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.

Cells that are required to withstand high mechanical stresses rely for their capacity to accommodate to distortion without structural damage on a membrane-associated complex of proteins. The archetypal example is the red cell membrane, which is subject to large shearing forces throughout its lifetime in the circulation, and responds to these by elastic deformation. The lipid bilayer itself is essentially devoid of elasticity and a protein-free bilayer membrane rapidly vesiculates under even mild shear stress. The red cell membrane skeleton, which gives the membrane its characteristic mechanical properties, is a roughly hexagonal lattice, composed of spectrin tetramers attached at their ends to junctional complexes consisting of several globular proteins (1). The spectrin tertramers are formed by the head-to-head association of pairs of ␣␤ heterodimers. The self-association of such dimers in free solution is weak (K a ϳ3 ϫ 10 5 M Ϫ1 at physiological temperature and ionic strength) (2,3), but the apposition of the association sites on the immobilized proteins in situ ensures that the spectrin remains overwhelmingly in the tetrameric state (some higher association states, especially the hexamer, appear also to exist in the network (4). We know little, however, about the dynamics of the spectrin dimer-dimer interaction in the intact red cell membrane; more especially, the possible effects of membrane deformation on this interaction have not been considered.
The origins of the elastic properties of the membrane remain a matter of debate. The end-to-end distance of the spectrin tetramers is constrained by the separation of the junction points to about half the equilibrium root-mean-square end-toend distance of the protein in solution (5,6), and this circumstance gave rise to the conjecture that the spectrin behaves as an entropy spring. More recent evidence, however, implied that this was not the predominant source of the elasticity (7), and there is indeed some structural evidence to suggest that spectrin may act as a helical Hookean spring (8). The maximum permitted local extension of the membrane, in the absence of unfolding of spectrin secondary structure (9), should be equivalent to the difference between the average separation of the junctions and the contour length of the tetramers, which is known from electron microscopy (10). This amounts to an extension of about 3-fold, sufficient to explain the large distortions that the cell undergoes in vivo (11,12) and which can be simulated in vitro (13). There are currently, however, no experimental data to discriminate between these or other kinds of local changes accompanying distortions of the membrane.
Here we show that under physiological conditions spectrin tetramers in the unstressed intact membrane exist in rapid equilibrium with dimers. Importantly, shear-induced membrane deformation markedly displaces the equilibrium in favor of the dimer. Based on these findings we suggest that such dissociation of spectrin tetramers is a primary part of the mechanism by which the membrane can accommodate the large reversible distortions that it suffers in the circulation. Such perturbation of protein-protein interactions under the action of external forces may be a general phenomenon.

Materials
Human venous blood was drawn, with informed consent, from healthy volunteers. Glutathione-Sepharose 4B was purchased from Amersham Biosciences, Dextran T40 from Amersham Biosciences AB (Uppsala, Sweden), electrophoresis reagents from Bio-Rad, and GelCode Blue Reagent from Pierce. All other chemicals were reagent grade and obtained from commercial sources. 1-154, were prepared by cloning and expression in Escherichia coli, as described by Nicolas et al. (14). The two ␣-spectrin constructs were cloned into the pGEX-2T vector, using BamHI and EcoRI restriction sites, upstream and downstream, respectively. The cDNAs were introduced into the BL21 (DE3) expression strain of the bacterium. The purification of the GST 1 fusion proteins and cleavage of the GST followed the established procedure (14). Peptide concentrations were determined spectrophotometrically. Materials were screened for purity by gel electrophoresis in the presence of SDS.

Methods
Introduction of Spectrin Fragments into Erythrocyte Ghosts-Red cells were isolated from freshly drawn blood by centrifugation and washed three with Tris-buffered isotonic saline (0.12 M potassium chloride, 10 mM Tris, pH 7.4). The cells were lysed with 35 volumes of ice-cold hypotonic buffer A (5 mM Tris, 5 mM potassium chloride, pH 7.4). The resulting ghosts were collected by centrifugation and washed once in cold lysis buffer. The ghosts (5 ϫ 10 9 cells/ml) were incubated for 40 min at 37°C with the required concentrations of the spectrin fragments, and 0.1 volume of 1.5 M potassium chloride, 50 mM Tris, pH 7.4, was added to restore isotonicity.
Measurement of Membrane Stability-To evaluate the effect of peptide incorporation on the resistance of the cells to shear, the resealed ghosts were suspended in 40% dextran, and membrane mechanical stability was quantitated using an ektacytometer, as described previously. The measure of membrane stability was taken as the rate of decrease of deformability index (DI) at a constant applied shear stress of 750 dynes cm Ϫ2 . To examine the effect of shear stress on the incorporation of peptides into the membrane skeletal network, the resealed ghosts, containing the peptide, were suspended in isotonic buffer, supplemented with 40% dextran, and sheared at a low stress of 250 dynes cm Ϫ2 at room temperature in the couette cell of the ektacytometer (15).
Extraction and Analysis of Spectrin from Resealed Ghosts-The resealed ghosts were washed with isotonic buffer. The binding of the peptide to the spectrin on the membrane, with and without a shearing step, was analyzed by re-lysing the ghosts with 30 volumes of ice-cold Buffer A, washing three times with the same buffer, and extracting the spectrin. Extraction was accomplished by suspension of the ghosts in 0.25 mM sodium phosphate, pH 7.4, and dialysis at 4°C overnight. The spectrin was collected by centrifugation (21,000 ϫ g) and examined by gel electrophoresis in the native state in a Tris-Bicine buffer system, run in the cold (16). The spectrin tetramer, dimer, and the complex of the dimer with the peptide fragment were well resolved, and the absence of a zone corresponding to the free fragment or of a trail of stained material showed that no dissociation had occurred during migration. The gels, stained with GelCode Blue, were evaluated by densitometry.
Interaction of Spectrin Peptides with Inside-out Vesicles-Inside-out vesicles (IOVs) were prepared as described previously (17). The binding of spectrin peptide fragments to IOVs was examined by pelleting the IOVs from the reaction mixture at 47,800 ϫ g. The pellet was analyzed by electrophoresis in SDS gels of 9% acrylamide, followed by staining with GelCode Blue.

RESULTS
Binding of ␣-Chain Peptide Fragment to Spectrin Self-association Sites in Situ-The dimer-tetramer equilibrium of spectrin is characterized by an unusually high activation energy (2). Thus at room temperature many hours are required to approach equilibrium, and in the cold the half-time is measured in weeks or months. The explanation of this phenomenon is that formation of the tetramer from its constituent ␣␤ dimers through a pair of intermolecular ␣-␤ bonds requires the prior rupture of two intramolecular ␣-␤ bonds, one in each of the antiparallel dimers (3,18). Because the intra-dimer bond has to open to allow the N-terminal ␣-chain fragment to bind to its C-terminal site on the ␤-chain of a dimer, the high activation energy persists in the fragment-dimer interaction. Therefore to approach binding equilibrium within a reasonable time the experiment must be carried out at elevated temperatures (30 -37°C) (16,19,20). Fig. 1A shows that at these temperatures (but not at the lower temperature of 24°C) incorporation of the ␣1-154 peptide into the spectrin in the membrane network does indeed occur. (A trace of spectrin dimer is always seen in the gel; its amount varies, and it is probably a consequence, at least in part, of proteolytic damage before or during extraction (21)). The peptides with and without the GST fusion domain were tested and no differences were found (data not shown). As a control, we also examined the short N-terminal ␣1-50 peptide, which does not enter the native fold and does not therefore bind to the ␤-chain in solution (14,22); this peptide was not incorporated into the membrane (Fig. 1B). We have further established that the long peptide does not bind to spectrin-and actin-free inside-out membrane vesicles (data not shown), which retain all other intrinsic and extrinsic membrane proteins. Thus any possibility that the peptide exerts its effect by binding to other proteins can be excluded.
From the kinetics of incorporation of the ␣1-154 peptide (Fig. 1C), it is clear that the binding is an equilibrium process,  2. Reversibility of peptide incorporation. Resealed ghosts containing the peptide were re-lysed, washed thoroughly before, and resealed without peptide. Spectrin extracts were analyzed by electrophoresis in 5% non-denaturing gels. Lane 1, spectrin extract from ghosts incubated and resealed in the presence of the ␣1-154 peptide; lanes 2-4, spectrin extracts from ghosts re-lysed and again resealed at 24, 30, and 37°C, respectively. The dimer-peptide complex largely disappeared at both 30 and 37°C, demonstrating reversibility of peptide incorporation.
effectively reaching completion after about 50 min at 37°C. The absence of a tetramer-fragment complex shows that the binding of a single peptide molecule causes dissociation of the tetramer into dimers, one or probably both (since the amount of free, uncomplexed dimer generated does not significantly increase) associated with the peptide. Fig. 2 reveals that the binding of the peptide to the spectrin in situ is reversible, for when the cells with incorporated peptide were washed free of unbound peptide and warmed to 37°C, the peptide was released from the membrane. Slower release ensued at 30°C, but none could be detected at 24°C over a period of 40 min.
Effect of Peptide Incorporation on Membrane Stability-The effect of increasing concentrations of the long peptide on the mechanical stability of the membrane was assessed by shearing in the ektacytometer at room temperature (15). As Fig. 3A shows, membrane stability is markedly reduced in ghosts con-taining the peptide, as reflected by a faster rate of decay of the DI. Increasing concentrations of the peptide in the resealing buffer resulted in a progressive decrease in membrane mechanical stability. The peptides with and without GST fusion domain were again tested and no differences were found. The decreased membrane mechanical stability was paralleled by a progressive increase in the incorporation of the peptide into the membrane skeleton (Fig. 3B). Fig. 3C shows that increasing concentrations of peptide in the resealing buffer led to a progressive accretion of the spectrin dimer-peptide complex, with a corresponding decrease in the concentration of spectrin tetramers. In Fig. 3D we show the impairment of membrane stability, measured by the half-time of breakdown under shear as a function of the extent of tetramer dissociation. A similar relationship between decreased membrane mechanical stability and elevated dimer content has previously been observed in red cells of subjects with hemolytic anemias, caused by spectrin mutations that result in defective dimer self-association (23). The short N-terminal ␣1-50 peptide, which did not incorporate into the membrane, had no effect on membrane mechanical stability at concentrations up to 100 M in the resealing buffer (data not shown).
Incorporation of the ␣-Chain Peptide into the Membrane under Shear-Resealed ghosts containing the ␣1-154 peptide were subjected to varying periods of low shear in the ektacytometer at room temperature. Because of the high activation energy of the binding and tetramer dissociation reactions, there is, on the time scale of these experiments, no detectable incorporation of the peptide into the membrane network in static cells (or of course binding to spectrin in free solution). Ghosts resealed with ␣1-154 peptide at 30°C were subjected to a constant low shear stress of 250 dynes cm Ϫ2 for the indicated periods of time. Spectrin extracts were analyzed as described above. The amount of spectrin dimer-peptide complex, reflecting the proportion of spectrin tetramers dissociated, is seen to increase with time of exposure to shear. Nevertheless, a time-dependent incorporation of the peptide was observed (Fig. 4). This striking effect demonstrates that mild shearing stress is sufficient to induce dissociation (presumably local) of spectrin tetramers, thus overcoming an activation energy of some 100 kcal mol Ϫ1 (2). DISCUSSION While the self-association of spectrin dimers in free solution is weak, especially at physiological temperature, the cohesion of the membrane skeletal network in situ is ensured by the close apposition of the binding sites. The strong linkage of the distal dimer ends to the network junctions, and especially the tight attachment of one dimer in each tetramer to membrane-bound ankyrin at a position close to the selfassociation site (24), can be assumed to restrict severely the excluded volume available to the dimer association sites. The dimer-tertramer equilibrium in situ is thus expected (and observed) to be grossly shifted in favor of the tetramer on entropic grounds. Binding of the univalent fragments at the dimer-dimer association sites was thus expected, if it occurred at all, to require a very large molar excess of the fragment, as was indeed found.
The association constant for binding of a univalent ␣-chain fragment to spectrin dimer in dilute solution is only a little lower than that for dimer self-association (16,19,20). This may be because only one intra-dimer interaction has to be broken to allow the fragment to bind. In any case, the fragment would in principle be expected to bind to any available dimers on the membrane. For dimers to become available, the spectrin in the network must undergo a continuous association-dissociation, or "breathing" process of a frequency compatible with entry of the fragment. The apparent association constant for the binding of the fragment to its sites in the network in situ should be defined by a simple Langmuir adsorption isotherm, formally equivalent to the Scatchard equation (25). However, the concentration of available dimers depends on the in situ dimer-tetramer equilibrium. This is concentration-independent, since no diffusion of the reactants on the membrane is permitted. It can thus be treated as a conformational equilibrium between an open and a sequestered state of the dimers. The system is therefore defined by two equilibria: S ϩ F ϭ SF and S ϭ S c , where S and S c represent the available and sequestered states of the spectrin dimer, respectively, and F the univalent fragment. Then if K and K s are the equilibrium constants for these two reactions, and writing ␣ for the fractional saturation of spectrin on the membrane with the fragment (expressing spectrin concentration in molar units of dimers), the binding of the fragment to the membrane is described by the relation: ␣ ϭ KK s f/(KK s f ϩ 1), where f is the concentration of the fragment. This equation was used to fit the data points of Fig. 3C and gave a value for KЈ ϭ KK s of 1.5 ϫ 10 4 M Ϫ1 . To extract K s we need to know K, but its value for the interaction in free solution cannot be equated with that for binding on the membrane, for it may well be grossly influenced by steric and electrostatic factors. A value in the range of those obtained from solution studies (16,19,20), say 10 6 m Ϫ1 , would lead to K s in the region of 0.015; that is about 1% of the tetramer population would be dissociated in the unperturbed cell. This proportion would almost certainly be further reduced by molecular crowding caused by hemoglobin (26). A more soundly based estimate must await a direct experimental determination of K.
The most striking outcome of this study is the observation that the dissociation of tetramers into dimers can be induced by shear, even at room temperature at which the equilibrium in solution is essentially frozen over the period of the experiment. This implies that a very modest mechanical force is sufficient to break the dimer-dimer interaction. It also implies that this association-dissociation, or breathing process operates continuously in the circulation, in which the cells are nearly always under shear. Our data do not as yet permit a rigorous quantitative description of this previously unsuspected effect. The influence of the membrane environment on the equilibrium and rate constants for the self-association of spectrin dimers and the interaction of spectrin dimers with a univalent fragment, as measured in dilute solution, is still uncertain. We can also not exclude that some additional dissociation of spectrin tetramers through entry of more peptide into the spectrin network could have occurred during the brief period of the highshear assay at room temperature. Thus the fractional dissociations of tetramers engendering the observed reductions in membrane stability should be regarded as minimum values. The likelihood of dissociation of bound peptide during the time of experimental manipulations after the cells were restored to the static state is remote. Various explanations have been advanced for the elasticity and stability of the membrane. One type of model is based on the stretching of spectrin from its relatively crumpled (27) or compressed (8) state at rest up to its fully extended length (but see also Ref. 7), allowing for an extension factor of about 3. Another suggestion is that the secondary structure of the protein, which is composed primarily of three-stranded ␣-helical elements (28), can be unfolded under the action of a tensile force (9). The question of whether protein-protein interactions in the network can be disrupted by mechanical forces has not previously been addressed. It is unlikely that dissociation would occur at the lattice junctions, for the ternary complex of spectrin, actin, and protein 4.1 (irrespective of contributions of other proteins present at the junctions) is very tightly associated (29). The results presented here indicate that rupture of spectrin tetramers is a likely mechanism for the capacity of the membrane to adapt to very large distortions.
These observations offer a rationale for the evolutionary advantage of the tetrameric structure of spectrin. If it functioned only as a simple elastic element of the network a fragile dimer-dimer link at the center would afford no advantage. It may be recalled that neuronal spectrin, fodrin, which is probably not exposed to high shearing forces during its lifetime in the cell, has the form of a stable tetramer, which cannot be dissociated into dimers by known physical means, short of denaturation (30).