The erythrocyte membrane skeleton A. Model of erythrocyte membrane skeleton. The spectrin‐actin subcortical lattice is anchored to the membrane via at least three different integral membrane proteins, as well as by at least one direct attachment. Which of these attachments drives membrane assembly is uncertain, although current evidence favors ankyrin‐independent linkages. B. Major proteins of the red cell membrane and the cortical membrane skeleton. SDS‐PAGE analysis of purified human red cell membranes yields approximately 16 major protein bands (lane 1). Extraction of such membranes with 0.1 mM EDTA at pH 8 or above liberates spectrin and actin and a fraction of the cell's protein 4.1, dematin, and adducin (lane 2). The membranes remaining after the cortical skeleton is removed retain all of the integral proteins and many of the linker proteins (lane 3). The nomenclature according to their order on SDS‐PAGE gels as well as their assigned names is given.

Four views of the spectrin membrane skeleton. A. The erythrocyte cytoskeleton in situ. This sample was prepared from paraformaldehyde‐fixed ghosts attached to coated coverslips and sheered to expose the skeleton, followed by quick‐freeze, deepetch, rotary‐replication (QFDERR). The arrows point to unquenched water left in the sample. The average filament length is 29–37 nm, which is about a third of the extended length of the spectrin heterodimer. This information together with immunogold labeling has suggested that in situ the skeleton is highly compact, with many side‐to‐side associations of spectrin as well as a large fraction of spectrin hexamers and octamers as well as tetramers 518. B. A view of the spread isolated skeleton prepared by extraction of ghosts with 2.5% Triton X‐100, followed by spreading onto a formvar film followed by QFDERR. After this treatment, the compact skeleton seen in situ is highly expanded. Small arrows point to apparent actin sites; large arrows point to spectrin hexamer junctions. Arrowheads point to laterally associated filaments. The double arrowhead designates an ankyrin‐AE1 complex 519. (Panels A and B were adapted with permission from the references cited.) C. Quick‐freeze deepetch replica of the inner face of a red cell membrane in situ that has been extracted with NP‐40 and NaCl to remove most of the ankyrin and protein 4.1, exposing the network of intertwined spectrin and 67 nm actin nuggets. Compared with (A), this view depicts a much looser skeleton. It is likely that the salt and detergent treatment used in this preparation has loosened the side‐to‐side associations of spectrin as well as removed many associated proteins. D. Image of an expanded skeleton prepared by negative staining. The stretched spectrin filaments (tetramers) predominate in these preparations, although hexamers are seen 279. The preparation shown here was used to determine by Fourier analysis the supercoiling of the spectrin heterotetramer (see Fig. 4).

Alterations in the spectrin skeleton alter the shape and stability of erythrocytes. Scanning electron microscopic images: A. Normal red cell. B. Defects in ankyrin or AE1 often lead to spherocytosis. C. Inherited defects in the self‐association of spectrin often lead to hereditary elliptocytosis. D. Increased intracellular Ca²⁺, lowered pH, or ATP depletion induce echinocytic transformations.

Fourier filtered images of the spectrin heterotetramer in situ. These images were derived from negative stained electron micrographs of the stretched spectrin cortical skeleton such as the micrograph shown in Figure 2D. This analysis revealed a coiled‐coil quaternary conformation of spectrin in situ. Note that the spectrin heterotetramer can be linearly deformed, accounting for its proposed “spring‐like” properties. Shown are three filtered images demonstrating how the pitch and length of the spectrin helix change with length. Each turn of the helix is estimated to contain four spectrin repeat units. Upon extraction from the membrane, spectrin heterodimers and heterotetramers assume a much more open and loose conformation (cf. Fig. 12).

Effects of embedded proteins as predicted by the bilayer couple hypothesis. Integral membrane proteins can have a major effect on the shape and stability of the bilayer. Control by a cortical skeleton ameliorates this potential effect. A. Cylindrical embedded proteins can impart curvature to the membrane by expanding one face preferentially. They thus will tend to cluster in regions of curvature that reduce their net interaction energy with the bilayer. A cortical skeleton aids in maintaining a homogeneous distribution of these molecules, and thereby contributes to membrane stability. C. Released of the constraints of a cortical skeleton, asymmetric embedded proteins can induce acute membrane curvatures and instability.

By controlling the distribution of embedded proteins, the cortical skeleton controls cell shape. The equilibrium erythrocyte shape and the lateral density of the embedded proteins and cortical skeleton has been calculated (A) without the stabilizing effect of the skeleton or (B) with a cortical skeleton. In these calculations, the relative volume was held constant at 0.6, and the relative surface areas of the two leaves of the bilayer was set at 1.0465; c_s = 4; k = 0.005; p = 500. Note that in case A, the equilibrium shape is elliptical, whereas in B the equilibrium shape is axisymmetric and biconcave. Also note the markedly inhomogeneous distribution of protein in the case without skeletal stabilization. Views are presented along the minor axis; the ellipse semiaxis ratio in A is 1.18.

Increasing the number of embedded molecules may induce instability in the cell membrane. The relative membrane free energy is calculated as a function of the number of embedded proteins for two different shapes, the discocyte and a shape consisting of a spherical mother cell and two spherical daughter vesicles. For both cells, v = 0.6; k_r/k_c = 3; c_s = 2, k = 0.005. For the discocyte, Δa_o = 1.038; for the mother cell and vesicles, net Δa = 1.7105, r_mother = 0.6985, and r_daughter = 0.5060. Note that as the number of embedded proteins (p) rises, the cell tends toward vesiculation, a consequence resisted by the presence of a cortical skeleton (cf. Fig. 6).

Spectrin typically is a multifunctional heterodimer that self‐associates. The functional unit is most often an αβ‐heterodimer. Each subunit contains non‐homologous NH₂‐ and COOH‐terminal domains (domains I and III, respectively), joined by a central domain (domain II) composed of multiple homologous repeats of ∼106 residues each. Within the molecules are many sites that interact with other proteins or regulator molecules.

The spectrins are the prototypical members of a gene superfamily that includes α‐actinin and dystrophin. The derived protein sequences of the repetitive unit found in the central domain of the four documented spectrin genes, α‐actinin, and dystrophin were first compared within each protein to arrive at a consensus repeat structure for each protein. These consensus repeats were then compared with each other to estimate their overall degree of relatedness. A. A relatedness tree in which the aggregate distance separating the different proteins is proportional to their similarity. This analysis indicates that the superfamily has three major branches, with the spectrins being most related to each other, then to α‐actinin, and only distantly to dystropin. B. Sequence comparison of the consensus repeat units for each protein in the superfamily. Note the strong conservation of the trp at position 18 in the repetitive unit. A similar residue occurs at position 21 in dystrophin, although the alignment algorithm does not score this in this comparison.

Spectrin contains many regions of sequence divergence associated with specialized function. The sequences of either (A) JI spectrin or (B) βI spectrin were compared with themselves by “dot blot” analysis. The parallel lines spaced at 106 residues are indicative of the repetitive structure of spectrin. Note the nonrepetitive structure in domains I and III of both spectrins, as well as the many regions of divergence even with domain II. Sites marked by * indicate the approximate location of several mutations that cause hemolytic disease (reviewed in 301). Note that all known functional domains (labeled), as well as disease causing mutations, are located in regions of sequence divergence. The role of many such regions remains undetermined, although it is likely that they may harbor activities necessary for some of spectrin's myriad functions (see Table 2).

The repetitive unit of spectrin is a stable triple helix, capable of refolding spontaneously. A. Spectrin's characteristic pattern of resistance to proteolytic digestion by trypsin is recovered spontaneously after denaturation with urea. This resistance derives from spectrin's secondary and tertiary structure, which limits access of the protease to over 300 potential sites in each subunit 473. In these experiments, carried out by Dr. William Knowles and Vincent Marchesi at Yale, αI/βI spectrin was denatured with increasing amounts of urea; then, for half the samples, the urea was diluted or dialyzed away, and the protein subjected to 60 min of trypsin proteolysis at 0°C. Note the spontaneous recovery of its resistance to trypsin, indicative that the protein can faithfully recover at least most aspects of its secondary and tertiary structure after denaturation (Drs. Knowles and Marchesi, personal communication). B. The putative structure of two spectrin repeat units as derived from the crystal coordinates of Drosophila αI spectrin's 14th repeat unit. It is probable that helix C joins helix A with minimal discontinuity.

The self‐association of αβ‐spectrin heterodimers to form tetramers is mediated by domain 1 of α‐spectrin and repeat 17 of β‐spectrin. A. Spectrin αβ heterodimers undergo a concentration‐dependent self‐association to form tetramers and larger oligomers. This process can be most clearly demonstrated by non‐denaturing gel electrophoresis, in which each oligomeric species migrates as a separate band. The different forms of spectrin can also be demonstrated by electron microscopy after rotary shadowing. As the concentration of spectrin incubated in vitro is diminished from 24 mg/ml (left lane) to 12, 6, and 3 mg/ml respectively, spectrin's state of self‐association changes from larger oligomeric species towards the dimer. Oligomerization to species beyond the tetramer is a property more pronounced in αI/βI spectrin than in αII/βII spectrin.

Domain maps of spectrin generated by trypsin digestion. αI/βI spectrin extracted from erythrocytes was subjected to mild trypsin digestion at 0°C, cleaving the protein into a reproducible number of well‐characterized large fragments. A. The alignment of the various tryptic fragments, as determined by high‐resolution peptide mapping 473. Also shown is the alignment of some of the fragments generated by 2‐nitro‐5‐thiocyanobenzoic acid (NTCB) digestion of αI/βI spectrin. B. The pattern following limited trypsin digestion. Peptide fragments are resolved by two‐dimensional IEF‐SDS‐PAGE analysis, and visualized by Coomassie blue. The pattern that results is of limited complexity, facilitating the identification of the sites of functional domains, post‐translational modifications, and inherited mutations in this very large protein.

The nomenclature of the spectrins. A. There are four known genes for spectrin; these encode spectrins αI, αII, βI, and βII. Multiple isoforms arise from each of these genes except αI (at least none have so far been discovered). The various isoforms for each spectrin are annotated by the addition of a Σ followed by arabic numbers. Non‐mammalian spectrins are referenced to this nomenclature by homology or, if not homologous, by special notation 307,543. B. Alternative transcripts in αII spectrin. Three sites of alternative mRNA splicing have been identified in αII spectrin 76,77,346. Note: numbering is based on the lung fibroblast sequence, which does not contain insert 2.

The calpain cleavage of αIIβII spectrin (fodrin) activates its susceptibility to regulation by Ca²⁺ and calmodulin. Diagram depicting the synergistic action of calpain and calmodulin on the self‐association and actin‐binding properties of spectrin. A. Intact αIIβII spectrin binds F‐actin and cross‐links actin filaments by forming a stable heterotetramer. Calcium and calmodulin are without effect on these interactions. B. Proteolysis of the α subunit, creating the breakdown products α‐bdp1 and α‐bdp2, has no immediate effect on the actin binding of spectrin or its self‐associative properties, and the spectrin molecule remains assembled and tetrameric under native conditions. The exact (calculated) size of the major proteolytic products of αII spectrin, α‐bdp1 and α‐bdp2, are 135,832 and 148,495 Da respectively. C. The binding of calmodulin to α‐cleaved spectrin dissociates the tetramer to dimers and reduces its ability to bind actin. This process is rapidly reversible and requires calcium. (Note: this is the only physiologic condition yet identified that will lead to dissociation of αIIβII spectrin heterotetramers.) D. Continued action of calpain in the presence of calmodulin leads to cleavage of the β‐subunit, generating β‐bdp1 and β‐bdp2. This cleavage causes dissociation of the heterodimer and irreversible loss of actin binding and crosslinking properties. The placement of these fragments relative to the intact molecule is as indicated. Note that the two fragments comprising the actin‐binding end of the molecule remain non‐covalently associated, while the other fragments dissociate.

Tripartite domain structure of ankyrin. Ankyrin isolated from erythrocytes (ANKI or Ank_R) contains three independently folded domains. The ankyrin repeats domain (ARD) consists of 24 nonidentical repeats of 33 residues each. The ARD motif is a common feature of many proteins. This domain in ankyrin is responsible for binding to AE1, Na,K‐ATPase, and typically other membrane proteins. The central domain is involved with ankyrin's interactions with spectrin. The region of the most critical residues are shaded black. The least well understood of ankyrin's domains is the putative regulator domain. Alternative transcripts involving this domain alter ankyrin's other interactions. Also shown is the derived structure of a recently cloned ankyrin with wide tissue distribution (Ank G, ANK3, ANKIII) 117,250,400.

Domain structure of protein 4.1. Many isoforms of protein 4.1 exist, all arising by a complex pattern of alternative mRNA spicing over a region encompassing 23 exons 94. The functional domain structure of the erythroid protein is shown here, based on several studies 75,93,94,98,203,204,205,272,284,391. The 10 kDa spectrin‐actin binding region appears to be an erythroid‐specific spiciform, suggesting that this activity may not be fundamental to protein 4.1's function. The other members of the 4.1 family (ezrin, moesin, radixin) share their strongest homologies in the 30 kD NH₂‐terminal region. The predicted secondary structure is derived from the algorithms of Garnier et al. 157.

Adducin isoforms and alternative transcripts. Multiple transcripts of both α‐adducin and β‐adducin have been identified. All of these so far involve alternative transcripts involving the COOH‐terminal third of the molecule (aka the “tail region”). This region is very proteolytically sensitive.

Stomatin contains a hydrophobic 28‐residue sequence thought to intercalate into the bilayer. A schematic view of stomatin suggests a typical transmembrane protein. However, both the NH2‐and COOH‐ termini are accessible from the cytoplasmic side of the membrane 199,442. Therefore, stomatin may assume an unusual structure with a putative hairpin loop inserting into the bilayer. Stomatin appears to regulate Na⁺/K⁺ leaks, and binds to adducin 465.

Spectrin in non‐erythroid cells is often highly organized. A. Rat myotubes after extraction with saponin and stained with rhodamine‐bungarotoxin, which stains the acetylcholine receptor (AChR) clusters. B. Same preparation stained with Mab VIIF7, which reacts with βI spectrin. Note the coincident distribution, indicating that this spectrin is codistributed with the AChR,. C. βIΣ2 spectrin is concentrated at the postsynaptic density in the molecular layer of the rat cerebellum. Immunoperoxidase labeling EM. Bar = 0.5. μm D. Immunofluorescent image of MDCK cell stained with an antibody to Ank _G119(ANKIIΣ4). The punctate eccentric cytoplasmic staining adjacent to the nucleus marks a form of βI spectrin associated with the Golgi apparatus 20,117.

A hypothetical model of the cadherin‐based transmembrane adhesion complex and its relationship to the associated cortical cytoskeleton. The Greek letters α and β indicate the corresponding catenins. Although there is evidence that α‐catenin can bind both spectrin 286 and actin 428 and that is exists as a dimer, the linkages shown are hypothetical. It is not known if catenin/spectrin and catenin/actin interactions can occur simultaneously. It is possible that α‐catenin not only links the cadherin complex to the cytoskeleton but also participates in actin bundling in other parts of the cell or in other cell types. There is no good evidence for a direct interaction between E‐cadherin and spectrin, but they co‐localize and are suspected to be joined by α‐catenin. The interactions between β‐ and α‐catenin are presented as an example of how β‐catenin (a member of the arm family) binds directly to both E‐cadherin and α‐catenin. It is not yet known which, if any, other members of the arm family can participate in this interaction. Plakoglobin, previously known as γ‐catenin, may participate, but cannot bind the same cadherin molecule (although theoretically both could bind a cadherin dimer). α‐Actinin, another member of the spectrin superfamily (see Fig. 9), has also been implicated in this complex, but its exact role remains uncertain; and hence it is not included in this figure. Both p120 (CAS) and Ptpaseμ also bind to cadherin, but neither appears to bind in same region as β‐catenin or to interact directly with spectrin.

The linked mosaic model of spectrin action. The fundamental role of the spectrin skeleton is to control lateral order in the plane of the membrane. Through its capacity to bind multiple ligands selectively, and to self‐associate through hetero‐and homo‐typic interactions, end‐on and side to side, spectrin alone can form ordered arrays of mosaics of limited size. Associated with these nascent arrays are various embedded and soluble proteins. It is hypothesized that the spectrin mosaics with their associated proteins are joined by linking interactions, most commonly involving actin or other filament systems, to form larger arrays. Collectively, these linked mosaics serve to control the lateral organization of integral membrane proteins, and to recruit selected cytoplasmic components (e.g., signal transduction molecules, kinases) in a way that enhances receptor efficiencies, creates signal transduction systems, and stabilizes the membrane against spontaneous and deleterious changes in the distribution of its integral membrane proteins. This proposed role applies with equal validity to internal as well as surface membranes. Perhaps for this reason spectrin and ankyrin are also associated with internal membranes.