Shlomo Levin

Membrane fluctuations in erythrocytes are linked to MgATP-dependent dynamic assembly of the membrane skeleton.

The observation of low-frequency fluctuations of the cell membrane in erythrocytes and in several nucleated cells suggests that this phenomenon may be a general property of the living cell. A study of these fluctuations in human erythrocytes and its ghosts has now been carried out using a novel optical method based on point dark field microscopy. We have demonstrated that the reestablishment of membrane fluctuations in erythrocyte ghosts is dependent on MgATP but does not necessarily require the restoration of the biconcave shape. The results imply that the dominant component of membrane fluctuations are metabolically dependent and suggest the existence of a dynamic mechano-chemical coupling within the membrane skeleton network induced by MgATP.

β‐Adrenergic agonists regulate cell membrane fluctuations of human erythrocytes

1 Mechanical fluctuations of the cell membrane (CMFs) in human erythrocytes reflect the bending deformability of the membrane‐skeleton complex. These fluctuations were monitored by time‐dependent light scattering from a small area (≈0.25 μm2) of the cell surface by a method based on point dark field microscopy. 2 Exposure of red blood cells (RBCs) to adrenaline (epinephrine) and isoproterenol (isoprenaline) resulted in up to a 45 % increase in the maximal fluctuation amplitude and up to a 35 % increase in the half‐width of the amplitude distribution. The power spectra of membrane fluctuations of control and treated cells revealed that adrenaline stimulated only the low frequency component (0.3‐3 Hz). Analysis of the dose‐response curves of β‐adrenergic agonists yielded an EC50 of 5 × 10−9 and 1 × 10−11 M for adrenaline and isoproterenol, respectively. Propranolol had an inhibitory effect on the stimulatory effect of isoproterenol. These findings show a potency order of propranolol > isoproterenol > adrenaline. 3 The stimulatory effect of adrenaline was a temporal one, reaching its maximal level after 20‐30 min but being abolished after 60 min. However, in the presence of 3‐isobutyl‐1‐methylxanthine, a partial stimulatory effect was maintained even after 60 min. Pentoxifylline and 8‐bromo‐cAMP elevated CMFs. However, exposure of ATP‐depleted erythrocytes to adrenaline or 8‐bromo‐cAMP did not yield any elevation in CMFs. These findings suggest that the β‐agonist effect on CMFs is transduced via a cAMP‐dependent pathway. 4 Deoxygenation decreased CMFs and filterability of erythrocytes by ≈30 %. The stimulatory effect of isoproterenol on CMFs was 2.2‐fold higher in deoxygenated RBCs than in oxygenated cells. 5 Exposure of RBCs to adrenaline resulted in a concentration‐dependent increase in RBC filterability, demonstrating a linear relationship between CMFs and filterability, under the same exposure conditions to adrenaline. These findings suggest that β‐adrenergic agonists may improve passage of erythrocytes through microvasculature, enhancing oxygen delivery to tissues, especially under situations of reduced oxygen tension for periods longer than 20 min.

Oxygenation-deoxygenation cycle of erythrocytes modulates submicron cell membrane fluctuations.

Low frequency submicron fluctuations of the cell membrane were recently shown to be characteristic for different cell types, nevertheless their physiological role is yet unknown. Point dark-field microscopy based recordings of these local displacements of cell membrane in human erythrocytes, subjected to cyclic oxygenation and deoxygenation, reveals a reversible decrease of displacement amplitudes from 290 +/- 49 to 160 +/- 32 nm, respectively. A higher rate of RBC adhesion to a glass substratum is observed upon deoxygenation, probably due to a low level of fluctuation amplitudes. The variation in the amplitude of these displacements were reconstituted in open RBC ghosts by perfusing them with composite solutions of 2,3 diphosphoglycerate, Mg+2, and MgATP, which mimic the intracellular metabolite concentrations in oxygenated and deoxygenated erythrocytes. The mere change in intracellular Mg+2 during oxygenation-deoxygenation cycle is sufficient to explain these findings. The results imply that the magnitude of fluctuations amplitude is directly connected with cell deformability. This study suggests that the physiological cycle of oxygenation-deoxygenation provides a dynamic control of the bending deformability and adhesiveness characteristics of the RBC via a Mg+2-dependent reversible assembly of membrane-skeleton proteins. The existing coupling between oxygenation-deoxygenation of the RBC and its mechanical properties is expected to play a key role in blood microcirculation and may constitute an example of a general situation for other circulating blood cells, where the metabolic control of cytoskeleton dynamics may modulate their dynamic mechanical properties.

Fast cell membrane displacements in B lymphocytes Modulation by dihydrocytochalasin B and colchicine

A novel type of cell membrane movement was characterized in B lymphocytes. Local submicron cell membrane displacements, within the frequency range 0.3–15 Hz, were registered in a murine lymphoma B cell line by a novel optical method based on point dark field microscopy. The cell membrane displacements were measured by monitoring changes in light scattering from very small illuminated areas (0.25 μm2) at the edge of the cell surface. B lymphocytes manifest a relative change in light scattering of 7.7 ± 1.3% (mean ± SD) which corresponds to cell membrane transverse displacement of 131 ± 22 nm. The confinement of cell membrane displacements to microdomains (≤0.2 μm2) emerged from the observed dependence of the displacement amplitude on the area size from which it is monitored. Colchicine (1 μM) decreased membrane fluctuations down to a value of 88 ± 14 nm, whereas dihydrocytochalasin B (2 μM) increased the amplitude of membrane displacements up to 184 ± 31 nm. These findings demonstrate the existence of a dynamic mechanical interaction between the cytoskeleton and the cell membrane in the frequency range of 0.3–15 Hz. The modulation of these interactions by the disruption of microfilaments or microtubules is explained in terms of the induced strain changes imposed on the cell membrane.

Correlation between local cell membrane displacements and filterability of human red blood cells

Local mechanical fluctuations of the cell membrane of human erythrocytes were shown to involve MgATP‐ and Mg2+‐driven fast membrane displacements. We propose that these local bending deformations of the cell membrane are important for cell passage through capillaries. In order to verify this hypothesis, we examined cell membrane fluctuations and filterability of erythrocytes over a wide range of medium osmolalities (180–675 mosmol/kg H2O). The results indicate the existence of a positive correlation between the amplitude of local cell membrane displacements and cell filterability. We suggest that the occurrence of metabolically driven membrane displacements on the side surface of the red blood cell diminishes its bending stiffness and enables it to fold more efficiently upon entrance into blood capillaries. Thus, local cell membrane displacements seem to play an important role in microcirculation.

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.

Local Bending Fluctuations of the Cell Membrane

Submicron mechanical fluctuations of the cell membrane are a newly recognized dynamical activity of the living cell (Krol et al., 1990; Levin and Korenstein, 1991; Mittelman et al., 1991; Tuvia et al., 1992a). These fluctuations consist of 300-20nm reversible displacements of the cell membrane in the frequency range of 0.3-30Hz, correspondingly. Submicron cell membrane fluctuations (CMF) were observed in different types of cells including red blood cells (Krol et al., 1990, Levin and Korenstein, 1991) monocytes, lymphocytes, 3T6 fibroblasts, cardiomyocytes (Krol et al., 1990) and murine lymphoma cells (Mittelman et al., 1991).

Human erythrocyte filterability at low driving pressure

In this study, the human RBC capillary flow has been modeled by passing 11 microl of RBC suspension (Hematocrit = 6%) in phosphate buffer solution (PBS) of a viscosity of 1 and 2.6 cP (in the presence of 2% Dextran) through 5 microm pore diameter polycarbonate Nuclepore filters. We have developed a digitally controlled experimental system for measuring the RBC filterability at a constant driving pressure, in the range of 10-400 Pa, producing a wall shear stress range of 1-50 Pa. The RBC filterability was evaluated by measuring the cell suspension flow rate normalized by the PBS flow rate. The RBC filterability has been found to be a nonlinear function of the driving pressure, having a single minimum locus at 25 Pa. Lowering the driving pressure below 25 Pa revealed an unexpected increase of the RBC filterability.The maximal RBC filterability (near unity) was detected at the lowest driving pressure (10 Pa) and the corresponding estimated RBC linear velocity while traveling through the capillary pore was as high as 800 microm/s. Increasing the driving pressure above 25 Pa confirmed previous results, where RBC filterability is monotonically and asymptotically increasing. Increasing the PBS medium viscosity from 1 to 2.26 cP significantly attenuated the RBC filterability and led to the anomalous increase of RBC deformability at the 10 Pa pressure range. We propose that the anomalous increase in RBC deformability was caused by RBCs undergoing spontaneous mechanical fluctuations.