αI-spectrin represents evolutionary optimization of spectrin for red blood cell deformability.

Spectrin tetramers of the membranes of enucleated mammalian erythrocytes play a critical role in red blood cell survival in circulation. One of the spectrins, αI, emerged in mammals with enucleated red cells after duplication of the ancestral α-spectrin gene common to all animals. The neofunctionalized αI-spectrin has moderate affinity for ßI-spectrin, whereas αII-spectrin, expressed in nonerythroid cells, retains ancestral characteristics and has a 10-fold higher affinity for ßI-spectrin. It has been hypothesized that this adaptation allows for rapid make and break of tetramers to accommodate membrane deformation. We have tested this hypothesis by generating mice with high-affinity spectrin tetramers formed by exchanging the site of tetramer formation in αI-spectrin (segments R0 and R1) for that of αII-spectrin. Erythrocytes with αIIßI presented normal hematologic parameters yet showed increased thermostability, and their membranes were significantly less deformable; under low shear forces, they displayed tumbling behavior rather than tank treading. The membrane skeleton is more stable with αIIßI and shows significantly less remodeling under deformation than red cell membranes of wild-type mice. These data demonstrate that spectrin tetramers undergo remodeling in intact erythrocytes and that this is required for the normal deformability of the erythrocyte membrane. We conclude that αI-spectrin represents evolutionary optimization of tetramer formation: neither higher-affinity tetramers (as shown here) nor lower affinity (as seen in hemolytic disease) can support the membrane properties required for effective tissue oxygenation in circulation.

Inter-subunit interactions in erythroid and non-erythroid spectrins.

Spectrins comprise α- and ß-subunits made up predominantly of a series of homologous repeating units of about 106 amino acids; the α- and ß-chains form antiparallel dimers by lateral association, and tetramers through head-to-head contacts between the dimers. Here we consider the first of these interactions. (1) We confirm earlier observations, showing that the first two paired repeats (ßIR1 with αIR21, and ßIR2 with αRI20) at one end of the erythroid spectrin (αIßI) dimer are necessary and sufficient to unite the chains; (2) we resolve a conflict in published reports by showing that the strength of the interaction is considerably increased on adding the adjoining pair of repeats (ßIR3-αIR19); (3) in brain (αIIßII) spectrin the first two pairs of repeats are similarly essential and sufficient for heterodimer formation; (4) this interaction is ~60-fold stronger than that in the erythroid counterpart, but no enhancement can be detected on addition of three further pairs of repeats; (5) formation of a tight αIßI dimer probably depends on structural coupling of the first two repeats in each chain; (6) an analysis of the sequences of the strongly interacting repeats, ßIR1, ßIIR1, αIR21 and αIIR20 and repeats in α-actinin, which also interact very strongly in forming an antiparallel dimer, affords a possible explanation for the different properties of the two spectrin isoforms in respect of the stability of the inter-chain interactions, and also suggests the evolutionary path by which the erythroid and non-erythroid sequences diverged.

Control of erythrocyte membrane-skeletal cohesion by the spectrin-membrane linkage.

Spectrin tetramer is the major structural member of the membrane-associated skeletal network of red cells. We show here that disruption of the spectrin-ankyrin-band 3 link to the membrane leads to dissociation of a large proportion of the tetramers into dimers. Noncovalent perturbation of the linkage was induced by a peptide containing the ankyrin-binding site of the spectrin beta-chain, and covalent perturbation by treatment with the thiol reagent, N-ethylmaleimide (NEM). This reagent left the intrinsic self-association capacity of the spectrin dimers unaffected and disturbed only the ankyrin-band 3 interaction. The dissociation of spectrin tetramers on the membrane into functional dimers was confirmed by the binding of a spectrin peptide directed against the self-association sites. Dissociation of the tetramers resulted, we infer, from detachment of the proximal ends of the constituent dimers from the membrane, thereby reducing their proximity to one another and thus weakening their association. The measured affinity of the interaction of the peptides with the free dimer ends on the membrane permits an estimate of the equilibrium between intact and dissociated tetramers on the native membrane. This indicates that in the physiological state the equilibrium proportion of the dissociated tetramers may be as high as 5-10%. These findings enabled us to identify an additional important functional role for the spectrin-ankyrin-band 3 link in regulating spectrin self-association in the red cell membrane.

Fluctuations of the red blood cell membrane: relation to mechanical properties and lack of ATP dependence.

We have analyzed the fluctuations of the red blood cell membrane in both the temporal ((omega(s(-1))) and spatial (q(m(-1))) frequency domains. The cells were examined over a range of osmolarities leading to cell volumes from 50% to 170% of that in the isotonic state. The fluctuations of the isotonic cell showed an approximately q(-3)-dependence, indicative of a motion dominated by bending, with an inferred bending modulus of approximately 9 x 10(-19) J. When the cells were osmotically swollen to just below the point of lysis (166% of physiological volume), a q(-1)-dependence of the fluctuations supervened, implying that the motion was now dominated by membrane tension; estimated as approximately 1.3 x 10(-4) nm(-1). When, on the other hand, the cells were osmotically dehydrated, the fluctuation amplitude progressively decreased. This was caused by a rise in internal viscosity, as shown by measurements on resealed ghosts containing a reduced hemoglobin concentration, which displayed no such effect. We examined, in addition, cells depleted of ATP, before the onset of echinocytosis, and could observe no change in fluctuation amplitude. We conclude that the membrane fluctuations of the red cell are governed by bending modulus, membrane tension, and cytosolic viscosity, with little or no dependence on the presence or absence of ATP.

[The Lyssenko affair: the eclipse of reason]. / L'affaire Lyssenko, une éclipse de la raison.

Trofim Lysenko was a Ukrainian peasant whose malign influence dominated biology in the Soviet Union and its imperium through most of Stalin's reign. Lysenko owed his ascendancy to repeated promises that he would rescue Soviet agriculture from the catastrophic state into which it had sunk, following Stalin's disastrous policy of collectivisation of the farms, and a succession of bad harvests. He claimed to have devised methods of imposing desirable hereditary characteristics on plants, and even of converting one species into another at will. He noisily denounced modern genetics as a bourgeois imposture, a view that resonated well with Marxist doctrine. As Lysenko's power grew he was able to smother scientific debate, and to crush all opposition through the arrest and often execution of many leading scientists. Lysenko's preposterous theories became the accepted orthodoxy in the academies and universities of Eastern Europe, and were greeted with enthusiasm by many Communist intellectuals in the West, not least in France. The Lysenko phenomenon is the most extreme, but by no means the only example of the perversion of science by ideology, often with the acquiescence of the scientific community. Nor can we be confident that nothing of the kind could happen today.

Tropomyosin modulates erythrocyte membrane stability.

The ternary complex of spectrin, actin, and 4.1R (human erythrocyte protein 4.1) defines the nodes of the erythrocyte membrane skeletal network and is inseparable from membrane stability under mechanical stress. These junctions also contain tropomyosin (TM) and the other actin-binding proteins, adducin, protein 4.9, tropomodulin, and a small proportion of capZ, the functions of which are poorly defined. Here, we have examined the consequences of selective elimination of TM from the membrane. We have shown that the mechanical stability of the membranes of resealed ghosts devoid of TM is grossly, but reversibly, impaired. That the decreased membrane stability of TM-depleted membranes is the result of destabilization of the ternary complex of the network junctions is demonstrated by the strongly facilitated entry into the junctions in situ of a beta-spectrin peptide, containing the actin- and 4.1R-binding sites, after extraction of the TM. The stabilizing effect of TM is highly specific, in that it is only the endogenous isotype, and not the slightly longer muscle TM that can bind to the depleted membranes and restore their mechanical stability. These findings have enabled us identify a function for TM in elevating the mechanical stability of erythrocyte membranes by stabilizing the spectrin-actin-4.1R junctional complex.

The ring-infected erythrocyte surface antigen (RESA) of Plasmodium falciparum stabilizes spectrin tetramers and suppresses further invasion.

The malaria parasite Plasmodium falciparum releases the ring-infected erythrocyte surface antigen (RESA) inside the red cell on entry. The protein migrates to the host cell membrane, where it binds to spectrin, but neither the nature of the interaction nor its functional consequences have previously been defined. Here, we identify the binding motifs involved in the interaction and describe a possible function. We have found that spectrin binds to a 108-amino acid fragment (residues 663-770) of RESA, and that this RESA fragment binds to repeat 16 of the beta-chain, close to the labile dimer-dimer self-association site. We further show that the RESA fragment stabilizes the spectrin tetramer against dissociation into its constituent dimers, both in situ and in solution. This is accompanied by enhanced resistance of the cell to both mechanical and thermal degradation. Resealed erythrocytes containing RESA(663-770) display resistance to invasion by merozoites of P falciparum. We infer that the evolutionary advantage of RESA to the parasite lies in its ability to prevent invasion of cells that are already host to a developing parasite, as well as possibly to guard the cell against thermal damage at the elevated body temperatures prevailing in febrile crises.

Mammalian alpha I-spectrin is a neofunctionalized polypeptide adapted to small highly deformable erythrocytes.

Mammalian red blood cells, unlike those of other vertebrates, must withstand the rigors of circulation in the absence of new protein synthesis. Key to this is plasma membrane elasticity deriving from the protein spectrin, which forms a network on the cytoplasmic face. Spectrin is a tetramer (alphabeta)(2), made up of alphabeta dimers linked head to head. We show here that one component of erythrocyte spectrin, alphaI, is encoded by a gene unique to mammals. Phylogenetic analysis suggests that the other alpha-spectrin gene (alphaII) common to all vertebrates was duplicated after the emergence of amphibia, and that the resulting alphaI gene was preserved only in mammals. The activities of alphaI and alphaII spectrins differ in the context of the human red cell membrane. An alphaI-spectrin fragment containing the site of head-to-head interaction with the beta-chain binds more weakly than the corresponding alphaII fragment to this site. The latter competes so strongly with endogenous alphaI as to cause destabilization of membranes at 100-fold lower concentration than the alphaI fragment. The efficacies of alphaI/alphaII chimeras indicate that the partial structural repeat, which binds to the complementary beta-spectrin element, and the adjacent complete repeat together determine the strength of the dimer-dimer interaction on the membrane. Alignment of all available alpha-spectrin N-terminal sequences reveals three blocks of sequence unique to alphaI. Furthermore, human alphaII-spectrin is closer to fruitfly alpha-spectrin than to human alphaI-spectrin, consistent with adaptation of alphaI to new functions. We conclude that alphaI-spectrin represents a neofunctionalized spectrin adapted to the rapid make and break of tetramers.

Thermal stabilities of brain spectrin and the constituent repeats of subunits.

The different genes that encode mammalian spectrins give rise to proteins differing in their apparent stiffness. To explore this, we have compared the thermal stabilities of the structural repeats of brain spectrin subunits (alphaII and betaII) with those of erythrocyte spectrin (alphaI and betaI). The unfolding transition midpoints (T(m)) of the 36 alphaII- and betaII-spectrin repeats extend between 24 and 82 degrees C, with an average higher by some 10 degrees C than that of the alphaI- and betaI-spectrin repeats. This difference is reflected in the T(m) values of the intact brain and erythrocyte spectrins. Two of three tandem-repeat constructs from brain spectrin exhibited strong cooperative coupling, with elevation of the T(m) of the less stable partner corresponding to coupling free energies of approximately -4.4 and -3.5 kcal/mol. The third tandem-repeat construct, by contrast, exhibited negligible cooperativity. Tandem-repeat mutants, in which a part of the "linker" helix that connects the two domains was replaced with a corresponding helical segment from erythroid spectrin, showed only minor perturbation of the thermal melting profiles, without breakdown of cooperativity. Thus, the linker regions, which tolerate few point mutations without loss of cooperative function, have evidently evolved to permit conformational coupling in specified regions. The greater structural stability of the repeats in alphaII- and betaII-spectrin may account, at least in part, for the higher rigidity of brain compared to erythrocyte spectrin.

Conformational stabilities of the structural repeats of erythroid spectrin and their functional implications.

The two polypeptide chains of the erythroid spectrin heterodimer contain between them 36 structural repeating modules, which can function as independently folding units. We have expressed all 36 and determined their thermal stabilities. These vary widely, with unfolding transition mid-points (T(m)) ranging from 21 to 72 degrees C. Eight of the isolated repeats are largely unfolded at physiological temperature. Constructs comprising two or more adjacent repeats show inter-repeat coupling with coupling free energies of several kcal mol(-1). Constructs comprising five successive repeats from the beta-chain displayed cooperativity and strong temperature dependence in forced unfolding by atomic force microscopy. Analysis of aligned sequences and molecular modeling suggests that high stability is conferred by large hydrophobic side chains at position e of the heptad hydrophobic repeats in the first helix of the three-helix bundle that makes up each repeat. This inference was borne out by the properties of mutants in which the critical residues have been replaced. The marginal stability of the tertiary structure at several points in the spectrin chains is moderated by energetic coupling with adjoining structural elements but may be expected to permit adaptation of the membrane to the large distortions that the red cell experiences in the circulation.

Phospholipid binding by proteins of the spectrin family: a comparative study.

Erythroid and neuronal spectrin (fodrin) are both known to interact strongly with the aminophospholipids that occur in the inner leaflet of plasma membranes. In erythroid spectrin the positions of the binding sites within the constituent (alphaI and betaI) polypeptide chains have been defined, and also the importance of the lipid interaction in regulating the properties of the membrane. Here we report the locations of the corresponding binding sites in the alphaII and betaII chains that make up the fodrin molecule. Of the 10 lipid-binding repeats in the erythroid spectrin chains 5 are conserved in fodrin; one cluster of 3 consecutive structural repeating units in alphaI erythroid spectrin (repeats 8-10) is displaced by one repeat in alphaII fodrin (repeats 9-11). Fodrin also contains one binding site at the N-terminus of the alphaII chain, not present in the erythroid protein. The regions of the two spectrins containing equivalent lipid-binding sites show a much higher degree of sequence identity than corresponding repeats that do not share this property. The evolutionary conservation of the distribution of a large proportion of strong lipid-binding sites in the polypeptide chains of these two proteins of disparate character argues for a specific function of fodrin-phospholipid interactions in the neuron.

Identification and functional characterization of protein 4.1R and actin-binding sites in erythrocyte beta spectrin: regulation of the interactions by phosphatidylinositol-4,5-bisphosphate.

The ternary complex of spectrin, F-actin, and protein 4.1R defines the erythrocyte membrane skeletal network, which governs the stability and elasticity of the membrane. It has been shown that both 4.1R and actin bind to the N-terminal region (residues 1-301) of the spectrin beta chain, which contains two calponin homology domains, designated CH1 and CH2. Here, we show that 4.1R also binds to the separate CH1 and CH2 domains. Unexpectedly, truncation of the CH2 domain by its 20 amino acids, corresponding to its N-terminal alpha helix, was found to greatly enhance its binding to 4.1R. The intact N terminus and the CH1 but not the CH2 domain bind to F-actin, but again, deletion of the first 20 amino acids of the latter exposes an actin-binding activity. As expected, the polypeptide 1-301 inhibits the binding of spectrin dimer to actin and formation of the spectrin-actin-4.1R ternary complex in vitro. Furthermore, the binding of 4.1R to 1-301 is greatly enhanced by PIP(2), implying the existence of a regulatory switch in the cell.

Phosphatidylserine binding sites in erythroid spectrin: location and implications for membrane stability.

The erythrocyte membrane is a composite structure consisting of a lipid bilayer tethered to the spectrin-based membrane skeleton. Two complexes of spectrin with other proteins are known to participate in the attachment. Spectrin has also been shown to interact with phosphatidylserine (PS), a component of the lipid bilayer, which is confined to its inner leaflet. That there may be multiple sites of interaction with PS in the spectrin sequence has been inferred, but they have not hitherto been identified. Here we have explored the interaction of PS-containing liposomes with native alpha- and beta-spectrin chains and with recombinant spectrin fragments encompassing the entire sequences of both chains. We show that both alpha-spectrin and beta-spectrin bind PS and that sites of high affinity are located within 8 of the 38 triple-helical structural repeats which make up the bulk of both chains; these are alpha8, alpha9-10, beta2, beta3, beta4, beta12, beta13, and beta14, and PS affinity was also found in the nonhomologous N-terminal domain of the beta-chain. No other fragments of either chain showed appreciable binding. Binding of spectrin and its constituent chains to mixed liposomes of PS and phosphatidylcholine (PC) depended on the proportion of PS. Binding of spectrin dimers to PS liposomes was inhibited by single repeats containing PS binding sites. It is noteworthy that the PS binding sites in beta-spectrin are grouped in close proximity to the sites of attachment both of ankyrin and of 4.1R, the proteins engaged in attachment of spectrin to the membrane. We conjecture that direct interaction of spectrin with PS in the membrane may modulate its interactions with the proteins and that (considering also the known affinity of 4.1R for PS) the formation of PS-rich lipid domains, which have been observed in the red cell membrane, may be a result.

Shear-response of the spectrin dimer-tetramer equilibrium in the red blood cell membrane.

The red cell membrane derives its elasticity and resistance to mechanical stresses from the membrane skeleton, a network composed of spectrin tetramers. These are formed by the head-to-head association of pairs of heterodimers attached at their ends to junctional complexes of several proteins. Here we examine the dynamics of the spectrin dimer-dimer association in the intact membrane. We show that univalent fragments of spectrin, containing the dimer self-association site, will bind to spectrin on the membrane and thereby disrupt the continuity of the protein network. This results in impairment of the mechanical stability of the membrane. When, moreover, the cells are subjected to a continuous low level of shear, even at room temperature, the incorporation of the fragments and the consequent destabilization of the membrane are greatly accentuated. It follows that a modest shearing force, well below that experienced by the red cell in the circulation, is sufficient to sever dimer-dimer links in the network. Our results imply 1) that the membrane accommodates the enormous distortions imposed on it during the passage of the cell through the microvasculature by means of local dissociation of spectrin tetramers to dimers, 2) that the network in situ is in a dynamic state and undergoes a "breathing" action of tetramer dissociation and re-formation.