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From www.bloodjournal.org by guest on June 16, 2017. For personal use only. Blood First Edition Paper, prepublished online November 4, 2015; DOI 10.1182/blood-2014-12-512772 Anatomy of the red cell membrane skeleton: unanswered questions Samuel E. Lux, IV1 1 Dana-Farber/Boston Children’s Cancer and Blood Disorders Center and Department of Medicine, Boston Children’s Hospital, Boston, MA 02115 Section: Review (Red Cells, Iron and Erythropoiesis) Left Running Head: LUX Right Running Head: ANATOMY THE RED CELL MEMBRANE SKELETON Copyright © 2015 American Society of Hematology From www.bloodjournal.org by guest on June 16, 2017. For personal use only. Abstract The red cell membrane skeleton is a pseudohexagonal meshwork of spectrin, actin, protein 4.1R, ankyrin, and actin-associated proteins that laminates the inner membrane surface and attaches to the overlying lipid bilayer via band 3-containing multi-protein complexes at the ankyrin and actin binding ends of spectrin. The membrane skeleton strengthens the lipid bilayer and endows the membrane with the durability and flexibility to survive in the circulation. In the 36 years since the first primative model of the red cell skeleton was proposed, many additional proteins have been discovered and their structures and interactions have been defined. But myriad questions about the skeleton’s structure remain and almost nothing is known of its physiology. Questions such as the structure of spectrin in situ, the way spectrin and other proteins bind to actin, how the membrane is assembled, the dynamics of the skeleton when the membrane is deformed or perturbed by parasites, the role lipids play, variations in membrane structure in unique regions like lipid rafts, and so on. This knowledge is important because the red cell membrane skeleton is the model for spectrin-based membrane skeletons in all cells, and because defects in the red cell membrane skeleton underlie multiple hemolytic anemias. 2 From www.bloodjournal.org by guest on June 16, 2017. For personal use only. History Modern knowledge of the red cell plasma membrane and its membrane skeleton began with Marchesi and Steer’s identification of spectrin in 1968.1 Prior to that year, almost the only thing known about membranes was that they contained a lipid bilayer.2 Indeed, there was a period in the 1960s when it was believed that red cell membranes contained only a single 22.5 kD protein called “structural protein” 3. The fertile decade following the discovery of spectrin and the more or less coincident introduction of SDS polyacrylamide gel electrophoresis as an analytic tool, led to the isolation and characterization of several major red cell membrane proteins. The first simple model of the membrane skeleton appeared 36 years ago (Figure 1).4 While it contained the core elements of the modern model, a great deal has been learned in the intervening decades (Figure 2). Hundreds of individuals have contributed to our knowledge of the red cell membrane skeleton but a few deserve special recognition, including: Peter Agre, David Anstee, G. Vann Bennett, Daniel Branton, Lesley Bruce, Jean Delaunay, David Discher, Velia Fowler, Patrick Gallagher, Walter Gratzer, Philip Low, Vincent Marchesi, Narla Mohandas, Jon Morrow, Jeri Palek, Luanne Peters, David Speicher, Ted Steck, and Michael Tanner. This review will focus on our current understanding of the anatomy of the membrane skeleton, particularly its spectrin-actin core, and on some of the key unanswered questions. Because of space limitations, many important aspects of the red cell membrane are not discussed. These include: integral membrane proteins that are not attached to the skeleton, the lipid bilayer, membrane biogenesis, posttranslational modifications of the membrane, red cell aging, and mouse and human membrane diseases. For interested readers, a few other recent reviews are recommended.6-10 The Red Cell Membrane Skeleton The red cell membrane contains about 20 major proteins and at least 850 minor ones11 Some of the important ones are described in Table 1. The integral membrane proteins are organized into macromolecular complexes centered on band 3, the anion exchange channel. Most of the peripheral 3 From www.bloodjournal.org by guest on June 16, 2017. For personal use only. membrane proteins form the membrane skeleton, a 40 nm to 90 nm thick protein meshwork that laminates the inner membrane surface. The skeleton is composed principally of spectrin, actin and its associated proteins (tropomyosin, tropomodulin, adducin and dematin), protein 4.1R and ankyrin. Spectrin Erythrocyte spectrin is a long, flexible, worm-like protein composed of two parallel chains (α- and βspectrin) oriented in opposite directions. Each chain contains multiple spectrin-type repeats with specialized functional domains at the “head” end for spectrin dimer-tetramer association and for ankyrin-1 binding, and domains at the “tail” end for binding to protein 4.1R, protein 4.2, short filaments of actin and other proteins. The details of spectrin structure are shown in Figure 3. On average, six spectrins bind per actin filament, leading to a pseudohexagonal arrangement.26 Isolated α- and β-spectrin chains bind to each other electrostatically at a pair of nucleation sites formed by repeats near the spectrin tail (Figure 3A). Once connected, the rest of the α- and β-chains zip together, coiling around each other roughly every four27 repeats. The side-to-side interactions beyond the nucleation site are relatively weak,28 allowing the two chains to slide past each other when the spectrin molecule flexes and extends during membrane deformation. Spectrin α- and β-chains associate with each other at the “head” end. Basically, the complementary partial repeats (α0 and β17) at the end of each chain join to form a complete three-helix repeat: α0/β17 (Figure 3A). In spectrin αβ-dimers, the longer α-chain folds back on itself, so the α0/β17 repeat interacts with repeat α4 and the α1 and α3 repeats contact each other (Figure 3A).13 In the α2β2-heterotetramers there are three such side-to-side interactions between the longer α-chains (Figure 3C). These lateral interactions, though weak, contribute to self-association. Higher oligomers (hexamers, octamers, etc) form by the same mechanism and can also be seen on the membrane.26,29,30 Even though nearly 95% of the spectrin is in the tetramer or oligomer forms,31 spectrin self-association is uniquely weak in the red cell.32 Tetramers dissociate and reform under physiologic conditions and this is greatly accentuated when the 4 From www.bloodjournal.org by guest on June 16, 2017. For personal use only. membrane is distorted by shear forces.33 This may be an evolutionary accommodation to permit the enormous distortions the red cell undergoes in parts of the microvasculature. Spectrin is a highly flexible molecule but it is not certain how it achieves its flexibility since crystal structures of tandem repeats shown a continuous helix across the junction between repeats (Figure 3B). There is evidence that the helical linker may bend in certain directions at the junction34 and that helices within some repeats may ‘melt’ and rearrange, shortening the repeat.34 In addition, some of the repeats are unstable at physiological temperatures (starred repeats in Figure 3A),23 and may partially unfold when red cells deform, which would contribute to flexibility.35 The Actin Protofilament and Its Associated Proteins Actin Red cells contain short, double helical, filaments of nonmuscle or β-actin, termed “protofilaments” (Figure 2). These lie roughly parallel to the membrane plane (±20 degrees) and are randomly oriented.36 There are about 30,000-40,000 protofilaments per red cell, with 6-8 actin monomers in each of the two strands. Not much is known about how these unique actin filaments form but some hints are emerging from defects in actin assembly. Mice that lack both Rac1 and Rac2 GTPases,37 or lack Hem-1, a member of the WAVE complex that regulates F-actin polymerization,38 develop hemolytic anemias with misshaped red cells, and clumped or irregular skeletons. This suggests that Rac GTPases and Hem-1 help organize or regulate the red cell membrane skeleton, acting through pathways that stimulate actin polymerization in many cell types. It is unclear whether such regulation occurs dynamically in the mature red cell or just during erythropoiesis. Tropomyosin Erythrocyte tropomyosin (TM) is a long rod-like dimer of α- and γ-TM isoforms (hTM5b and hTM5, respectively).10 One TM dimer binds to each of the two strands in the actin protofilament (Figure 2). 5 From www.bloodjournal.org by guest on June 16, 2017. For personal use only. Binding is Mg2+-dependent. The red cell TM isoforms also bind to tropomodulin (see below). Red cell membranes that lack TM are markedly more fragile than normal39 and only endogenous TM, not the longer muscle isoforms, can restore stability. Red cell TM is just long enough (~34 nm) to cover the estimated 7 actin monomers in one strand of the F-actin protofilament, which supports the idea that tropomyosin serves as a molecular ruler, along with actin capping proteins, in assembling the filament40 and strengthens the filament after assembly. In yeast, formins—a family of proteins that nucleate actin filaments—direct which TM isoform associates with an actin filament.41 It will be interesting to see if that is also true in erythroid precursors, and if formins play a role in protofilament formation. Tropomodulin-1 Tropomodulin-1 (TMod1) has two functions: it caps the pointed or slow-growing end of actin filaments (Figure 2) and binds tropomyosin, which greatly strengthens actin capping.42 There are 30,000 copies per red cell or one per actin protofilament. One TMod1 binds to both actin strands and both TMs in the actin protofilament.42 Mice that lack TMod1 in their red cells have variable actin protofilament lengths and irregular holes in the membrane skeleton.43 These mice also have increased TMod3 in their red cells, which may partially compensate. TMod3 is a protein that plays multiple roles in definitive erythropoiesis, erythroblast binding to so-called “nurse” macrophages, and enucleation44 but is not normally present in mature red cells. Protein 4.1R Protein 4.1R promotes spectrin-actin binding and helps attach the membrane skeleton to the membrane. The first of these functions is especially important. Under physiologic conditions, erythrocyte spectrin binds very weakly to F-actin. Protein 4.1R binds to both spectrin and actin and generates a high affinity ternary complex.45 The cofactor activity is contained in a 10-kd spectrin-actin binding domain (SABD) in the middle of the molecule46 (Figure 4). The actin-binding site in the SABD is straddled by a two-part 6 From www.bloodjournal.org by guest on June 16, 2017. For personal use only. spectrin-binding site in a single linear peptide (Figure 4C).48 Red cells that lack protein 4.1R are very fragile, but their membrane strength is normalized by the SABD peptide.49 Unfortunately, the threedimensional structure of the SABD within protein 4.1R is unknown. Solving the complete structure of this important protein is a major need in the field. The other important 4.1R domain is the N-terminal, globular membrane binding domain (MBD). It binds glycophorin C, protein p55, band 3 and calmodulin in separate, well-defined areas47 (Figure 4B). These interactions are discussed briefly in the section on the Actin Junctional Complex. Adducin Adducin is a complex, multifunctional protein containing an α-subunit, and either a β-subunit or less often a related γ-subunit.50 The adducin subunits contain a globular head domain and an extended, flexible tail.50 The predominant form is probably the αβ heterodimer.51 There are 30,000 copies of the dimer per red cell. As suggested by the number of copies, adducin caps the fast growing or “barbed” end of the actin protofilaments in red cells (Figure 2).51 It also recruits spectrin to neighboring sites on actin,52 which enhances the capping about 10-fold.49 These interrelated functions require a domain at the end of the adducin tail.50 Both functions are regulated by calmodulin and by several protein kinases.53 The effects of phosphorylation are complex and it isn’t clear if they are physiological important.54 Indeed, the importance of adducin itself is uncertain, since mice that lack the protein have only mild hemolysis and spherocytosis.55 Adducin also helps attach the membrane skeleton to the lipid bilayer through interactions with the anion exchange channel (band 3)56 and glucose transporter-1 (Figure 2).57 The band 3 binding site is located in the tails of α- and β-adducin.56 Whether adducin binds dimers or tetramers of band 3 and whether it binds one or two of each is unknown. 7 From www.bloodjournal.org by guest on June 16, 2017. For personal use only. Dematin Dematin is a monomeric, actin binding protein with two actin binding sites: one in its headpiece and one in its tail.58 Dematin was first identified by it’s ability to bundle actin filaments into cables,59 but in mature red cells, where there are no such cables, dematin has two functions. First, unmodified dematin binds to spectrin and enhances spectrin-actin binding.60 This function is lost when dematin is phosphorylated by protein kinase A.58,60 Second, dematin binds to the glucose transporter57 and helps attach actin to the membrane. How Does Spectrin Bind to the Actin Protofilament? Surprisingly little is known about this central question. Actin and protein 4.1 bind to two tandem calponin homology (CH) domains at the N-terminal- or tail-end of β-spectrin (Figure 3A). The CH1 domain is constitutively active but the CH2 domain is incipient.18 It is unmasked by removal of a blocking helix or by phosphatidylinositol-4,5-bisphosphate (PIP2), a lipid involved in many signaling pathways.18 How, or even if, PIP2 regulates the CH2 domain in the native red cell is not known. The neighboring calmodulin-like EF hand domain of α-spectrin is also important. Deletion of the last 13 amino acids of the domain in a mini-spectrin or in the severely hemolytic sph1J mouse nearly ablates actin binding.21 The relationships of the EF hand domain, the calponin homology domain and F-actin are not known in spectrin, but probably resemble those in better studied spectrin family members such as α-actinin,24,25,61 utrophin,62 dystrophin, fimbrin, and plectin (Figure 3C). The problem is, these proteins bind actin in different ways. Sometimes the CH domains are in contact with each other (closed position), and sometimes they are extended and separated from each other (open position). In some analyses only CH1 binds to actin;25 in others both CH1 and CH2 bind.24,61 The EF hand domain is always in contact with the CH domains and not in contact with actin, so it presumably regulates the CH domains. The fact that the αactinin CH domains adopt both open and closed positions24 and that utrophin also has two modes of 8 From www.bloodjournal.org by guest on June 16, 2017. For personal use only. binding62 suggests that the CH domains in spectrin might also do so and that this might be regulatory (convert weak binding to strong binding, for instance). Perhaps protein 4.1R and/or the EF domains act by converting the CH domains to a high affinity conformation. Spectrin could occupy up to two actins in the actin filament given the stoichiometry (6 spectrins/~14 actin monomers – see Table 1), so a variety of arrangements are possible. In any event, these are clearly key questions that need to be answered going forward. Another obvious problem that is rarely mentioned is that spectrin is in a planar array, but the spectrin binding sites on the helical actin protofilament must be in a helical array. Spectrin doesn't hang down into the cytoplasm or stick up into the lipid bilayer, so if all the spectrin binding sites on actin are saturated, either the actin-binding end of spectrin must be flexible enough to curve around the actin filament or spectrin must be able to bind to actin in more than one way. What is the Structure of Spectrin on the Membrane? Electron micrographs of purified spectrin tetramers63 or spectrin filaments in fully stretched skeletons26 (Figure 5A) show a spaghetti-like molecule with an end-to-end length of up to 200 nm. This is how spectrin is usually depicted in membrane models. However, the filaments of native, unstretched skeletons are much more compact (Figure 5B) and simple calculations show that the average distance between actin filaments—i.e., the length of a spectrin tetramer—is only 60-70 nm in vivo.8,66 This corresponds roughly to the length of presumed spectrin filaments in native skeletons.29,30,65 It isn’t known how spectrin folds in this resting state. The best evidence comes from visually enhanced electron micrographs of spectrin in partially expanded skeletons (Figure 5C and D). These suggest that α- and β-spectrin are coiled about each other in a two-start helix with approximately 10 turns per spectrin tetramer27 (Figure 5E). Some micrographs show relatively straight spectrin filaments;27 others show some kinking.30 This may reflect whether the filaments are under tension. In this model, spectrin molecules extend and contract along the helical axis by varying the pitch and diameter of the double strand (Figure 5E).27 This spring-like behavior is presumably critical to the elastic behavior of the membrane. 9 From www.bloodjournal.org by guest on June 16, 2017. For personal use only. However, there is evidence for other models—such as having spectrin molecules adopt a random worm-like configuration,67 or having helical segments of the repeats that ‘melt’ and extend under tension.34 or that simply unfold when stressed.35 The fact that previously occluded cysteines can be labelled when red cells are deformed by shear is evidence that some repeats unfold and that spectrin may even detach from ankyrin or actin when stretched.68 Since the structure of spectrin affects its mechanical properties as well as the spatial relationships of all the other proteins that bind to it, defining its structure in vivo, at rest and when deformed, is a high priority. Attachment of the Membrane Skeleton Ankyrin Erythrocyte ankyrin (ankyrin-1 or ankyrin-R) links spectrin to a complex of band 3 and other proteins in the lipid bilayer. The protein has three domains69 (Figure 6). The membrane domain is composed entirely of ankyrin repeats. It is a remarkably open, spiral structure, somewhat like a twisted sickle, that encircles and binds band 3 tetramers (Figure 6B). Whether band 3 spontaneously forms tetramers and then attracts ankyrin, stabilizing the tetramers, or whether ankyrin, which has two binding sites for band 3 (Figure 6A), independently binds two band 3 dimers (i.e., a “dimer of dimers”), is unresolved. The fact that a stable subpopulation of band 3 tetramers can be isolated from nonionic detergent extracts of ghosts74 argues for the former. But, the structure of a band 3 tetramer binding to the full membrane domain has not been solved and at least one recent paper argues that band 3 dimers must bind independently,75 so the question remains. The ankyrin-spectrin binding site is well defined. The ZU5A subdomain of ankyrin binds to a special site created by the junction of the 14th and 15th repeats within β-spectrin (Figure 6C). Only one ankyrin binds per tetramer, perhaps for steric reasons. Ankyrin binding promotes spectrin tetramer and oligomer formation by about 10-fold and vice-versa.16 Ankyrin attachment to band 3 also strengthens spectrin self- 10 From www.bloodjournal.org by guest on June 16, 2017. For personal use only. association.76 Presumably, both of these mechanisms help regulate the relatively tenuous self-association interaction. Band 3 Erythrocyte band 3, or officially SLC4A1, is the major red cell membrane protein, with about 1.2 million copies per cell. Functionally, it is two proteins:77 an N-terminal, cytoplasmic, peripheral membrane protein that is a key attachment site for the membrane skeleton, glycolytic enzymes and deoxyhemoglobin; and a C-terminal integral membrane protein that forms the red cell anion exchange channel and aids CO2 transport (Figure 7). The glycolytic enzymes form a metabolic complex (“metabolon”) extending from phosphofructokinase (PFK) through lactic dehydrogenase (LDH). Three of the enzymes bind to the Nterminus of band 3: PFK, aldolase and glyceraldehyde-3-phosphate dehydrogenase (G3PD). The others bind indirectly as part of the complex.79 The enzymes are inactive when bound79 but they are displaced and activated by deoxyhemoglobin, which also binds to the N-terminus,80 or by phosphorylation of two tyrosines within the binding sites79 (Figure 7). The enzymes79 and their ATP product86 are located in discrete clumps along the membrane, which supports the idea of a physical metabolon. Conditions that displace the enzymes from the membrane activate glycolytic fluxes by 45% in intact red cells, which supports a functional one.87 As noted earlier, ankyrin binds to band 3 tetramers. The acidic N-terminus and two loops on the surface of band 3 are involved in the binding70 (Figure 7). As with the glycolytic metabolon, ankyrin is displaced from band 3 by deoxyhemoglobin.81 The affinity of deoxyhemoglobin is weak, but the concentration of hemoglobin is so high in red cells that roughly half of the band 3 molecules are bound to deoxyhemoglobin when red cells are deoxygenated.81 Loosened ankyrin constraints could improve blood flow in hypoxic areas, but with the risk that red cells subject to prolonged deoxygenation, such as those trapped in the spleen, might suffer membrane vesiculation. (One wonders if this is the long sought mechanism for splenic conditioning88 in hereditary spherocytosis?) 11 From www.bloodjournal.org by guest on June 16, 2017. For personal use only. The Ankyrin Complex Ankyrin and band 3 are part of the multiprotein ankyrin complex (Figure 2). From the amounts of each protein in the red cell (Table 1), the complex is estimated to contain: one ankyrin, one band 3 tetramer, two glycophorin A or B dimers or heterodimers, two protein 4.2 molecules, and one Rh complex (a trimer of RhAG combined with Rh D and RhCE). CD47, LW and the proteins in the glycolytic metabolon are also components but they are present in less than stoichiometric amounts (Table 1) and so must only contribute to a subset of complexes. One wonders how many such subsets there are? Do they have constant composition or are they in a dynamic equilibrium? And are the rare complexes that contain CD47 and LW localized to specific membrane subdomains? As detailed in Table 1 and Figure 2, there are multiple interactions among proteins in the ankyrin complex. Some of these are critical. Red cells lacking band 3 also lack protein 4.2 and glycophorin A, for example.89 And, red cells lacking protein 4.2 nearly lack CD47.90 A consequence of this promiscuity is that missense mutations that interfere with a single interaction within the ankyrin complex have little effect. Hereditary spherocytosis is caused by defects in spectrin, ankyrin, band 3 or protein 4.2 that impair ankyrin complex formation, and almost all the known mutations diminish the concentration of these proteins rather than block their binding functions. The Actin Junctional Complex The actin protofilament and its associated proteins are also attached to the membrane. For many years this was believed to occur exclusively via a ternary complex of protein 4.1R, p55 and glycophorin C or D. Each of these proteins interacts with the other two and the interaction sites are well mapped.47,91,92 However, it was never clear why, if this was an important skeleton attachment and the sole attachment at the actin binding end of spectrin, patients with complete absence of glycophorin C and D had, at most, very mild hereditary elliptocytosis. During the past few years it has become clear that the actin junctional complex is more complex and, like the ankyrin complex, is centered on band 3 (Figure 2). First, protein 4.1R and ankyrin compete for 12 From www.bloodjournal.org by guest on June 16, 2017. For personal use only. binding to band 3,93 which suggests they bind to separate band 3 populations. The interaction with 4.1R is known to be important since zebrafish lacking band 3 can be rescued by expression of mouse band 3 but only if the mouse band 3 contains the 4.1R binding sites.94 Second, protein 4.2, which requires band 3 for incorporation into the membrane, binds to the EF hand domain at the actin-binding end of spectrin. Third, adducin binds to band 3,56 which definitely localizes a subpopulation of band 3 to the neighborhood of actin (Figure 2). The latter interaction is important since the membrane fragments if the connection is severed.56 The stoichiometry of the actin junctional complex is uncertain. On average, six spectrins and six proteins 4.1R bind per actin (Figure 8), and there is enough band 3, glycophorin A, glycophorin C/D (GPC/D), and probably enough Glut1 and stomatin to supply six complexes per actin filament (Table 1). But, there is only one copy of adducin per actin filament and only three copies of dematin if it is a monomer as recent studies indicate.58 Similarly, while protein 4.1R, GPC/D and protein p55 are usually pictured as a three part complex, there is only enough p55 for one copy per actin filament if it is a dimer, as some data suggest.95 And there is only about one copy of protein 4.2 available if 240,000 copies (one per band 3 dimer) are tied up in the ankyrin complex. The actin junctional complex also contains enzymes in the glycolytic metabolon78 and the blood group proteins Kx/Kell and DARC/Duffy,97 though the amounts of these proteins are insufficient for even one copy per complex. So, it is uncertain at the moment whether the actin junctional complex contains only a single band 3 dimer, interacting with just one or two of the six spectrin-4.1R dyads, or whether it is a larger complex containing three to six band 3 dimers that interact with all the spectrins and proteins 4.1R. Both possibilities are depicted in Figure 8. Recent evidence favors a larger complex,96 but the data are not conclusive. If so, it is not clear whether all or just a subset of band 3 molecules bind to adducin. In either case, as shown in Figure 8, one of the interesting and under appreciated facts is that the ankyrin and actin junctional complexes are quite close to each other in situ on the membrane and must often collide, especially during red cell deformation, but perhaps even when the cell is at rest. This raises 13 From www.bloodjournal.org by guest on June 16, 2017. For personal use only. the interesting possibility that proteins like band 3, protein 4.1R, protein 4.2, and adducin, which have binding partners in both complexes, may sometimes switch allegiances. Since some proteins compete for the same binding sites—for example, as noted, protein 4.1R displaces ankyrin from band 393—this could be a regulatory process. Distribution of Band 3 Of the ~1.2 million band 3 monomers per red cell, 40% are tetramers bound to ankyrin.96 Depending on whether the actin junctional complex contains an average of one or six band 3 dimers, an additional 7% to 40% of the band 3 dimers are located there. Recent experiments comparing normal mouse erythrocytes to α-adducin-deficient erythrocytes suggest that roughly 33% of the band 3 molecules are immobilized by adducin, which favors a larger complex.96 The remaining band 3 dimers (25-30%) are believed to be diffusing freely in the lipid bilayer, constrained for the most part by the boundaries of the spectrin corrals, which average less than 100 nm in size.96 This is remarkably similar to the values of 25% to 29% freely diffusing band 3 measured in normal red cells using different methods.96,98,99 It is important to remember that some of the minor proteins found in band 3 complexes can only exist in a subset of the complexes—probably multiple different subsets—and that the lipid bilayer is heterogeneous, with areas like the lipid rafts where certain proteins concentrate. So, the band 3 complexes must be more heterogeneous than these three states imply. Analyses of band 3 mobilities support this conclusion.96 The Future A great deal has been learned about the structure of the red cell membrane and the membrane skeleton in the 36 years since the first model was published (Figure 1). I sometimes hear people say that the red cell membrane is no longer an active area of hematology research, but I disagree. We have the broad outlines of how the membrane is organized (Figure 2), but we know few of the details, and what we know is about a static structure. Many questions remain (Table 2). We don’t know how the skeleton is constructed 14 From www.bloodjournal.org by guest on June 16, 2017. For personal use only. during erythropoiesis. We know very little about the dynamics of the skeleton—how labile individual bonds are, how the skeleton responds to capillary deformation, how the skeleton is disassembled and reassembled during invasion by malaria or other parasites, and so forth. And as discussed above, we don’t know how spectrin is folded in the intact skeleton or how the proteins that form the actin junctional complex are organized. Or how spectrin binds to actin. We don’t know if band 3-binding proteins like 4.1R, 4.2 and adducin switch from one band 3 complex to another given the apparent proximity of the complexes at each end of spectrin (Figure 8). We don’t understand why the same repeats in different spectrins are so similar if they just serve as spacers. Do they have binding or mechanical functions that we don’t yet know about? We don’t know what roles post-translational changes like phosphorylation, fatty acylation, methylation, glycation, hydroxylation and ubiquitination, or Ca2+-binding and ATP-binding play. And, we don’t understand the interactions between the membrane skeleton and the overlying lipids, which will likely have regulatory as well as structural functions. This is only a partial list—some other questions are itemized in Table 2—but I hope my point is clear. Since the red cell membrane skeleton is the paradigm for studies of spectrin-based membrane skeletons in all cells, and since we now know that spectrin and other membrane skeletal proteins invest internal organelles and some transport vesicles as well as plasma membranes, and even play a role in nuclear organization, it is more important than ever to understand the structure and function of the red cell membrane skeleton. 15 From www.bloodjournal.org by guest on June 16, 2017. For personal use only. Acknowledgments The author regrets that important papers from many talented individuals who have made significant contributions to red cell research could not be discussed or cited due to space limitations. I gratefully acknowledge the camaraderie and friendship of a large number of colleagues in the red cell membrane field, who over many years have enriched my career and aided my research. S. E. Lux wrote the review and designed the figures and tables. The author has no conflict of interest to declare. 16 From www.bloodjournal.org by guest on June 16, 2017. For personal use only. References 1. Marchesi VT, Steers E Jr. Selective solubilization of a protein component of the red cell membrane. Science. 1968;159(3811):203-204. 2. Danielli JF, Davson H. A contribution to the theory of permeability of thin films. J Cell Comp Physiol. 1935;5(4):495-508 3. Bakerman S, Wasemiller G. Studies on structural units of human erythrocyte membrane. I. Separation, isolation, and partial characterization. Biochemistry. 1967;6(4):1100-1113. 4. Lux SE. Dissecting the red cell membrane skeleton. Nature 1979;281(5731):426-429. 5. Korsgren C, Peters LL, Lux SE. Protein 4.2 binds to the carboxyl-terminal EF-hands of erythroid alpha-spectrin in a calcium- an calmodulin-dependent manner. J Biol Chem. 2010;285(7):47574770. 6. Lux SE. The red cell membrane. In: Orkin SH, Ginsburg D, Nathan DG, Look AT, Fisher DE, Lux SE, eds. Nathan and Oski's Hematology and Oncology of Infancy and Childhood, Chapter 15, 8th ed. Philadelphia:Elsevier; 2015, pp 445-514. 7. Burton NM, Bruce LJ. Modelling the structure of the red cell membrane. Biochem Cell Biol. 2011;89(2):200-215. 8. Mankelow TJ, Satchwell TJ, Burton NM. Refined views of multi-protein complexes in the erythrocyte membrane. Blood Cells Mol Dis. 2012;49(1):1-10. 9. Fowler VM. The human erythrocyte plasma membrane: a Rosetta Stone for decoding membranecytoskeleton structure. Curr Top Membr. 2013;72:39-88. 10. Mohandas N, Gallagher PG. Red cell membrane: past, present, and future. Blood. 2008;112(10):3939-3948. 11. Pesciotta EN, Sriswasdi S, Tang HY, Mason PJ, Bessler M, Speicher DW. A label-free proteome analysis strategy for identifying quantitative changes in erythrocyte membranes induced by red cell disorders. J Proteomics. 2012;76 Spec No.:194-202. 17 From www.bloodjournal.org by guest on June 16, 2017. For personal use only. 12. Yan Y, Winograd E, Viel A, Cronin T, Harrison SC, Branton D. Crystal structure of the repetitive segments of spectrin. Science. 1993;262(5142):2027-2030. 13. Sriswasdi S, Harper SL, Tang HY, Gallagher PG, Speicher DW. Probing large conformational rearrangements in wild-type and mutant spectrin using structural mass spectrometry. Proc Natl Acad Sci U S A. 2014;111(5):1801-1806. 14. An X, Gauthier E, Zhang X, et al. Adhesive activity of Lu glycoproteins is regulated by interaction with spectrin. Blood. 2008;112(13):5212-5218. 15. Ipsaro JJ, Mondragón A. Structural basis for spectrin recognition by ankyrin. Blood. 2010;115(20):4093-4101. 16. Giorgi M, Cianci CD, Gallagher PG, Morrow JS. Spectrin oligomerization is cooperatively coupled to membrane assembly: a linkage targeted by many hereditary hemolytic anemias. Exp Mol Pathol. 2001;70(3):215-230. 17. Li D, Tang HY, Speicher DW. A structural model of the erythrocyte spectrin heterodimer initiation site determined using homology modeling and chemical cross-linking. J Biol Chem. 2008;283(3):1553-1562. 18. An X, Debnath G, Guo X, et al. 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. Biochemistry. 2005;44(31):10681-10688. 19. Li X, Bennett V. Identification of the spectrin subunit and domains required for formation of spectrin/adducin/actin complexes. J Biol Chem. 1996;271(26):15695-15702. 20. Pei X, Guo X, Coppel R, Mohandas N, An X. Plasmodium falciparum erythrocyte membrane protein 3 (PfEMP3) destabilizes erythrocyte membrane skeleton. J Biol Chem. 2007;282(37):26754-26758. 21. Korsgren C, Lux SE. The carboxyterminal EF domain of erythroid alpha-spectrin is necessary for optimal spectrin-actin binding. Blood. 2010;116(14):2600-2607. 18 From www.bloodjournal.org by guest on June 16, 2017. For personal use only. 22. An X, Guo X, Sum H, Morrow J, Gratzer W, Mohandas N. Phosphatidylserine binding sites in erythroid spectrin: location and implications for membrane stability. Biochemistry. 2004;43(2):310-315. 23. An X, Guo X, Zhang X, et al. Conformational stabilities of the structural repeats of erythroid spectrin and their functional implications. J Biol Chem. 2006;281(15):10527-10532. 24. Liu J, Taylor DW, Taylor KA. A 3-D reconstruction of smooth muscle alpha-actinin by CryoEm reveals two different conformations at the actin-binding region. J Mol Biol. 2004;338(1):115-125. 25. Galkin VE, Orlova A, Salmazo A, Djinovic-Carugo K, Egelman EH. Opening of tandem calponin homology domains regulates their affinity for F-actin. Nat Struct Mol Biol. 2010;17(5):614-616. 26. Liu SC, Derick LH, Palek J. Visualization of the hexagonal lattice in the erythrocyte membrane skeleton. J Cell Biol. 1987;104(3):527-536. 27. McGough AM, Josephs R. On the structure of erythrocyte spectrin in partially expanded membrane skeletons. Proc Natl Acad Sci U S A. 1990;87(13):5208-5212. 28. Li D, Harper S, Speicher DW. Initial propagation of spectrin heterodimer assembly involves distinct energetic processes. Biochemistry. 2007;46(37):10585-10594. 29. Ursitti JA, Wade JB. Ultrastructure and immunocytochemistry of the isolated human erythrocyte membrane skeleton. Cell Motil Cytoskeleton. 1993;25(1):30-42. 30. Nans A, Mohandas N, Stokes DL. Native ultrastructure of the red cell cytoskeleton by cryoelectron tomography. Biophys J. 2011;101(10):2341-2350. 31. Liu SC, Windisch P, Kim S, Palek J. Oligomeric states of spectrin in normal erythrocyte membranes: biochemical and electron microscopic studies. Cell. 1984;37(2):587-594. 32. Salomao M, An X, Guo X, Gratzer WB, Mohandas N, Baines AJ. Mammalian alpha I-spectrin is a neofunctionalized polypeptide adapted to small highly deformable erythrocytes. Proc Natl Acad Sci U S A. 2006;103(3):643-648. 33. An X, Lecomte MC, Chasis JA, Mohandas N, Gratzer W. Shear-response of the spectrin dimer19 From www.bloodjournal.org by guest on June 16, 2017. For personal use only. tetramer equilibrium in the red blood cell membrane. J Biol Chem. 2002;277(35):31796-31800. 34. Grum VL, Li D, MacDonald RI, Mondragon A. Structures of two repeats of spectrin suggest models of flexibility. Cell. 1999;98(4):523-535. 35. Johnson CP, Tang HY, Carag C, Speicher DW, Discher DE. Forced unfolding of proteins within cells. Science. 2007;317(5838):663-666. 36. Picart C, Discher DE. Actin protofilament orientation at the erythrocyte membrane. Biophys J 1999;77(2):865-878. 37. Kalfa TA, Pushkaran S, Mohandas N, et al. Rac GTPases regulate the morphology and deformability of the erythrocyte cytoskeleton. Blood. 2006;108(12):3637-3645. 38. Chan MM, Wooden JM, Tsang M, et al. Hematopoietic protein-1 regulates the actin membrane skeleton and membrane stability in murine erythrocytes. PLoS One. 2013;8(2):e54902. 39. An X, Salomao M, Guo X, Gratzer W, Mohandas N. Tropomyosin modulates erythrocyte membrane stability. Blood. 2007;109(3):1284-1288. 40. Fowler VM. Tropomodulin: A cytoskeletal protein that binds to the end of erythrocyte tropomyosin and inhibits tropomyosin binding to actin. J Cell Biol. 1990;111(2):471–481. 41. Johnson M, East DA, Mulvihill DP. Formins determine the functional properties of actin filaments in yeast. Curr Biol. 2014;24(13):1525-1530. 42. Yamashiro S, Gokhin DS, Kimura S, Nowak RB, Fowler VM. Tropomodulins: pointed-end capping proteins that regulate actin filament architecture in diverse cell types. Cytoskeleton (Hoboken). 2012;69(6):337-370. 43. Moyer JD, Nowak RB, Kim NE, et al. Tropomodulin 1-null mice have a mild spherocytic elliptocytosis with appearance of tropomodulin 3 in red blood cells and disruption of the membrane skeleton. Blood. 2010;116(14):2590-2599. 44. Sui Z, Nowak RB, Bacconi A, et al. Tropomodulin3-null mice are embryonic lethal with anemia due to impaired erythroid terminal differentiation in the fetal liver. Blood. 2014;123(5):758-767. 20 From www.bloodjournal.org by guest on June 16, 2017. For personal use only. 45. Ohanian V, Wolfe LC, John KM, Pinder JC, Lux SE, Gratzer WB. Analysis of the ternary interaction of the red cell membrane skeletal proteins spectrin, actin, and 4.1. Biochemistry. 1984;23(19):4416-4420. 46. Correas I, Speicher DW, Marchesi VT. Structure of the spectrin-actin binding site of erythrocyte protein 4.1. J Biol Chem. 1986;261(28):13362-13366. 47. Han BG, Nunomura W, Takakuwa Y, Mohandas N, Jap BK. Protein 4.1R core domain structure and insights into regulation of cytoskeletal organization. Nat Struct Biol. 2000;7(10):871-875. 48. Gimm JA, An X, Nunomura W, Mohandas N. Functional characterization of spectrin-actinbinding domains in 4.1 family of proteins. Biochemistry. 2002;41(23):7275-7282. 49. Discher DE, Winardi R, Schischmanoff PO, Parra M, Conboy JG, Mohandas N. Mechanochemistry of protein 4.1’s spectrin-actin-binding domain: ternary complex interactions, membrane binding, network integration, structural strengthening. J Cell Biol. 1995;130(4):897907. 50. Joshi R, Gilligan DM, Otto E, McLaughlin T, Bennett V. Primary structure and domain organization of human alpha and beta adducin. J Cell Biol. 1991;115(3):665-675. 51. Kuhlman PA, Hughes CA, Bennett V, Fowler VM. A new function for adducin. Calcium/calmodulin-regulated capping of the barbed ends of actin filaments. J Biol Chem. 1996;271(14):7986-7991. 52. Li X, Matsuoka Y, Bennett V. Adducin preferentially recruits spectrin to the fast growing ends of actin filaments in a complex requiring the MARCKS-related domain and a newly defined oligomerization domain. J Biol Chem. 1998;273(30):19329-19338. 53. Matsuoka Y, Hughes CA, Bennett V. Adducin regulation. Definition of the calmodulin-binding domain and sites of phosphorylation by protein kinases A and C. J Biol Chem. 1996;271(41):25157-25166. 54. Manno S, Takakuwa Y, Mohandas N. Modulation of erythrocyte membrane mechanical function 21 From www.bloodjournal.org by guest on June 16, 2017. For personal use only. by protein 4.1 phosphorylation. J Biol Chem. 2005;280(9):7581-7587. 55. Robledo RF, Ciciotte SL, Gwynn B, et al. Targeted deletion of alpha adducin results in absent beta- and gamma-adducin, compensated hemolytic anemia, and lethal hydrocephalus in mice. Blood. 2008;112(10):4298-4307. 56. Anong WA, Franco T, Chu H, et al. Adducin forms a bridge between the erythrocyte membrane and its cytoskeleton and regulates membrane cohesion. Blood. 2009;114(9):1904-1912. 57. Khan AA, Hanada T, Mohseni M, et al. Dematin and adducin provide a novel link between the spectrin cytoskeleton and human erythrocyte membrane by directly interacting with glucose transporter-1. J Biol Chem. 2008;283(21):14600-14609. 58. Chen L, Brown JW, Mok YF, et al. The allosteric mechanism induced by protein kinase A (PKA) phosphorylation of dematin (band 4.9). J Biol Chem. 2013;288(12):8313–8320. 59. Siegel DL, Branton D. Partial purification and characterization of an actin-bundling protein, band 4.9, from human erythrocytes. J Cell Biol. 1985;100(3):775-785. 60. Koshino I, Mohandas N, Takakuwa Y. Identification of a novel role for dematin in regulating red cell membrane function by modulating spectrin-actin interaction. J Biol Chem. 2012;287(42):35244-35250. 61. Tang J, Taylor DW, Taylor KA. The three-dimensional structure of alpha-actinin obtained by cryoelectron microscopy suggests a model for Ca2+-dependent actin binding. J Mol Biol. 2001;310(4):845-858. 62. Galkin VE, Orlova A, VanLoock MS, Rybakova IN, Ervasti JM, Egelman EH. The utrophin actinbinding domain binds F-actin in two different modes: implications for the spectrin superfamily of proteins. J Cell Biol. 2002;157(2):243-251. 63. Shotton DM, Burke BE, Branton D. The molecular structure of human erythrocyte spectrin. Biophysical and electron microscopic studies. J Mol Biol. 1979;131(2):303-329. 64. Byers T, Branton D. Visualization of the protein associations in the erythrocyte membrane 22 From www.bloodjournal.org by guest on June 16, 2017. For personal use only. skeleton. Proc Natl Acad Sci U S A 1985;82(18):6153-6157. 65. Ursitti JA, Pumplin DW, Wade JB, Bloch RJ. Ultrastructure of the human erythrocyte cytoskeleton and its attachment to the membrane. Cell Motil Cytoskeleton. 1991;19(4):227-243 66. Shen BW, Josephs R, Steck TL. Ultrastructure of the intact skeleton of the human erythrocyte membrane. J Cell Biol. 1986;102(3):997-1006. 67. Coleman, T. R., D. J. Fishkind, M. S. Mooseker, and J. S. Morrow. Functional diversity among spectrin isoforms. Cell Motil Cytoskeleton. 1989;12(4):225-247. 68. Krieger CC, An X, Tang HY, Mohandas N, Speicher DW, Discher DE. Cysteine shotgun-mass spectrometry (CS-MS) reveals dynamic sequence of protein structure changes within mutant and stressed cells. Proc Natl Acad Sci U S A. 2011;108(20):8269-8274. 69. Lux SE, John KM, Bennett V. Analysis of cDNA for human erythrocyte ankyrin indicates a repeated structure with homology to tissue-differentiation and cell-cycle control proteins. Nature. 1990;344(6261):36-43. 70. Grey JL, Kodippili GC, Simon K, Low PS. Identification of contact sites between ankyrin and band 3 in the human erythrocyte membrane. Biochemistry. 2012;51(34):6838-6846. 71. Stabach PR, Simonović I, Ranieri MA, et al. The structure of the ankyrin-binding site of betaspectrin reveals how tandem spectrin-repeats generate unique ligand-binding properties. Blood. 2009;113(22):5377-5384. 72. Davis LH, Davis JQ, Bennett V. Ankyrin regulation: an alternatively spliced segment of the regulatory domain functions as an intramolecular modulator. J Biol Chem. 1992;267(26):1896618972. 73. Michaely P, Tomchick DR, Machius M, Anderson RG. Crystal structure of a 12 ANK repeat stack from human ankyrinR. EMBO J. 2002;21(23):6387-6396. 74. Hanspal M, Golan DE, Smockova Y, et al. Temporal synthesis of band 3 oligomers during terminal maturation of mouse erythroblasts. Dimers and tetramers exist in the membrane as 23 From www.bloodjournal.org by guest on June 16, 2017. For personal use only. preformed stable species. Blood. 1998;92(1):329-338. 75. Kim S, Brandon S, Zhou Z, et al. Determination of structural models of the complex between the cytoplasmic domain of erythrocyte band 3 and ankyrin-R repeats 13-24. J Biol Chem. 2011:286(23):20746-20757. 76. Blanc L, Salomao M, Guo X, et al. Control of erythrocyte membrane-skeletal cohesion by the spectrin-membrane linkage. Biochemistry. 2010;49(21):4516-4523. 77. Lux SE, John KM, Kopito RR, Lodish HF. Cloning and characterization of band 3, the human erythrocyte anion-exchange protein (AE1). Proc Natl Acad Sci U S A. 1989;86(23):9088-9093. 78. Puchulu-Campanella E, Chu H, Anstee DJ, Galan JA, Tao WA, Low PS. Identification of the components of a glycolytic enzyme metabolon on the human red blood cell membrane. J Biol Chem. 2013;288(2):848-858. 79. Campanella ME, Chu H, Low PS. Assembly and regulation of a glycolytic enzyme complex on the human erythrocyte membrane. Proc Natl Acad Sci U S A. 2005;102(7):2402-2407. 80. Chu H, Breite A, Ciraolo P, Franco RS, Low PS. Characterization of the deoxyhemoglobin binding site on human erythrocyte band 3: implications for O2 regulation of erythrocyte properties. Blood. 2008;111(2):932-938. 81. Stefanovic M, Puchulu-Campanella E, Kodippili G, Low PS. Oxygen regulates the band 3-ankyrin bridge in the human erythrocyte membrane. Biochem J. 2013;449(1):143-150. 82. Lombardo CR, Willardson BM, Low PS. Localization of the protein 4.1-binding site on the cytoplasmic domain of erythrocyte membrane band 3. J Biol Chem. 1992;267(14):9540-9546. 83. Bonar P, Schneider HP, Becker HM, Deitmer JW, Casey JR. Three-dimensional model for the human Cl-/HCO3- exchanger, AE1, by homology to the E. coli ClC protein. J Mol Biol. 2013;425(14):2591-2608. 84. Sterling D, Alvarez BV, Casey JR. The extracellular component of a transport metabolon. Extracellular loop 4 of the human AE1 Cl-/HCO3- exchanger binds carbonic anhydrase IV. J Biol 24 From www.bloodjournal.org by guest on June 16, 2017. For personal use only. Chem. 2002;277(28):25239-25246. 85. Vince JW, Reithmeier RA. Carbonic anhydrase II binds to the carboxyl terminus of human band 3, the erythrocyte Cl-/HCO3- exchanger. J Biol Chem. 1998;273(43):28430-28437. 86. Chu H, Puchulu-Campanella E, Galan JA, Tao WA, Low PS, Hoffman JF. Identification of cytoskeletal elements enclosing the ATP pools that fuel human red blood cell membrane cation pumps. Proc Natl Acad Sci U S A. 2012;109(31):12794-12799. 87. Lewis IA, Campanella ME, Markley JL, Low PS. Role of band 3 in regulating metabolic flux of red blood cells. Proc Natl Acad Sci U S A. 2009;106(44):18515-18520. 88. Griggs RC, Weisman R Jr, Harris JW. Alterations in osmotic and mechanical fragility related to in vivo erythrocyte aging and splenic sequestration in hereditary spherocytosis. J Clin Invest. 1960;39(1):89-101. 89. Peters LL, Shivdasani RA, Liu SC, et al. Anion exchanger 1 (band 3) is required to prevent erythrocyte membrane surface loss but not to form the membrane skeleton. Cell. 1996;86(6):917927. 90. Bruce LJ, Ghosh S, King MJ, et al. Absence of CD47 in protein 4.2-deficient hereditary spherocytosis in man: an interaction between the Rh complex and the band 3 complex. Blood. 2002;100(5):1878-1885. 91. Marfatia SM, Leu RA, Branton D, Chishti AH. Identification of the protein 4.1 binding interface on glycophorin C and p55, a homologue of the Drosophila discs-large tumor suppressor protein. J Biol Chem. 1995;270(2):715-719. 92. Chishti AH. Function of p55 and its nonerythroid homologues. Curr Opin Hematol. 1998;5(2):116-121 93. An XL, Takakuwa Y, Nunomura W, Manno S, Mohandas N. Modulation of band 3-ankyrin interaction by protein 4.1. Functional implications in regulation of erythrocyte membrane mechanical properties. J Biol Chem. 1996;271(52):33187-33191. 25 From www.bloodjournal.org by guest on June 16, 2017. For personal use only. 94. Paw BH, Davidson AJ, Zhou Y, et al. Cell-specific mitotic defect and dyserythropoiesis associated with erythroid band 3 deficiency. Nat Genet. 2003;34(1):59-64. 95. Ruff P, Speicher DW, Husain-Chishti A. Molecular identification of a major palmitoylated erythrocyte membrane protein containing the src homology 3 motif. Proc Natl Acad Sci U S A. 1991;88(15):6595-6599. 96. Kodippili GC, Spector J, Hale J, et al. Analysis of the mobilities of band 3 populations associated with ankyrin protein and junctional complexes in intact murine erythrocytes. J Biol Chem. 2012;287(6):4129-4138. 97. Salomao M, Zhang X, Yang Y, et al. Protein 4.1R-dependent multiprotein complex: new insights into the structural organization of the red blood cell membrane. Proc Natl Acad Sci U S A. 2008;105(23):8026-8031. 98. Dahl KN, Parthasarathy R, Westhoff CM, Layton DM, Discher DE. Protein 4.2 is critical to CD47-membrane skeleton attachment in human red cells. Blood. 2004;103(3):1131-1136. 99. Mirchev R, Lam A, Golan DE. Membrane compartmentalization in Southeast Asian ovalocytosis red blood cells. Br J Haematol. 2011;155(1):111-121. 26 Table 1. Some Important Erythrocyte Membrane Proteins Protein Oligomeric ular mers/ State Mass Cell (kd)* x 10-3 † Oligomers in Oligomers in Functions in Mature Erythrocyte Membranes Ankyrin Actin Jct Complex Complex Com- Unit Complex plex Cell‡ and Unit Cell‡ β-Actin 41.6 500 Oligomer (~14) 1 Forms ~37 nm long filaments that serve as a molecular junction in the membrane skeleton. Binds spectrin with the aid of protein 4.1R. Binds tropomyosin, tropomodulin, adducin, dematin and chorein α-Adducin 81.0 30 Heterodimer/ β-Adducin 80.7 30 tetramer 1 Caps the fast-growing (barbed) end of actin and recruits spectrin to nearby (dimer) sites on actin. Links actin junction to band 3, Glut1 and maybe stomatin. Binds chorein. Aldolase A 39.4 20 Tetramer <1 <1 <1 Forms glycolytic complex on band 3 with PFK, enolase, G3PD, PK and LDH Ankyrin-1§ 206.0 124 ± 11 Monomer 1 3 Attaches β -spectrin and the membrane skeleton to band 3 and to protein 4.2 and RhAG in the ankyrin-linked band 3 complex Band 3 (SLC4A1) 101.8 1200 Dimer or tetramer 2 6 (dimer) (dimer) 1-6 Anion exchange channel (especially HCO - and Cl-) with the aid of CAII and 3 (dimer) CAIV. Forms membrane complexes with GPA, GPB, the Rh complex, Glut1, stomatin and other proteins that are linked to spectrin via ankyrin-1 and protein 4.2, and to the actin junctional complex via protein 4.1R, protein 4.2, p55, adducin, and Glut1. Binds peroxiredoxin. Forms a glycolytic metabolon with G3PD, PFK, PGK, aldolase, LDH and PK. Binds deoxyhemoglobin. Carries Ii and many other blood group antigens. From www.bloodjournal.org by guest on June 16, 2017. For personal use only. Molec- Mono- Carbonic anhydrase II 29.1 Carbonic anhydrase IV 33.1 100 Unknown Attaches to the outside (CAIV) and inside (CAII) surfaces of band 3, the Unknown HCO3- channel. Catalyzes interconversion of CO2 and HCO3-, which aids in HCO3- transport and CO2 elimination. 37.1 6-10 Unknown <1 Binds to protein 4.1R and maybe to ankyrin. Multifunctional protein in nonerythroid cells. Function in the red cell is unknown. CD47 33.3 17 Monomer <1 <1 Binds to protein 4.2 and to the Rh-complex (RhAG/RhD/RhCE) within the ankyrin-linked band 3 complex. Inhibits phagocytosis. DARC/Duffy 35.6 6-13 Unknown <1 Chemokine receptor. Carries the Duffy (Fy) blood group antigen. Receptor for P. vivax. Function in normal red cell unknown. Dematin 43.1 & 130 Monomer 3 Binds and bundles actin filaments. Facilitates spectrin-actin binding. Helps 45.5 Glyceraldehyde-3- 35.9 attach actin to the membrane via Glut1 500 Tetramer 1 3 phosphate dehydrogenase Glucose transporter-1 1-3 Forms glycolytic metabolon on band 3 with aldolase, PFK, enolase, PK and LDH. 54.1 200-700 Dimer & tetramer ? ? 1-6 Transports glucose. Transports L-dehydroascorbic acid when bound to (dimer) stomatin. Links actin to the membrane via interactions with dematin, adducin and band 3. Glycophorin A 14.3 1000 Glycophorin B 7.7 170-250 Homo or heterodimer 2 6 1-6 Binds band 3 in the ankyrin-linked band 3 complex, and maybe to RhAG. May facilitate band 3 transfer to the membrane. Carries MNSsU blood group antigens and much of red cell’s negative surface charge. Glycophorin C 13.8 143 Monomer? Homo or Glycophorin D 11.5 82 heterodimer? 6 Links actin junctional complex to membrane via interactions with protein 4.1R, p55 and band 3. Carries Gerbich blood group antigens. 28 From www.bloodjournal.org by guest on June 16, 2017. For personal use only. CD44 Kell 82.8 Kx 50.9 3-18 Heterodimer with Kx <1 The Kell-Kx complex may reside in the actin-linked band 3 complex. Kell Heterodimer with Kell <1 carries the Kell blood group antigen. Kx probably has an undiscovered transport function. Lu/BCAM 64.3 1.5-8 Unknown Adhesion molecule that carries the Lutheran (Lu) blood group antigen. Binds LW glycoprotein/ICAM4 27.0 3-5 Monomer <1 <1 Intracellular adhesion molecule that carries the Landsteiner-Wiener (LW) blood group antigen. Interacts with RhAG/RhD/RhCE and probably resides in the ankyrin-linked band 3 complex. p55/MPP1 52.3 80 Dimer? 1 Links actin junctional complex to membrane via interactions with some protein 4.1R and GPC/D molecules. Heavily palmitoylated. May contribute to organization of lipid rafts. Peroxiredoxin 2 21.8 200 Dimer or decamer ? ? ? Abundant antioxidant protein that is partially linked to band 3 on the membrane. Also binds stomatin and activates the Ca2+-dependent K+ (Gardos) channel. Phosphofructokinase-liver 85.0 Phosphofructokinase- 85.2 6 Heterotetramer <1 <1 <1 Forms glycolytic metabolon on band 3 with aldolase, G3PD, enolase, PK and LDH. muscle Protein 4.1R 66.4 ~240 6 Binds to both β-spectrin and actin and greatly strengthens the spectrin-actin Monomer interaction. Links actin to the membrane via interactions with GPC/D, p55 and band 3. Competes with ankyrin for binding to band 3. Protein 4.2 79.8/ 76.9h ~280 Monomer? 2 6 1 Binds to band 3, ankyrin and CD47 in the ankyrin-linked band 3 complex. Also binds to the EF hand domain of α-spectrin. 29 From www.bloodjournal.org by guest on June 16, 2017. For personal use only. to α-spectrin. RhAG 44.2 100-200 Heterotrimer of RhCE 45.4 100-200 RhAG, RhD & the ankyrin-linked band 3 complex. RhAG transports NH3/NH4+ and maybe RhD 45.1 100-200 RhCE? CO2 α-Spectrin 280.0 242 β-Spectrin 246.4 242 Heterodimer/tetra- 2 3 6 (dimer) (dimer) The RhAG/RhD/RhCE trimer binds to CD47, LW, ankyrin, CD47 and GPB in 6 Crosslinks actin filaments into a hexagonal lattice at the tail end with the (dimer) help of protein 4.1R, adducin, and dematin. Binds protein 4.2 via the αspectrin EF hand domain. Self-associates into tetramers and oligomers and binds to ankyrin-1 and Lu/BCAM at the head end. Stomatin 31.7 200-400? Oligomer (9-12)? ? ? 1 Binds to Glut1 and converts it into an ascorbic acid transporter. Also binds band 3, adducin and peroxiredoxin. Located in lipid rafts. Tropomodulin 1 40.6 30 Monomer 1 Caps slow-growing (pointed) end of actin. Binds tropomyosin, which strengthens capping. Tropomyosin 1 (αTM) 32.9 Tropomyosin 3 (γTM) 32.9 140i Homodimer 2 Binds to and stabilizes actin filaments in a Mg2+-dependent manner. May help specify actin filament lengths in the red cell. * Size of the mature protein chain without any signal peptide, propeptides or initiator methionine, but not including posttranslational modifications. † References for the numbers of copies of each protein are listed in the footnotes to Table 2 in reference 6. It must be emphasized that the numbers of many red cell membrane proteins are roughly estimated but not accurately measured, which affects the estimates of the composition of the ankyrin complex and actin junctional complex. ‡ The unit cell is defined in Figure 8. § Data shown are for full length ankyrin (band 2.1), the major isoform, except for the number of copies/cell, which includes the smaller isoforms: bands 2.2, 2.3 and 2.6. Abbreviations: CAII, carbonic anhydrase-II; CAIV, carbonic anhydrase-IV; G3PD, glyceraldehyde-3-phosphate dehydrogenase; Glut1, glucose transporter-1; GPA, glycophorin A; GPB, glycophorin B; GPC/D, glycophorins C and D; LDH, lactic dehydrogenase; PFK, phosphofructokinase; PGK, phosphoglycerate kinase; PK, pyruvate kinase; RhAG, Rh-associated glycoprotein. 30 From www.bloodjournal.org by guest on June 16, 2017. For personal use only. mer/oligomer 1 From www.bloodjournal.org by guest on June 16, 2017. For personal use only. Table 2. Some Unanswered Questions General • What are the amounts and stoichiometries of the membrane proteins (only a few are accurately measured)? Spectrin • What is the structure of spectrin in vivo? How does it change when the membrane is deformed? • How does spectrin bind to the actin protofilament? What is the stoichiometry? Where do the CH1 and CH2 domains and protein 4.1R bind on actin? Is there more than one binding conformation? If so, how do these differ functionally and how are they regulated? • Why is the EF hand domain essential for normal spectrin-actin binding? How does it work? • How do spectrin molecules, which lie in a plane, accommodate different orientations of their binding sites on the helical actin protofilament? • Higher order spectrin oligomers (hexamers, octamers, etc) also serve as molecular junctions in the skeleton. Are there significant numbers of these and do they have a unique functional role? • If the available spectrin repeat structures are correct and there is a continuous alpha helix across the interrepeat junction, how is spectrin flexibility achieved? • Why are the same repeats in different spectrins so similar? Do they have binding or mechanical functions that we don’t yet know about? • What binds to the spectrin SH3 domain? What are the consequences? • There is evidence that some of the spectrins in mature red cells contain the muscle isoform of β1-spectrin, which has a lipid-interacting pleckstrin homology domain. Is this true, and if so, what is its function? Actin • How are actin protofilaments formed? How do they achieve their uniform size? Do formins play a role? • Are the actin protofilaments stable or do they turnover, as the presence of a critical concentration of G-actin in the red cell cytoplasm suggests? If so, how is this regulated? • How are the various proteins arranged on the actin protofilament? Is the arrangement the same in all the protofilaments? If there are open binding sites on actin, do molecules like spectrin move from site to site with membrane deformation? • Do red cell myosin and caldesmon have a function in the membrane of mature red cells? Ankyrin • Does ankyrin bind a band 3 tetramer or two dimers? If a tetramer, is it preformed before binding or do two dimers bind and convert to a tetramer on ankyrin. What is the structure of the band 3 tetramer-ankyrin complex? • What is the structure of the ankyrin molecule? How do the various domains relate to each other spatially? • Where do protein 4.2 and RhAG bind? • What is the function of the death domain in ankyrin? • What are the functions of the many ankyrin spliceoforms? From www.bloodjournal.org by guest on June 16, 2017. For personal use only. Band 3 • What is the overall structure of band 3? • Where do protein 4.2 and adducin bind on band 3? • Since the numbers of integral membrane proteins vary by orders of magnitude (Table 1), the band 3 multiprotein complexes must vary in composition. How many such complexes are there? Do they have specific, stable compositions or are they in equilibrium and continually changing their composition? Are the sub-stoichiometric proteins (e.g., CD44, CD47, LW, Kx/Kell, DARC) uniformly distributed or localized to lipid rafts or other membrane lipid or protein subdomains? • How are the proteins in the band 3 multiprotein complexes arranged relative to each other? • Do protein interactions within the band 3 multiprotein complexes allosterically affect the interactions or functions of other proteins in the complexes? • Does protein 4.1R bind to band 3 in vivo? If so, does band 3 bind to each of the spectrin-actin-4.1R complexes or only to a subset, such as the subset that interacts with adducin (Figure 8)? • Are any band 3 molecules “unbound” (i.e., not attached to the membrane skeleton directly or indirectly). If so, how many? Are they also part of multiprotein complexes? Do they have a unique function? Glycolytic Metabolon • The enzymes that comprise the proposed glycolytic metabolon vary in concentration by orders of magnitude. Are the estimates correct? If so, do some complexes lack rare components (but then how would they function?) or are they gigantic so as to include at least one copy of each enzyme? Are other enzymes in the glycolytic pathway part of the complex? • How is the glycolytic metabolon regulated in vivo? What is the purpose of activating glycolysis by deoxyhemoglobin? Do products of glycolysis, such as ATP cause vasodilation, directly or indirectly? Does nitric oxide play a role? Is the rate of dissociation and activation of the glycolytic metabolon rapid enough to be relevant during normal circulatory transits? Protein 4.1R • What is the structure of intact protein 4.1R and the spectrin-actin-4.1R complex? • Calmodulin binds to and regulates protein 4.1R. The EF hand domain is a calmodulin-like structure that resides near 4.1R in the actin junctional complex. In addition, the EF domain binds calmodulin. Does 4.1R bind to the EF domain or do they bind each other through calmodulin? If so is this a regulatory interaction? • Does phosphatidyl-4,5-bisphosphate regulate protein 4.1R binding to actin or other proteins in vivo? • Since there is insufficient protein p55/MPP to make a 1:1:1 molar complex with protein 4.1R and glycophorin C/D, how is the complex constructed? Protein 4.2 • What is the function of protein 4.2 when attached to the spectrin EF hand domain? Does it actually occupy the domain in vivo? If so, does it bind to protein 4.1R? 32 From www.bloodjournal.org by guest on June 16, 2017. For personal use only. • What is the function of the ATP-binding site on protein 4.2? Adducin • Is adducin a dimer or tetramer? • What is the structure of adducin? How does the C-terminal tail relate to the head end? • Band 3, spectrin and actin all bind to the C-terminal tail. What is the structure of this complex? How are the multiple interactions regulated? Can they all occur at once on the same molecule or do some interactions interfere with others? When adducin binds spectrin and actin does it also interact with nearby proteins 4.1R or 4.2? Does adducin bind band 3 dimers or tetramers and does it bind one or two molecules of each? Dematin • How does dematin contribute to spectrin binding in vivo? • Is dematin’s ability to bundle actin filaments important in the mature red cell? • Where does dematin bind relative to spectrin and adducin on the actin protofilament? Does the number of dematins per protofilament vary? Tropomyosin • Do the red cell isoforms of tropomyosin determine the unique structure of the short actin protofilaments? Do specific actin-nucleating formins play a role in this process? Posttranslational • What do post-translational modifications like phosphorylation, palmitoylation, myristoylation, hydroxylation, methylation, glycation and ubiquitination do? Are the effects important in vivo? Modifications • How are the multiple effects of Ca2+ and calmodulin integrated in vivo? Dynamics • Do the actin junctional complex and ankyrin complex contact each other in vivo? Do proteins like band 3, proteins 4.1R and 4.2, and adducin, which have potential binding partners in both complexes, sometimes switch allegiances? If so, is this be a regulatory process? • How labile are the individual bonds in the membrane skeleton? Which bonds, other than the spectrin dimertetramer interaction, break when the skeleton is deformed? Are the rates of dissociation and reassociation such that the bonds dissociate during red cell deformation in the circulation? • How is the skeleton disassembled and reassembled during invasion by malaria and other parasites? Lipids • What are the interactions between the membrane skeleton and the overlying lipids? Do these interactions have regulatory as well as structural functions? Does the membrane skeleton vary in unique regions of the lipid bilayer, such as lipid rafts? 33 From www.bloodjournal.org by guest on June 16, 2017. For personal use only. Legends Figure 1. The first model of the red cell membrane skeleton, published 36 years ago. (Reprinted from Lux SE4 with permission.) Figure 2. Current model of the red cell membrane. Most of the known protein contacts are shown, but the relative positions of the proteins to each other within the various complexes are mostly not known. The major proteins are drawn roughly to scale but the shapes are mostly imaginary. Roughly 40% of the band 3 molecules are tetramers in a complex with ankyrin and other integral proteins near the spectrin selfassociation site (“Ankyrin Complex”). A roughly similar fraction of the band 3 molecules, probably dimers, are located near the spectrin-actin junction and bind to spectrin via protein 4.1R, protein 4.2 and adducin (“Actin Junctional Complex”). As described later in the text. it is likely these two complexes, with their associated proteins, are large enough that they sometimes contact each other. The remaining band 3 dimers float untethered within the lipid bilayer (“Unbound Band 3”). The actin protofilament lies parallel to the membrane. The complexes of proteins associated with band 3 are not constant—some proteins are present in much smaller numbers than others (e.g., Kell, Kx, CD44, CD47, DARC/Duffy, LW, PFK and aldolase). The amounts of p55, adducin and dematin are also insufficient to interact with all the spectrin-4.1-actin complexes. As a consequence, the ankyrin and actin junctional complexes must vary in composition and mobility. For visual clarity, peroxiredoxin-2 is shown attached just to unbound band 3 but there is no evidence for this selectivity. Ank, ankyrin; CH1 and CH2, actin binding domains of β-spectrin; EF, Ca2+binding EF hand domain of α-spectrin; GEC, glycolytic enzyme complex (G3PD, PFK, LDH, PK, aldolase and enolase); GPA, glycophorin A; GPB, glycophorin B; GPC, glycophorin C; Prx2, peroxiredoxin-2; RhAG, Rh-associated glycoprotein. (Professional illustration by Somersault18:24. Adapted from Korsgren C, Peters LL, Lux SE,5 with permission.) 34 From www.bloodjournal.org by guest on June 16, 2017. For personal use only. Figure 3. Spectrin. (A) Organization of erythrocyte spectrin and the dimer-tetramer equilibrium. Spectrin is a long, flexible, worm-like protein composed of two chains (α- and β-spectrin). Each chain contains a tandem array of ~5.0 nm, ~106-amino acid, triple helical spectrin-type repeats with specialized functional domains for self-association and ankyrin-1 binding at the “head” end, and for binding to actin, protein 4.1R and other associated proteins at the “tail” end. Each spectrin repeat is formed by three α-helices (A, B & C) with short connecting loops that are folded like a flattened Z into a triple helical bundle.12 α-Spectrin contains 21 numbered repeats (α1-α21), plus a partial repeat (α0) at the N-terminus that contains a single Chelix. One of the 21 is really an SH3 domain (α10), but is numbered as a repeat by convention. β-Spectrin contains 16 true repeats (β1-β16) plus a partial repeat (β17) at the C-terminus that contains just the A and B helices. Note that for the spectrin αβ-dimer to convert to the α2β2-tetramer it must first cleave the internal linkage between the partial spectrin repeats α0 and β17 and then unfold (open dimer).13 This is the ratelimiting step in the dimer-tetramer equilibrium. Dimer-tetramer self-association occurs at the “head” end of the spectrin dimer where the adhesion protein Lu/BCAM also attaches.14 Ankyrin-1 binds nearby to spectrin repeats β14-β15.15 These two reactions cooperate: ankyrin-binding favors spectrin tetramer formation and vice-versa.16 The isolated α- and β-spectrin chains join to form spectrin heterodimers at a nucleation site near the “tail” end of spectrin (repeats α-21 pairs with β-1 and α-20 with β-2) and then zip together in a cooperative manner.17 Actin and protein 4.1R bind to CH domains at the N-terminal end of β-spectrin, just beyond the nucleation site.18 Binding to the CH2 domain is activated by phosphatidylinositol-4,5bisphosphate (PIP2).18 Adducin binds in the same region.19 Protein 4.2 and calcium bind to a neighboring EF-hand domain on α-spectrin.5 Both the CH and EF-hand domains are needed for full actin binding.20,21 PS denotes spectrin repeats that bind phosphatidyl serine.22 Blue stars mark repeats that are relatively unstable at physiological temperatures.23 (B) Structure of spectrin repeats β8 and β9 (Protein Data Bank: 1S35). Note that each repeat is formed by three α-helices (A, B & C) in a Z-configuration. Note also that helix C in β8 and helix A in β9 form a continuous, α-helix that spans the junction between the repeats. (C) Hypothetical model of the tail end of spectrin based on recent structures of α-actinin.24,25 Note that the first actin binding 35 From www.bloodjournal.org by guest on June 16, 2017. For personal use only. domain (CH1) binds to F-actin in an extended (open) conformation.25 The intimate relationship between the EF hand and CH domains and recent evidence that the EF domain is required for optimal spectrin-actin binding,20,21 suggest the EF hands regulate actin binding. Figure 4. Protein 4.1R. (A) Domain map of protein 4.1R and the location of erythrocyte binding partners. (B) Structure of the protein 4.1R membrane binding domain (MBD).47 Subdomains where specific proteins bind are colored and labelled. (C) Schematic representation of the protein 4.1R spectrin-actin binding domain (SABD, blue). Two spectrin binding regions straddle an actin binding peptide.48 In this hypothetical model, each of the spectrin binding regions is assumed to interact with a different one of the two calponin homology (CH) domains of β-spectrin and both CH domains are assumed to interact with actin.48 Neither of these things has been proved. (Panel B adapted from Han BG, et al.47 with permission.) Figure 5. Negatively-stained electron micrographs of red cell membrane skeletons and spectrin. (A) A membrane skeleton that was stretched during preparation. Note the pseudohexagonal organization of the skeleton and the location of various proteins. Inset, bottom left. Example of a 37 nm long F-actin protofilament. Sp4, spectrin tetramer; Sp6, spectrin hexamer. (B) Skeleton in situ in a red cell prepared by a minimally perturbing quick-freeze, deep etch, rotary replication procedure. The dense network of filaments average 29 ± 9 nm between intersections. (C) and (D) Spectrin from partially stretched skeletons. Some of the spectrin molecules show a helical periodic substructure, as noted by the region between the arrowheads in C or by the single arrowhead in D. (E) Right handed double-helical models of spectrin periodicity obtained from the experiments in D by visual filtering of the periodic regions from multiple spectrin molecules. In this model, the spring-like spectrins extend and contract by varying their pitch and diameter. Native spectrin tetramer has approximately 10 turns with a pitch of about 7 nm (70 Å) and a diameter of about 5.9 nm (59 Å), ((A) Reprinted from Liu SC et al.26 Inset from Byers T and Branton D.64 (B) From 36 From www.bloodjournal.org by guest on June 16, 2017. For personal use only. Ursitti et al.65 (C) from Ursitti JA and Wade JB.29 (D) and (E) From McGough AM and Josephs R.27 All with permission.) Figure 6. Ankyrin. (A) Schematic of erythroid ankyrin (Ank1 or AnkR) structure.69 The Membrane Domain contains 24 ankyrin repeats, grouped functionally into four subdomains of six repeats. There are two binding sites for band 3, one involving repeats 7 to 12 (D2, domain 2), and one involving repeats 13 to 24 (D34). The ankyrin binding loops on band 3 are predicted to interact with ankyrin repeats 19 and 20 (light blue) of D4.70 The interaction site within D2 is not known. The Spectrin Domain contains three subdomains of which ZU5A (light blue) contains the binding site for spectrin.15,71 The C-Terminal (Regulatory) Domain is thought to modulate the binding functions of the other two domains.69,72 It exists in numerous spliced variants of mostly unknown function. The function of the conserved death domain (DD) is also a mystery. (B) Hypothetical model73 of the interaction between the membrane domain of ankyrin (green) and a band 3 tetramer (red, blue, cyan and purple subunits). Note how the ankyrin repeats form a large (9 nm diameter) twisted helical spiral. In this deduced model, the concave surface of the D34 region interacts with the red subunit in one band 3 dimer. One subunit in the second band 3 dimer contacts the concave surface of the D2 region, which contains a second band 3 binding site. (C) Spectrin-ankyrin interaction. Note how ZU5-A, the spectrin-binding subdomain within ankyrin, binds in the notch created by the sharp angle between spectrin repeats β14 and β15. (Panel B from Michaely P, et al73 and panel C from Ipsaro JJ and Mondragón A15 with permission.) Figure 7. Band 3. Organizational model of human erythrocyte band 3 (anion exchange protein). The protein contains two structurally and functionally distinct domains: a cytoplasmic binding domain (amino acids (aa) 1-359) and a transmembrane domain (aa 360-911) that forms the anion exchange channel.77 Cytoplasmic Domain: The glycolytic enzymes phosphofructokinase (PFK), aldolase, and glyceraldehyde-3-phosphate (G3PD) bind to amino acids 1-23 at the N-terminus of band 3 and contact amino acids 356-384,78 which are 37 From www.bloodjournal.org by guest on June 16, 2017. For personal use only. nearby in the folded protein. The enzymes are inactive when bound79 but they are displaced and activated by deoxyhemoglobin, which also binds to the N-terminus,80 or by phosphorylation of two tyrosines within the binding sites.79 Enolase, pyruvate kinase (PK) and lactic dehydrogenase (LDH) also localize to the membrane and are displaced by deoxyhemoglobin, but don't bind to band 3.79 This suggests that many of the enzymes in the glycolytic pathway form a functional complex (or ‘metabolon’) that efficiently generates ATP, particularly under hypoxic conditions. Ankyrin also interacts with the N-terminus,81 but the main ankyrin sites are aa 63-73 and 175-185, which loop out from the surface.70 Protein 4.1 binds to two sites with the (I/L)RRRY motif, near the end of the domain.82 Ankyrin and protein 4.1R inhibit each other’s binding. The binding sites for protein 4.2 and adducin have not been identified. Membrane Domain: The best current model83 for the anion exchange channel is based on the structure of the ClC bacterial chloride channel. The intra- and trans-membrane helices are lettered as they are in the ClC channel. A complex carbohydrate structure is attached to Asn 642. Carbonic anhydrase IV binds to the extracellular loop of band 3 between helices I and J,84 and CAII may bind to the C-terminal segment.85 Both are perfectly positioned to create - HCO3 from CO2 and shuttle the ions to or from the plasma through band 3. Figure 8. Model of the unit cell of the membrane skeleton. (A) The membrane skeleton is a quasihexagonal lattice centered around ~40,000 F-actin protofilaments. Each 37 nm, double helical protofilament is capped by adducin (A) and tropomodulin (T) and contains about 14 G-actin subunits. The red cell surface area is about 140 μm2 so each hexagonal unit cell is 3500 nm2 and the average length of a spectrin dimer is about 32 nm, which is one-third it’s length when fully stretched. Available data suggests the spectrin chains are coiled about each other, and expand and contract by changing their pitch and diameter27 (Figure 5E). They are shown here as straight, though they are sometimes kinked.30 Spectrin oligomers are not shown in this model but exist in vivo. In addition, the skeleton is not as regularly arrayed in vivo as in this averaged model. (B) A recent model of the ankyrin complex (green ovals) assembled from known structures of the proteins by Burton and Bruce7 has a cross-sectional area of 17.5 nm x 14 nm, assuming the proteins are 38 From www.bloodjournal.org by guest on June 16, 2017. For personal use only. closely packed. They estimate the cross-sectional area of the actin junctional complex is about 20 nm x 17 nm if it only contains a single band 3 dimer7 (small tan ovals). Such a complex would presumably lie toward the end of the actin protofilament where adducin resides. This is shown in the two unit cells on the right. Alternatively, if the actin junctional complex contains six band 3 dimers interacting with all six 4.1R proteins, it would probably be more centered and roughly approximate the area of the large tan oval in the unit cell on the left. Recent data favor the larger arrangement,96 but in either case it is likely the ankyrin complex and actin junctional complex would be close to each other and probably often touch, particularly during red cell deformation. Band 3 dimers that are not associated with either the ankyrin or actin junctional complexes (small blue ovals) diffuse in the lipid bilayer within the spectrin corrals. There will be three to eight of these per unit cell depending on the number of band 3’s in the actin junctional complex. (From Lux SE6 with permission.) 39 Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 From www.bloodjournal.org by guest on June 16, 2017. For personal use only. Prepublished online November 4, 2015; doi:10.1182/blood-2014-12-512772 Anatomy of the red cell membrane skeleton: unanswered questions Samuel E. 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