Spectrin, human erythrocyte shapes, and mechanochemical properties.

Physical studies of human erythrocyte spectrin indicate that isolated spectrin dimers and tetramers in solution are worm-like coils with a persistence length of approximately 20 nm. This finding, the known polyelectrolytic nature of spectrin, and other structural information about spectrin and the membrane skeleton molecular organization have lead us to the hypothesis that the human erythrocyte membrane skeleton constitutes a two-dimensional ionic gel (swollen ionic elastomer). This concept is incorporated in what we refer to as the protein gel-lipid bilayer membrane model. The model accounts quantitatively for red elastic shear modulus and the maximum elastic extension ratio reported for the human erythrocytes membrane. Gel theory further predicts that depending on the environmental conditions, the membrane skeleton modulus of area compression may be small or large relative to the membrane elastic shear modulus. Our analyses show that the ratio between these two parameters affects both the geometry and the stability of the favored cell shapes and that the higher the membrane skeleton compressibility the smaller the values of the gel tension needed to induce cell shape transformations. The main virtue of the protein gel-lipid bilayer membrane model is that it offers a novel theoretical and molecular basis for the various mechanical properties of the membrane skeleton such as the membrane skeleton modulus of area compression and osmotic tension, and the effects of these properties on local membrane skeleton density, cell shape, and shape transformations.     

Elastic energy of curvature-driven bump formation on red blood cell membrane.

Model calculations were performed to explore quantitative aspects of the discocyte-echinocyte shape transformation in red blood cells. The shape transformation was assumed to be driven by changes in the preferred curvature of the membrane bilayer and opposed by the elastic shear rigidity of the membrane skeleton. The energy required for echinocyte bump formation was calculated for a range of bump shapes for different preferred curvatures. Energy minima corresponding to nonzero bump heights were found when the stress-free area difference between the membrane leaflets or the spontaneous curvature of the membrane became sufficiently large, but the calculations predict that the membrane can tolerate significant differences in the resting areas of the inner and outer leaflets or significant spontaneous curvature without visible changes in shape. Thus, if the cell is near the threshold for bump formation, the calculations predict that small changes in membrane properties would produce large changes in cellular geometry. These results provide a rational framework for interpreting observations of shape transformations in red cells and for understanding the mechanism by which small changes in membrane elastic properties might lead to significant changes in geometry.     

A conserved region of the MSP-1 surface protein of Plasmodium falciparum contains a recognition sequence for erythrocyte spectrin.

The major surface protein MSP-1 of Plasmodium falciparum blood-stage malaria parasites contains notably conserved sequence blocks with unknown function. The recombinant protein 190L, which represents such a block, exhibits a high affinity for red blood cell membranes. We demonstrate that both 190L and native MSP-1 protein bind to the inner red blood cell membrane skeleton protein spectrin. By using overlapping peptides covering the 190L molecule, we show that the spectrin contact site of 190L is included in a linear sequence of 30 amino acid residues. Association of 190L with naturally occurring spectrin deficient red blood cells is drastically reduced. In the same cells parasite invasion is normal, but the intracellular parasite development arrests late in the trophozoite stage. A similar situation arises when synthetic peptides covering the spectrin recognition sequence of 190L are added to P.falciparum cultures. These data and the cellular localization of MSP-1 suggest the possibility that MSP-1 associates with spectrin under natural conditions.     

Membrane orientation of sheep spectrin

Based primarily on studies of human erythrocytes, current theories of the structure and organization of erythrocyte membrane localize spectrin to the membrane cytoplasmic surface. Affinity purified anti-sheep spectrin antibodies were used in indirect immunofluorescence studies of intact erythrocytes from various vertebrate species and inside-out and right-side-out impermeable sheep erythrocyte vesicles. This investigation detected immunologically reactive external and potentially transmembranal determinant(s) of the sheep erythrocyte spectrin "assembly." Parallel studies using anti-sheep and anti-human spectrin antibodies, as well as 125I surface-labelling studies of intact sheep and human erythrocytes, indicated that this particular membrane orientation of spectrin was evident in sheep but not in human erythrocytes. Antisera containing antibodies to the external portion of this spectrin "assembly" demonstrated external fluorescence to a variable degree on some, but not all, vertebrate erythrocytes surveyed, confirming that the sheep erythrocyte was not the only exception. It is suggested that there may be subtle species variability in the intermolecular associations of the spectrin "assembly" with(in) the erythrocyte membrane not requiring alterations of the spectrin molecule itself.     

Structural and functional studies of interaction between Plasmodium falciparum knob-associated histidine-rich protein (KAHRP) and erythrocyte spectrin

Plasmodium falciparum dramatically modifies the structure and function of the membrane of the parasitized host erythrocyte. Altered membrane properties are the consequence of the interaction of a group of exported malaria proteins with host cell membrane proteins. KAHRP (the knob-associated histidine-rich protein), a member of this group, has been shown to interact with erythrocyte membrane skeletal protein spectrin. However, the molecular basis for this interaction has yet to be defined. In the present study, we defined the binding motifs in both KAHRP and spectrin and identified a functional role for this interaction. We showed that spectrin bound to a 72-amino-acid KAHRP fragment (residues 370-441). Among nine-spectrin fragments, which encompass the entire alpha and beta spectrin molecules (four alpha spectrin and five beta spectrin fragments), KAHRP bound only to one, the alpha N-5 fragment. The KAHRP-binding site within the alpha N-5 fragment was localized uniquely to repeat 4. The interaction of full-length spectrin dimer to KAHRP was inhibited by repeat 4 of alpha spectrin. Importantly, resealing of this repeat peptide into erythrocytes mislocalized KAHRP in the parasitized cells. We concluded that the interaction of KAHRP with spectrin is critical for appropriate membrane localization of KAHRP in parasitized erythrocytes. As the presence of KAHRP at the erythrocyte membrane is necessary for cytoadherence in vivo, our findings have implications for the development of new therapies for mitigating the severity of malaria infection.     

Widespread occurrence of avian spectrin in nonerythroid cells

We have prepared an antibody against chicken erythrocyte α spectrin, using as immunogen protein purified by two-dimensional polyacrylamide gel electrophoresis. One- and two-dimensional immunoautoradiography show that this antiserum reacts only with α spectrin in chicken erythrocytes and crossreacts with α spectrin in erythrocytes from various mammals. Immunofluorescence reveals that this antiserum reacts with a plasma membrane component in erythrocytes as well as in most nonerythroid avian and mammalian cells. Intense staining is seen at or near the plasma membrane in neurons, lens cells, endothelial and epithelial cells of the gastrointestinal and respiratory tracts, skeletal and cardiac muscle, as well as skeletal myotubes grown in tissue culture. Immunoautoradiography indicates that the crossreactive antigen in these nonerythroid tissues has the same molecular weight and isoelectric point as the chicken erythrocyte antigen. Smooth muscle, tracheal cilia, myelin and mature sperm stain weakly or not at all. These results suggest that spectrin is more extensively distributed than previously recognized, and that the functions of spectrin elucidated for erythrocytes may apply to other cell types as well.     

Relationship between membrane lipid mobility and spectrin distribution in lymphocytes.

We have previously established that T and B lymphocytes in situ are remarkably heterogeneous with respect to the cytoskeletal protein spectrin. Since in erythrocytes spectrin is known to play an important role in the regulation of membrane fluidity, lipid organization and lateral mobility of membrane proteins, we have sought to determine if the heterogeneous patterns of spectrin distribution that we have observed are related to possible differences in membrane lipid organization in these various subsets. To this end, we have utilized a fluorescent pyrene-labelled phospholipid as a probe of the lipid lateral mobility and have examined two related T cell systems maintained in vitro, DO.11.10 cells and a spontaneously arising variant, DO.11.10V. In these (and other cloned in vitro systems) we have previously observed that the cells homogeneously express one of the kinds of spectrin distribution patterns observed in situ. Thus the uniformity of staining of these systems permits us to address whether the various patterns of spectrin distribution may be predictive of differences in membrane lipid properties. Here we show that in cells in which there is little or nor spectrin at the plasma membrane (DO.11.10) that the lipids in the plasma membrane are considerably less mobile than in its related variant in which spectrin is diffusely distributed within the cell and at the plasma membrane. From this and previous results, we conclude that differences in the distribution of the cytoskeletal protein spectrin among lymphocytes may be a useful parameter in helping to predict the status of membrane lipid organization.     

Weak interaction of spectrin with phosphatidylcholine-phosphatidylserine multilayers: a 2H and 31P NMR study

Spectrin from human erythrocytes binds to bilayer dispersions of both DMPC and DMPS:DMPC (1:1, w/w). However, no effect of bound spectrin on the conformation of the lipid head groups, as measured from the deuterium quadrupolar splittings of DMPC or DMPS specifically deuterated in the polar head groups, was detected in 1:1 mixtures of the two lipids containing either deuterated DMPC or DMPS. Neither the phase transition of the DMPS:DMPC mixtures, nor the spin-lattice relaxation time (T1) of the deuterated DMPS head group, was affected by spectrin. These results argue against any strong interaction of spectrin with phosphatidylserine and rule out the possibility that spectrin is responsible for the maintenance of PS in the inner monolayer of the erythrocyte membrane during the whole life-span of this cell.     

Disruption of phospholipid asymmetry in erythrocyte vesicles deficient in spectrin

The transbilayer distribution of phospholipids in right-side-out and inside-out vesicles derived from human erythrocytes was studied by phospholipase A2 digestion assays and by staining with the fluorescent dye merocyanine 540. In both types of vesicles, the normal asymmetric distribution of phospholipids characteristic of intact cells was disrupted. Because both types of vesicles are deficient in spectrin, the major protein of the cytoskeletal network which normally underlies the membrane, these results support the contention that spectrin is involved in the maintenance of phospholipid asymmetry.     

Control of band 3 lateral and rotational mobility by band 4.2 in intact erythrocytes: release of band 3 oligomers from low-affinity binding sites.

Band 4.2 is a human erythrocyte membrane protein of incompletely characterized structure and function. Erythrocytes deficient in band 4.2 protein were used to examine the functional role of band 4.2 in intact erythrocyte membranes. Both the lateral and the rotational mobilities of band 3 were increased in band 4.2-deficient erythrocytes compared to control cells. In contrast, the lateral mobility of neither glycophorins nor a fluorescent phospholipid analog was altered in band 4.2-deficient cells. Compared to controls, band 4.2-deficient erythrocytes manifested a decreased ratio of band 3 to spectrin, and band 4.2-deficient membrane skeletons had decreased extractability of band 3 under low-salt conditions. Normal band 4.2 was found to bind to spectrin in solution and to promote the binding of spectrin to ankyrin-stripped inside-out vesicles. We conclude that band 4.2 provides low-affinity binding sites for both band 3 oligomers and spectrin dimers on the human erythrocyte membrane. Band 4.2 may serve as an accessory linking protein between the membrane skeleton and the overlying lipid bilayer.     

Immunofluorescent patterns of spectrin in lymphocyte cell lines

Spectrin, a membrane-associated cytoskeletal protein, has been observed in all of 45 lymphoid and myeloid cell lines examined. For these experiments, formalin-fixed cells from randomly selected lines propagated by using conventional tissue culture procedures were examined by immunofluorescence, using an antibody directed against chicken erythrocyte alpha-spectrin. Two distinct immunofluorescent patterns of spectrin distribution were identified. In most lines examined (16 mouse and 18 human lymphoid or myeloid lines), spectrin was symmetrically distributed near the submembranous region of the plasma membrane. In the remainder of the cell lines examined, a second pattern was observed; in these cultures, the cells contain a polar submembranous aggregate of spectrin with little staining at the rest of the plasma membrane. Long-term T lymphocyte cell lines in which greater than 60% of the cells expressed a polar submembranous aggregate of spectrin (PSA-S) include mouse cell lines EL-4, LBRM-33, CT-6X, NIXT, 22CM-37, and 7ON-2 and human lines JM and PEER. Other established cultures in which PSA-S were observed included the human macrophage-like line U-937 and gibbon T cell line MLA-144. Phorbol myristate acetate or mezerin caused a reversible alteration in the distribution of spectrin in these cell lines. These drugs, which increase membrane fluidity, caused a complete but temporary symmetrical redistribution of the spectrin aggregate. Our results indicate that the pattern of spectrin distribution, either aggregated or evenly dispersed, is a stable characteristic (but one that can be altered) in various cell lines, and that because similar variations in pattern have been noted in situ, it is likely that the pattern present in any given cell line reflects a characteristic associated with a particular stage of a cell's maturation. It is anticipated that these cell lines, positive and negative for the expression of natural polarity of spectrin distribution, will provide useful models for future studies to define further the role of spectrin in lymphocyte plasma membrane functions.     

Relation between the organization of spectrin and of membrane lipids in lymphocytes

In lymphocytes, the cytoskeletal protein spectrin exhibits two organizational states. Because the plasma membrane lipids of lymphocytes also display two organizational states, it was asked whether there is a relation between the organization of spectrin and of membrane lipids. When mouse thymocytes were stained with merocyanine 540 (MC540), a fluorescent lipophilic probe that binds preferentially to loosely packed, disorganized lipid bilayers, some cells fluoresced brightly and some only dimly or not at all. When the same population was stained for spectrin by indirect immunofluorescence, the spectrin in some cells was uniformly distributed, while in others it was concentrated in a unipolar aggregate. Techniques enriching for mature thymocytes selected for cells displaying low MC540 fluorescence and aggregated spectrin, the same characteristics found in peripheral blood lymphocytes. Flow cytometric sorting of thymocytes based on MC540 phenotype simultaneously sorted them by spectrin phenotype. Finally, treatment with agents that alter the distribution of spectrin caused mature lymphocytes to display high MC540 fluorescence and uniform spectrin. Thus, a relation exists between the organizational states of spectrin and of membrane lipids in lymphocytes: aggregated spectrin is found in cells with tightly organized membrane lipids, uniform spectrin in those with loosely organized lipids. Spectrin may thus be involved in modulating membrane lipid organization in lymphocytes as it is in erythrocytes. Since loosely organized lipids may promote adhesion of blood cells to reticuloendothelial cells, spectrin may thereby be involved in transducing an internally generated adhesion signal to the lymphocyte surface.     

Red Blood Cell Shape and Fluctuations: Cytoskeleton Confinement and ATP Activity

We review recent theoretical work that analyzes experimental measurements of the shape and fluctuations of red blood cells. Particular emphasis is placed on the role of the cytoskeleton and cell elasticity and we contrast the situation of elastic cells with that of fluid-filled vesicles. In red blood cells (RBCs), the cytoskeleton consists of a two-dimensional network of spectrin proteins. Our analysis of the wave vector and frequency dependence of the fluctuation spectrum of RBCs indicates that the spectrin network acts as a confining potential that reduces the fluctuations of the lipid bilayer membrane. However, since the cytoskeleton is only sparsely connected to the bilayer, one cannot regard the composite cytoskeleton membrane as a polymerized object with a shear modulus. The sensitivity of RBC fluctuations and shapes to ATP concentration may reflect the transient defects induced in the cytoskeleton network by ATP.     

Erythrocyte Membrane Model with Explicit Description of the Lipid Bilayer and the Spectrin Network

The membrane of the red blood cell (RBC) consists of spectrin tetramers connected at actin junctional complexes, forming a two-dimensional (2D) sixfold triangular network anchored to the lipid bilayer. Better understanding of the erythrocyte mechanics in hereditary blood disorders such as spherocytosis, elliptocytosis, and especially, sickle cell disease requires the development of a detailed membrane model. In this study, we introduce a mesoscale implicit-solvent coarse-grained molecular dynamics (CGMD) model of the erythrocyte membrane that explicitly describes the phospholipid bilayer and the cytoskeleton, by extending a previously developed two-component RBC membrane model. We show that the proposed model represents RBC membrane with the appropriate bending stiffness and shear modulus. The timescale and self-consistency of the model are established by comparing our results with experimentally measured viscosity and thermal fluctuations of the RBC membrane. Furthermore, we measure the pressure exerted by the cytoskeleton on the lipid bilayer. We find that defects at the anchoring points of the cytoskeleton to the lipid bilayer (as in spherocytes) cause a reduction in the pressure compared with an intact membrane, whereas defects in the dimer-dimer association of a spectrin filament (as in elliptocytes) cause an even larger decrease in the pressure. We conjecture that this finding may explain why the experimentally measured diffusion coefficients of band-3 proteins are higher in elliptocytes than in spherocytes, and higher than in normal RBCs. Finally, we study the effects that possible attractive forces between the spectrin filaments and the lipid bilayer have on the pressure applied on the lipid bilayer by the filaments. We discover that the attractive forces cause an increase in the pressure as they diminish the effect of membrane protein defects. As this finding contradicts with experimental results, we conclude that the attractive forces are moderate and do not impose a complete attachment of the filaments to the lipid bilayer.     

Brain spectrin(240/235) and brain spectrin(240/235E): two distinct spectrin subtypes with different locations within mammalian neural cells

Adult mouse brain contains at least two distinct spectrin subtypes, both consisting of 240-kD and 235-kD subunits. Brain spectrin(240/235) is found in neuronal axons, but not dendrites, when immunohistochemistry is performed with antibody raised against brain spectrin isolated from enriched synaptic/axonal membranes. A second spectrin subtype, brain spectrin(240/235E), is exclusively recognized by red blood cell spectrin antibody. Brain spectrin(240/235E) is confined to neuronal cell bodies and dendrites, and some glial cells, but is not present in axons or presynaptic terminals.     

The human erythrocyte membrane skeleton may be an ionic gel

In the first paper in this series (Stokke et al. Eur Biophys J 1986, 13:203-218) we developed the general theory of the mechanochemical properties and the elastic free energy of the protein gel--lipid bilayer membrane model. Here we report on an extensive numerical analysis of the human erythrocyte shapes and shape transformations predicted by this new cell membrane model. We have calculated the total elastic free energy of deformation of four different cell shape classes: disc-shaped cells, cup-shaped cells, crenated cells, and cells with membrane invaginations. We find that which of these shape classes is favoured depends strongly on the spectrin gel osmotic tension, IIGu, and the surface tensions, IIEu and IIPu, of the extracellular and protoplasmic halves of the membrane lipid bilayer, respectively. For constant ratio IIEu/IIPu greater than O large negative or positive values of IIGu favour respectively the crenated and invaginated cell shape classes. For small absolute values of IIGu, IIEu, and IIPu, biconcave or cup-shaped cells are the stable ones. Our numerical analysis shows that the higher the membrane skeleton compressibility is, the smaller are the values of IIGu needed to induce cell shape transformation. We find that the stable and metastable shapes of discocytes and stomatocytes generally depend both on the shape of the stressfree membrane skeleton and the membrane skeleton compressibility.     

Crenation and cupping of the red cell: A new theoretical approach. Part I. Crenation

Crenation can be thought of as a surface instability caused by intrinsic precurvature of the membrane. Mathematical modeling, on the presupposition that the red blood cell is a thin shell consisting of a connected (coupled) bilayer having uniformly distributed elastic properties shows that crenation can be initiated by negative precurvature, that is, intrinsic curvature having its concavity directed towards the outside of the cell. This is contrary to the currently accepted view which attributes the effect to positive precurvature of an unconnected bilayer. Crenation and the biconcave shape can coexist in the red cell. This suggests that the bilayer must be connected even when the cell is crenated because the biconcave shape could not otherwise be maintained. The progressive development of crenation to more advanced stages, such as the echinocyte type III and the spheroechinocyte can be accounted for if the outer layer of the membrane is stressed beyond the range where strain is proportional to stress. This is consistent with the extremely small radius of curvature at the tips of the crenations.Certain small variations in the uncrenated biconcave shape of the red cell can be interpreted mathematically as due either to negative intrinsic curvature or to shear resistance. Since, however, a small amount of negative precurvature has been shown to be capable of inducing crenation, it is unlikely to be the cause of the variations in the biconcave shape. These must therefore be due to shear resistance.In the light of this new approach, membrane molecular models based on the assumption that crenation is due to positive precurvature need reconsideration.     

Effect of the level of ATP and of the state of spectrin on osmotic properties of bovine erythrocytes

The effect of the intracellular level of ATP and of the state of spectrin on the critical cell volume of bovine erythrocyte was studied. The state of spectrin was changed by thermal denaturation, which for the bovine red cell took place at similar temperature as for the human erythrocyte. The increase of the ATP level and the spectrin denaturation increased the critical cell volume, while metabolic starvation decreased it. The changes of the ATP level did not influence the critical volume after the denaturation of spectrin. The results suggest that the ATP-dependent effect on the critical cell volume was caused by an alteration of the membrane extensibility due to the change of the membrane skeleton-lipid bilayer interaction(s).