with decreased deformability and hemoglobin content following sepsis

With the use of the cecal ligation and puncture model in mice, this study tested whether sepsis-induced decreased erythrocyte deformability is restricted to a subpopulation of cells. Erythrocyte subpopulations were isolated by centrifugal elutriation. Lineweaver-Burk conversion of deformability-response curves to shear stress was used to determine the shear stress at half-maximal cell elongation (K(EI)) and maximal cell elongation (EI(max)). Sepsis decreased erythrocyte deformability in whole blood. K(EI) values were elevated (2.7 vs. 2.1 Pa) and EI(max) values decreased (0.56 vs. 0.50) in sepsis compared with sham mice. K(EI) values for cells eluted at 7 ml/min (smallest and oldest cells) were similar; however, K(EI) values for cells eluted at 8 ml/min were greater in septic than sham animals (2.50 vs. 2.10). Younger and larger subpopulations of erythrocytes (eluted at 9, 10, and 11 ml/min) also showed a tendency of decreased deformability in sepsis. Mean corpuscular hemoglobin content was decreased in cells eluted at 7 and 8 ml/min in sepsis (4.5 and 10.2 pg) compared to sham (7.4 and 11.4 pg) mice. This study indicates that an erythrocyte subpopulation that represents 20% of circulating cells shows the most pronounced decrease in cell deformability during sepsis. Increased rigidity together with decreased corpuscular hemoglobin content in these cells may contribute to microcirculatory dysfunction and immune modulation during sepsis.