Accumulation and drainage of hemin in the red cell membrane

The subject of hemin intercalation in red cell membranes and the correlation of the accumulated hemin level with the membrane pathology was studied. Methods which made use of dioxan and octan-2-ol mixtures to quantitate small amounts of hemin in membranes were developed. Applying these methods, hemin levels were measured in the cytoskeleton and the remaining lipid core of various red cell membranes. The amount of hemin, in both membrane fractions, was higher in pathological cells of sickle cell anemia and beta-thalassemia as compared to normal circulating cells. Correlation exists between the amount of the membrane-accumulated hemin and the severity of the disease. The level of hemin in the membrane was found to be age dependent, old cells in circulation accumulating more hemin than young cells. The level of hemin in all cells tested was much lower than the amount found previously to cause immediate hemolysis when applied externally (Kirschner-Zilber, I., Rabizadeh, E. and Shaklai, N. (1982) Biochim. Biophys. Acta 690, 20-30). This was explained by the differences between the process leading to immediate lysis and membrane changes recognized as pathological by the in-vivo sequestration mechanism. In search of a physiological mechanism which may drain the cell membrane from the hazardeous hemin, albumin, the main serum protein, was found capable of serving as an efficient agent for extracting hemin trapped in red cell membranes. It is suggested that under normal conditions albumin extracts enough hemin to leave the erythrocyte with unharmful hemin amounts, however, under pathological conditions greater amounts accumulate leading to a shorter cell life span.     

The red cell membrane and its cytoskeleton.

Gel-filtration (Sephadex G-75) analysis of hepatic cytosol reveals both qualitative and quantitative sex differences in oestrogen-binding proteins. The elution profile of [³H]oestradiol-labelled cytosol shows four species of oestrogen-binding proteins (peaks I, II, IV and V) common to both sexes. The amount of [³H]oestradiol binding in peak I is equivalent in both males and females and corresponds quantitatively to the specific oestrogen receptor. The amount of binding in the remaining three peaks is greater in males than females. In addition, an oestrogen-binding protein (peak III) is present that is unique to male cytosol. Proteinase-inhibition studies demonstrate that the observed multiplicity of oestrogen-binding proteins is not an artefact of proteolytic breakdown. Sex differences in oestrogen-binding proteins are absent in immature male and female animals; the oestrogen-binding protein profile in immature rats resembles that of an adult female. Gonadectomy of adult animals does not affect the oestrogen-binding-protein profile. In contrast, neonatal (day 1) castration results in partial feminization of the characteristic oestrogen-binding protein profile seen in the adult male; the appearance of Peak III is suppressed and marked decreases in the amount of oestradiol binding occurs in the remaining peaks. Hypophysectomy of adult animals results in near abolishment of the observed sex differences; the male oestrogen-binding protein profile is partially feminized and the female profile is partially masculinized, as characterized by the appearance of [³H]oestradiol binding in the region of peak III and increased amounts of binding in peaks IV and V. The present studies demonstrate a multiplicity of oestrogen-binding proteins in liver cytosol and raise the possibility that the presence of some of these proteins may be imprinted at birth through the hypothalamic–pituitary axis, by a mechanism requiring neonatal androgen exposure.     

Composition of the ternary protein complex of the red cell membrane cytoskeleton

The red cell membrane skeletal network is constructed from actin, spectrin and protein 4.1 in a molar ratio of actin subunits/spectrin heterodimer/protein 4.1 of 2:1:1. This represents saturation of the actin filaments, since incubation with extraneous spectrin and protein 4.1 leads to no binding of additional spectrin, either to the inner surface of ghost membranes or to lipid-free membrane cytoskeletons. Partial extraction of spectrin from the membrane is accompanied by release of actin under all conditions. Regardless of the proportion of spectrin extracted, the molar ratio of spectrin dimers/actin subunits is constant at 1:2. This is not the result of release or cooperative breakdown of whole lattice junctions from the network, for the number of actin filaments, judged by capacity to nucleate polymerisation of added G-actin, remains unchanged even when as much as 60% of the total spectrin has been lost. A similar 1:2:1 stoichiometry characterises the complex formed when G-actin is allowed to polymerise in the presence of varying amounts of spectrin and protein 4.1. When this complex is treated with the depolymerising agent, 1 M guanidine hydrochloride, it breaks down into smaller units of the same stoichiometry. After cross-linking these can be recovered from a gel-filtration column. Complexes prepared starting from G-actin appear to be much more stable than those formed when spectrin and protein 4.1 are bound to F-actin.     

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.     

An examination of the soluble oligomeric complexes extracted from the red cell membrane and their relation to the membrane cytoskeleton

A part of the spectrin extracted from red cell membranes at low ionic strength occurs in the form of a high-molecular weight oligomeric complex with actin and proteins 4.1 and 4.9. When the extraction is performed at 35 degrees, the spectrin is present in this complex as the dimer, all higher forms being dissociated. We have been unable to establish any correlation between the fraction of the spectrin thus complexed and the metabolic state of the cell. At least a large part of the complex appears to be a defined monodisperse species, sedimenting at 31S. The actin is present as short protofilaments. The average number of spectrin molecules associated with each molecule of complex has been studied by cytochalasin binding and electron microscopy. The complexes present the appearance in the electron microscope of spiders, in which the legs are spectrin dimers, attached to a globular element, containing by inference, actin and proteins 4.1 and 4.9; they are active in nucleating the polymerization of G-actin. The complexes are extremely stable, being resistant to dissociation under the conditions of the deoxyribonuclease assay, even after treatment with trypsin to degrade the actin-associated proteins. It is suggested that the complexes represent intact junctions of the membrane cytoskeletal network. Relevant structural features of the network are revealed by electron microscopy. The results lead to inferences concerning the mechanism of dissociation of the network from the membrane.     

Modulation of red cell K transport by membrane lipids

Alterations in the state of the membrane lipids affect human red cell K⁺ transport. Depletion of membrane cholesterol by 29–34% significantly inhibited both total K⁺ influx and ouabain-sensitive K⁺ influx. Addition of the hydrophobic anesthetic, chlorpromazine, in concentration from 2 · 10⁻⁵ to 2 · 10⁻⁴ M increased both total K⁺ influx and ouabain-sensitive K⁺ influx. In each case the effect on both processes was almost identical which indicates a linkage between K⁺ “pump” and “leak”. Further, these results demonstrate that red cell K⁺ transport can be modulated by local conditions in the micro-environment of the transport system.     

Modulated red blood cell survival by membrane protein clustering

Human and murine blood cells treated with ZnCl₂ and bis(sulfosuccinimidyl)suberate (BS³) (a cross linking agent) undergo band 3 clustering and binding of hemoglobin to red blood cell membrane proteins. These clusters induce autologous IgG binding and complement fixation, thus favouring the phagocytosis of ZnCl₂/BS³ treated cells by macrophages. The extension of red blood cell opsonization can be easily modulated by changing the ZnCl₂ concentration in the 0.1–1.0 mM range thus providing an effective way to affect blood cell recognition by macrophages. In fact, murine erythrocytes treated with increasing ZnCl₂ concentrations have proportionally reduced survivals when reinjected into the animal. Furthermore, the organ sequestration of ZnCl₂/BS³ treated cells strongly resembles the typical distribution of the senescent cells. Since the ZnCl₂/BS³ treatment can also be performed on red blood cells loaded with drugs or other substances, this procedure is an effective drug-targeting system to be used for the delivery of molecules to peritoneal, liver and spleen macrophages.     

Interactions among red cell membrane proteins

Interactions between human red band 2.1 with spectrin and depleted inside-out vesicles were studied by fluorescence resonance energy transfer and batch microcalorimetry. The band 2.1-spectrin binding isotherm is consistent with a one to one mole ratio. The association constant of 1.4 X 10(8) M-1 corresponds to the association free energy of -11.1 kcal/mol. Under our experimental conditions, the enthalpy of interaction of band 2.1-spectrin was found to be -10.8 kcal/mol and is independent of the protein mole ratio. The calculated entropic factor (-T delta S = 0.3 kcal/mol) strongly suggests a predominantly enthalpic character of the reaction. In addition, we investigated the role of band 2.1 on the binding of band 4.1 to spectrin [Podgorski, A., & Elbaum, D. (1985) Biochemistry 24, 7871-7876] and concluded that only small, if any, alterations of binding of band 4.1 to spectrin have taken place in the presence or absence of band 2.1. This suggests thermodynamic independence of the binding sites. Although the attachment of the cytoskeletal network to the membrane takes place through, at least, two different interactions, band 2.1-band 3 and 4.1-glycophorin, the relative enthalpy values suggest that band 2.1 contributes significantly more than band 4.1 to the energy of the interaction. In addition, we observed that polymerization of actin is modulated by the cytoskeletons as judged by their effect on the rate of actin polymerization.     

Regulation of plasma membrane blebbing by the cytoskeleton

When neuroblastoma cells are exposed to lysophosphatidic acid (LPA), they undergo a vigorous, but transient blebbing phase. The effect is sensitive to inhibition by staurosporine, KT 5926 (an inhibitor of myosin light chain kinase), and cytochalasin B, suggesting that LPA activates the phosphorylation of myosin light chain and increases the contractile activity of the actomyosin network. Cell contractions increase the intracellular pressure driving bleb formation. Calyculin, an inhibitor of protein phosphatase2A, also causes blebbing which continues as long as the drug is present, presumably by keeping myosin light chain in the phosphorylated state. Blebbing of neuroblastoma cells is regulated by the status of all three cytoskeletal systems: disassembly of microtubules by nocodazole and of intermediate filaments by acrylamide increased the number of blebbing cells. Cytochalasin B, on the other hand, prevents bleb retraction and, after prolonged incubation, bleb formation. These results are discussed in terms of a model viewing the cytoskeleton as an integrated network transmitting force throughout the cell. Bleb retraction was studied by transfecting neuroblastoma cells with a vector containing the gene for gamma-cytoplasmic actin fused to the green fluorescent protein EGFP (EGFP-actin). EGFP-actin was not detected on the membranes of extending blebs, but started accumulating along the cytoplasmic surface of blebs as soon as the extension phase came to an end and retraction set in. These results confirm earlier suggestions that actin polymerization is required for bleb retraction and for the first time directly relate the two events.     

Thermoelasticity of red blood cell membrane.

The elastic properties of the human red blood cell membrane have been measured as functions of temperature. The area compressibility modulus and the elastic shear modulus, which together characterize the surface elastic behavior of the membrane, have been measured over the temperature range of 2-50 degrees C with micropipette aspiration of flaccid and osmotically swollen red cells. In addition, the fractional increase in membrane surface area from 2-50 degrees C has been measured to give a value for the thermal area expansivity. The value of the elastic shear modulus at 25 degrees C was measured to be 6.6 X 10(-3) dyne/cm. The change in the elastic shear modulus with temperature was -6 X 10(-5) dyne/cm degrees C. Fractional forces were shown to be only on the order of 10-15%. The area compressibility modulus at 25 degrees C was measured to be 450 dyne/cm. The change in the area compressibility modulus with temperature was -6 dyne/cm degrees C. The thermal area expansivity for red cell membrane was measured to be 1.2 X 10(-3)/degrees C. With this data and thermoelastic relations the heat of expansion is determined to be 110-200 ergs/cm2; the heat of extension is 2 X 10(-2) ergs/cm2 for unit extension of the red cell membrane. The heat of expansion is of the order anticipated for a lipid bilayer idealized as twice the behavior of a monolayer at an oil-water interface. The observation that the heat of extension is positive demonstrates that the entropy of the material increases with extension, and that the dominant mechanism of elastic energy storage is energetic. Assuming that the red cell membrane shear rigidity is associated with "spectrin," unit extension of the membrane increases the configurational entropy of spectrin by 500 cal/mol.     

Serum proteins as mediators of hemin efflux from red cell membranes: specificity of hemopexin

The involvement of the serum heme-binding proteins hemopexin and albumin in the clearance of erythrocyte membranes from toxic hemin was compared. In the presence of hemopexin initial rates of hemin efflux from resealed ghosts were faster and the amount of extracted hemin larger. When hemin-containing ghosts were treated with a protein mixture of 1:45 hemopexin to albumin, as present in serum, most of the hemin was extracted in the form of heme-hemopexin. It was concluded that hemopexin is the serum protein responsible for heme extraction from cell membranes.     

Haemoglobin and the red cell membrane.

The results presented here indicate that haemoglobin is an integral part of the red cell membrane. The haemoglobin content of the membrane is highly dependent on the Ca++ content of the membrane in health and disease. Changes in the red cell interior alter the whole organization of the membrane and are even reflected in the binding of immunoglobulins to the red cell surface. The preferential binding of Hb-s A2 and S to the membrane has been confirmed. This phenomenon cannot be explained by differences in the charge between these haemoglobins and Hb A.     

Regulation of red cell membrane deformability and stability by skeletal protein network

The skeletal protein network of the red blood cell is thought to be important in regulating such membrane functions as deformability and stability. In the present study, we measured membrane deformability and stability of the resealed ghosts using an ektacytometer, a laser diffraction method, and identified the functional role of protein 4.1 and that of Ca2+ and calmodulin in maintaining membrane stability. To obtain direct evidence for a crucial role of protein 4.1 in maintaining membrane stability, we reconstituted protein 4.1-deficient membranes with purified protein 4.1. Although native membranes deficient in protein 4.1 had marked reduction in membrane stability, reconstitution with increasing concentrations of purified protein 4.1 resulted in progressive restoration of membrane stability, providing direct evidence that protein 4.1 is essential for normal membrane stability. To determine if Ca2+ and calmodulin could modulate membrane properties, we measured membrane stability and deformability of resealed ghosts prepared in the presence of varying concentrations of Ca2+ and physiologic concentrations of calmodulin. Our data show that Ca2+ concentrations in the range of 1 to 100 microM can markedly decrease membrane stability only in the presence of calmodulin, but not in its absence. In contrast, deformability decreased only at Ca2+ concentrations higher than 100 microM, and calmodulin had no effect. Examination of the the effects of Ca2+ and calmodulin on various membrane protein interactions has enabled us to suggest that the observed changes in membrane stability may be partly related to the effects of Ca2+ and calmodulin on spectrin-protein 4.1-actin interaction.     

Modelling the structure of the red cell membrane

The red cell membrane has long been the focus of extensive study. The macromolecules embedded within the membrane carry the blood group antigens and perform many functions including the vital task of gas exchange. Links between the intramembrane macromolecules and the underlying cytoskeleton stabilize the biconcave morphology of the red cell and allow deformation during microvascular transit. Much is now known about the proteins of the red cell membrane and how they are organised. In many cases we have an understanding of which proteins are expressed, the number of each protein per cell, their oligomeric state(s), and how they are collected in large multi-protein complexes. However, our typical view of these structures is as cartoon shapes in schematic figures. In this study we have combined knowledge of the red cell membrane with a wealth of protein structure data from crystallography, NMR, and homology modelling to generate the first, tentative models of the complexes which link the membrane to the cytoskeleton. Measurement of the size of these complexes and comparison with known cytoskeletal distance parameters suggests the idea of interaction between the membrane complexes, which may have profound implications for understanding red cell function and deformation.