membrana eritrocitaria

doi:10.1182/blood-2008-07-1611662008 112: 3939-3948

Narla Mohandas and Patrick G. Gallagher Red cell membrane: past, present, and future

http://bloodjournal.hematologylibrary.org/content/112/10/3939.full.htmlUpdated information and services can be found at:

(1174 articles)Red Cells � (1569 articles)Free Research Articles �

(34 articles)ASH 50th Anniversary Reviews �Articles on similar topics can be found in the following Blood collections

http://bloodjournal.hematologylibrary.org/site/misc/rights.xhtml#repub_requestsInformation about reproducing this article in parts or in its entirety may be found online at:

http://bloodjournal.hematologylibrary.org/site/misc/rights.xhtml#reprintsInformation about ordering reprints may be found online at:

http://bloodjournal.hematologylibrary.org/site/subscriptions/index.xhtmlInformation about subscriptions and ASH membership may be found online at:

Copyright 2011 by The American Society of Hematology; all rights reserved.Washington DC 20036.by the American Society of Hematology, 2021 L St, NW, Suite 900, Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly

For personal use only. by guest on January 10, 2013. bloodjournal.hematologylibrary.orgFrom

http://bloodjournal.hematologylibrary.org/content/112/10/3939.full.html

http://bloodjournal.hematologylibrary.org/cgi/collection/ash_50th_anniversary_reviews

http://bloodjournal.hematologylibrary.org/cgi/collection/free_research_articles

http://bloodjournal.hematologylibrary.org/cgi/collection/red_cells

http://bloodjournal.hematologylibrary.org/site/misc/rights.xhtml#repub_requests

http://bloodjournal.hematologylibrary.org/site/misc/rights.xhtml#reprints

http://bloodjournal.hematologylibrary.org/site/subscriptions/index.xhtml

http://bloodjournal.hematologylibrary.org/subscriptions/ToS.dtl

http://bloodjournal.hematologylibrary.org/


Red cell membrane: past, present, and futureNarla Mohandas1 and Patrick G. Gallagher2

1Red Cell Physiology Laboratory, New York Blood Center, New York, NY; and 2Department of Pediatrics, Yale University School of Medicine, New Haven, CT

As a result of natural selection driven bysevere forms of malaria, 1 in 6 humans inthe world, more than 1 billion people, areaffected by red cell abnormalities, makingthem the most common of the inheriteddisorders. The non-nucleated red cell isunique among human cell type in that theplasma membrane, its only structuralcomponent, accounts for all of its diverseantigenic, transport, and mechanical char-acteristics. Our current concept of the red

cell membrane envisions it as a compos-ite structure in which a membrane enve-lope composed of cholesterol and phos-pholipids is secured to an elastic networkof skeletal proteins via transmembraneproteins. Structural and functional charac-terization of the many constituents of thered cell membrane, in conjunction withbiophysical and physiologic studies, hasled to detailed description of the way inwhich the remarkable mechanical proper-

ties and other important characteristicsof the red cells arise, and of the manner inwhich they fail in disease states. Currentstudies in this very active and excitingfield are continuing to produce new andunexpected revelations on the function ofthe red cell membrane and thus of the cellin health and disease, and shed new lighton membrane function in other diversecell types. (Blood. 2008;112:3939-3948)

History

Introduction

Jan Swammerdam, a Dutch biologist and microscopist, first observedand described red cells in 1668 but it was some years before hisobservations, recorded in his notebooks, were publicly disseminated.Antonie van Leeuwenhoek, another brilliant Dutch microscopist, wasthe first to publish, in Philosophical Transactions of the Royal Society in1675, a remarkable description of the unique features of human redblood cells.1 He stated, “when he was greatly disordered, the globules ofhis blood appeared hard and rigid, but grew softer and more pliable ashis health returned: whence he infers that in a healthy body they should besoft and flexible, that they may be capable of passing through the capillaryveins and arteries, by easily changing their round figures into ovals, andalso reassuming their former roundness when they come into vesselswhere they find larger room.” This striking observation made more than300 years ago has proven both prescient and accurate.An excellent reviewby Bessis and Delpech provides a comprehensive description of prioritiesand credits for the discovery and description of red cells.2

George Gulliver, following the work of William Hewson,published the primary features of red cell membranes in Blood ofVertebrata in 1862, “Not withstanding the current observations thatthe red corpuscle is absolutely homogeneous, it is really composedof 2 very different parts. One of these is membranous, colourlessand insoluble in water; the other is semifluid or viscid, containingthe color, and very soluble in water.”3 Gorter and Grendel in 1925provided the first insights into the structure of the membrane, andindeed biologic membranes generally, by the brilliant deductionthat there are “bimolecular layers of lipids on the chromocytes ofblood.”4 This model has continually evolved over the past 80 years,thanks to a succession of seminal contributions that includedoutlining of the fluid mosaic model of the structure of cellmembranes by Singer and Nicolson,5 isolation of spectrin byMarchesi and Steers,6 and the definition of the topology of red cellmembrane proteins by Steck and colleagues.7 This progress hasbenefited greatly from key discoveries that included methods for

isolation of membranes (ghosts); protein component analysisthrough the development of gel electrophoresis and mass spectrom-etry; advances in imaging technologies; biochemical, structural,and functional characterization of the various protein componentsof the membrane; defining of asymmetric distribution of phospho-lipids in the membrane; and delineating the nature of interactionsamong various membrane proteins and between proteins and lipids.While a very large number of investigators contributed to the manyexciting advances made in our understanding of the structuralorganization of the red cell membrane during these interveningyears, a few deserve special mention including Peter Agre, JaneBarker, Daniel Branton, Vann Bennett, Jean Delaunay, BernardForget, Walter Gratzer, Joseph Hoffman, Philip Low, Samuel Lux,Vincent Marchesi, Jon Morrow, Jiri Palek, Eric Ponder, andTheodore Steck.

The non-nucleated erythrocyte is unique among human cells in thatthe plasma membrane, its only structural component, accounts for all ofits diverse antigenic, transport, and mechanical characteristics. Thediscoid shape of the red cell evolves from the multilobulated reticulocyteduring 48 hours of maturation first in the bone marrow and then in thecirculation (Figure 1). An important and distinguishing feature of thediscoid human red cell is its ability to undergo large passive deforma-tions during repeated passage through the narrow capillaries of themicrovasculature, with cross-sections one-third its own diameter, through-out its 120-day life span. The most dramatic manifestations of thisproperty are seen during red cells’ transit in vivo from the splenic cords tothe splenic sinus and during flow-induced deformation in vitro (Figure 1).Several elegant studies from the 1940s through the 1960s by, amongothers, John Dacie, Lawrence E. Young, Thomas Hale Ham, James H.Jandl, and William B. Castle, documented that splenic sequestration ofabnormal red cells with reduced deformability accounts for the decreasedlife span and resulting hemolytic anemia in several red cell disorders.

Although a key role for cellular deformability in regulating red cellfunction and survival has long been recognized, quantitative character-ization of deformability began in 1964 with the publication of the

Submitted July 30, 2008; accepted August 15, 2008. DOI 10.1182/blood-2008-07-161166.

© 2008 by The American Society of Hematology

ASH 50th anniversary review

3939BLOOD, 15 NOVEMBER 2008 � VOLUME 112, NUMBER 10




seminal study by Rand and Burton, based on the micropipette aspirationtechnique to measure “stiffness” of the membrane.8 Subsequently, avariety of experimental strategies including rheoscopy, flow channels,ektacytometry, and optical trapping have been used to generate quantita-tive descriptions of the material characteristics that regulate the ability ofred cells to undergo deformation, and of the role of various membranecomponents in this process. Paul LaCelle, Evan Evans, Robert Hoch-muth, Dennis Discher, and several other investigators developed bothquantitative measures of deformability and the essential theoreticalunderpinnings.

Studies during the past 3 decades on red cells from healthypeople and from patients with various inherited red cell disordershave illuminated the molecular processes underlying normal andaberrant red cell membrane function.9-13 We will survey the currentstate of understanding of the structural organization of the normalred cell membrane and describe how anomalous structural organi-zation accounts for altered features of membrane and cell in theknown red cell membrane disorders.

Present view of structural organization of normal red cellmembrane

The structural organization of the human red cell membraneenables it to undergo large reversible deformations while maintain-ing its structural integrity during its 4-month sojourn in thecirculation. The red cell membrane exhibits unique material

behavior. It is highly elastic (100-fold softer than a latex membraneof comparable thickness), rapidly responds to applied fluid stresses(time constants in the range of 100 milliseconds), and is strongerthan steel in terms of structural resistance. While a normal red cellcan deform with linear extensions of up to approximately 250%, a3% to 4% increase in surface area results in cell lysis. Hence animportant feature of induced red cell deformations, both in vitroand in vivo, is that they involve no significant change in membranesurface area. These unusual membrane material properties are theresult of an evolution-driven “engineering” process resulting in acomposite structure in which a plasma membrane envelope com-posed of cholesterol and phospholipids is anchored to a 2-dimensional elastic network of skeletal proteins through tetheringsites on cytoplasmic domains of transmembrane proteins embed-ded in the lipid bilayer (Figure 2).11 Direct interaction of severalskeletal proteins with the anionic phospholipids affords additionalattachments of the skeletal network to the lipid bilayer.14,15

Membrane lipids. The lipid bilayer is composed of equalproportions by weight of cholesterol and phospholipids.16 Whilecholesterol is thought to be distributed equally between the2 leaflets, the 4 major phospholipids are asymmetrically disposed.Phosphatidylcholine and sphingomyelin are predominantly locatedin the outer monolayer, while most phosphatidylethanolamine andall phosphatidylserine (PS), together with the minor phosphoinosi-tide constituents, are confined to the inner monolayer.17,18 Several

Figure 1. Multilobular reticulocyte (top left panel), the precursor of the mature discoid red cell (top right panel). A red cell traversing from the splenic cord to splenicsinus (bottom left panel). Note the marked deformation the cell undergoes during its passage through the narrow endothelial slit separating the cord from the sinus. Ellipsoidalcells generated in vitro by flow-induced deformation in vitro of discoid cells (bottom right panel).

3940 MOHANDAS and GALLAGHER BLOOD, 15 NOVEMBER 2008 � VOLUME 112, NUMBER 10




different types of energy-dependent and energy-independent phos-pholipid transport proteins have been implicated in generating andmaintaining phospholipid asymmetry.19,20 “Flippases” move phos-pholipids from the outer to the inner monolayer while “floppases”do the opposite against a concentration gradient in an energy-dependent manner. In contrast, “scramblases” move phospholipidsbi-directionally down their concentration gradients in an energy-independent manner. Although several different membrane pro-teins have been said to exert these different lipid transport activitiesin human red cells, there is still considerable debate about theiridentity. Recent studies have described “lipid rafts” enriched incholesterol and sphingolipids in association with specific mem-brane proteins that include flotillins, stomatin, G-proteins, and�-adrenegic receptors in the red cell membrane.21,22

The maintenance of asymmetric distribution of phospholipids,in particular exclusive localization of PS and phosphoinositides tothe inner monolayer, has several functional implications. Becausemacrophages recognize and phagocytose red cells that expose PS attheir outer surface, the confinement of this lipid in the innermonolayer is essential if the cell is to survive its frequent encounterwith macrophages of the reticuloendothelial system, especially thespleen. Loss of lipid asymmetry leading to exposure of PS on theouter monolayer has been suggested to play a role in prematuredestruction of thalassemic and sickle red cells.23-25 Furthermore,the restriction of PS to the inner monolayer also inhibits theadhesion of normal red cells to vascular endothelial cells, therebyensuring unimpeded transit through the microvasculature.26 Byreason of their interactions with skeletal proteins, spectrin, andprotein 4.1R, both PS and phosphatidylinositol-4,5-bisphosphate(PIP2) can regulate membrane mechanical function.27,28 Recentstudies have established that binding of spectrin to PS enhancesmembrane mechanical stability.27 PIP2 enhances the binding of4.1R to glycophorin C but decreases its interaction with band 3,and thereby may modulate the linkage of the bilayer to themembrane skeleton.29 Lipid rafts that have been implicated in cellsignaling events in nonerythroid cells have been shown in erythroidcells to mediate �2-adregenic receptor signaling and increasecAMP levels, and thus regulating entry of malarial parasites intonormal red cells.30

Membrane proteins. More than 50 transmembrane proteins ofvarious abundance ranging from a few hundred to a million copiesper red cell have been well characterized. A large fraction—some25—of transmembrane proteins define the various blood groupantigens. The membrane proteins exhibit diverse functional hetero-geneity, serving as transport proteins, as adhesion proteins involvedin interactions of red cells with other blood cells and endothelialcells, as signaling receptors, and other still undefined activities.

Membrane proteins with transport function include band 3(anion transporter), aquaporin 1 (water transporter), Glut1 (glucoseand L-dehydroascorbic acid transporter), Kidd antigen protein(urea transporter), RhAG (gas transporter, probably of carbondioxide), Na�-K�-ATPase, Ca�� ATPase, Na�-K�-2Cl� cotrans-porter, Na�-Cl� cotransporter, Na�-K� cotransporter, K�-Cl�

cotransporter, and Gardos Channel. Membrane proteins withadhesive function include ICAM-4, which interacts with integrinsand Lu, the laminin-binding protein. Detailed discussions of thedefined as well as presumptive functions of various membraneproteins can be found in several excellent recent reviews.31-34 Ofdirect relevance to structural integrity of the membrane are2 macromolecular complexes of membrane proteins, one ankyrin-based, and the other protein 4.1R-based. Band 3 and RhAG link thebilayer to the membrane skeleton through the interaction of theircytoplasmic domains with ankyrin, and glycophorin C, XK, Rh,and Duffy through their interaction with protein 4.1R.35-39 Recentstudies have indicated that 2 other members of the spectrin-actin-protein 4.1R junctional complex, adducin and dematin, can alsoserve as linking proteins by interacting with band 3 and Glut1,respectively.39,40 These membrane protein linkages with skeletalproteins may play a role in regulating cohesion between lipidbilayer and membrane skeleton and thus enable the red cell tomaintain its favorable membrane surface area by preventingmembrane vesiculation. In addition to its linking function, band 3also assembles various glycolytic enzymes, the presumptive CO2

transporter, and carbonic anhydrase into a macromolecular com-plex termed a “metabolon,” which may play a key role in regulatingred cell metabolism and ion and gas transport function.41

Skeletal proteins. The principal protein constituents of the2-dimensional spectrin-based membrane skeletal network are �-and �-spectrin, actin, protein 4.1R, adducin, dematin, tropomyosin,and tropomodulin.16,42-44 A unique structural feature of the longfilamentous spectrin is its large number of triple-helical repeats of106 amino acids, 20 in �-spectrin and 16 in �-spectrin. Firstdiscovered in erythrocytes,45 these triple-helical bundles define aspectrin super family of proteins that includes dystrophin, actinin,and utrophin.46 �- and �-spectrin form an antiparallel heterodimerthrough strong lateral interaction between repeats 19 and 20 nearthe C-terminus of �-spectrin with repeats 1 and 2 near the N-terminus of �-spectrin. The 36 triple-helical repeats of spectrin arestructurally heterogeneous in terms of their thermal stability.47

Spectrin tetramer, the major structural component of the 2-dimen-sional skeletal network, is formed by the lateral interaction of asolitary helix at the N-terminus of the �-chain from 1 dimer with2 helices at the C-terminus of the �-chain from the other to create astable triple-helical repeat (Figure 3).48-50 While the spectrin dimer-dimer interaction was long thought to be static, recent studies haverevealed that dissociation of spectrin tetramers can be induced bymembrane deformation.51 The other end of the 100 nm-longspectrin dimer forms a junctional complex with F-actin and protein4.1R.28,52,53 While actin interacts weakly with N-terminus of�-spectrin; the interaction is greatly enhanced by protein 4.1R.54

The length of the actin filaments in the red cell membrane appears

Ankyrin complex 4.1R complex

Dematin

Adducin

4.1R

Tropomodulin

Tropomyosin

Actin protofilament

GPALW

Band 3 Band 3Band 3

α-spectrin

Protein 4.2

β-spectrin

Ankyrin

Glut1

CD47

GPA

RhAGRh

RhDuffy

Kell

XKGPC

p55

Figure 2. A schematic representation of red cell membrane. The membrane is acomposite structure in which a plasma membrane envelope composed of amphiphiliclipid molecules is anchored to a 2-dimensional elastic network of skeletal proteinsthrough tethering sites (transmembrane proteins) embedded in the lipid bilayer.Illustration by Paulette Dennis.

RED CELL MEMBRANE 3941BLOOD, 15 NOVEMBER 2008 � VOLUME 112, NUMBER 10




to be tightly regulated, probably by tropomyosin, and is made up of14 to 16 actin monomers.55 Adducin and tropomodulin cap actinfilaments at opposite ends, while the function of the actin bundlingprotein dematin in the junctional complex has yet to be fullydefined.56-58 The spectrin dimer-dimer interaction and the spectrin-actin-protein 4.1R junctional complex are key regulators of mem-brane mechanical stability and play a critical role in preventingdeformation-induced membrane fragmentation as the cell encoun-ters high fluid shear stresses in circulation.

While much progress has indeed been made in our understand-ing of the structural organization of the various lipid and proteincomponents of the normal red cell membrane, the current modelsare far from comprehensive and continue to evolve. A recent studyusing state-of-the-art proteomic approaches has generated a compre-hensive catalog of red cell proteins and has identified more than300 proteins including 105 integral membrane proteins.59 Thecurrent membrane models account for fewer than 15% of thesemolecules!

Implications of the structural organization of variousmembrane components for normal red cell function

In performing its primary function of oxygen delivery to thetissues, the red cell must absorb continuous mechanical punish-ment throughout its lifetime without structural deterioration. Exten-sive biophysical studies have identified 3 constitutive features asthe primary regulators of the ability of the cell’s capacity toundergo the necessary deformations. These are: (1) the geometry ofthe cell, particularly cell surface area to volume ratio; (2) thecytoplasmic viscosity determined by intracellular hemoglobinconcentration; and (3) membrane deformability.9-11

Cell geometry. The normal biconcave human red cell with avolume of 90 fL and surface area of 140 �m2 possesses an excesssurface area of 40% compared with a sphere of the same volume.Without excess surface area to volume ratio the cell cannot deform,for any deviation from the spherical state at constant volumeimplies an increase in surface area, which is forbidden by the lipidbilayer properties. Maintenance of membrane surface area ismediated by strong cohesion between the bilayer and the mem-brane skeleton that prevents membrane vesiculation, and by amechanically stable spectrin-based membrane skeleton that pre-vents membrane breakup. Maintenance of cell volume is mediatedby various membrane-associated ion transporters. Surface area lossas a result of membrane vesiculation due to decreased membranecohesion, or cell fragmentation as a consequence of reducedmembrane mechanical stability, as well as increase in cell volume

due to defective ion transporters, will all compromise the ability ofthe cell to deform and lead to its premature removal fromcirculation.

The determinant of normal membrane cohesion is the system of“vertical” linkages between bilayer and membrane skeleton, formedby the interactions of the cytoplasmic domains of various mem-brane proteins with the spectrin-based skeletal network (Figure 2).Band 3 and RhAG provide such links by interacting with ankyrin,which in turn binds to �-spectrin. Protein 4.2 binds to both band 3and ankyrin and can regulate the avidity of the interaction betweenband 3 and ankyrin. Glycophorin C, band 3, XK, Rh, and Duffy allbind to protein 4.1R, the third member of the ternary junctionalcomplex with �-spectrin and actin.35-39 Recent studies reveal thatband 3 and Glut1 can also link the bilayer to the skeleton throughtheir interactions with the skeletal proteins, adducin and dematin,respectively.39,40 Of the various linkages documented, those medi-ated by band 3 appear to be the dominant determinant of membranecohesion followed by the linkage mediated by RhAG.

A dominant regulator of membrane mechanical stability thatenables the red cell to maintain its structural integrity through thevicissitudes of the circulation is the stability of the network.Network stability is critically dependent on both the avidity ofinteraction between spectrin dimers60,61 and the interactions thatdefine the junctional complex at the distal ends of spectrintetramers.62,63 While the spectrin tetramer is formed through theassociation of the solitary helix at the N-terminus of the �-chainfrom one dimer with 2 helices at the C-terminus of the �-chainfrom the other to create a stable triple helical repeat, the adjacenttriple helical repeats are needed for effective interaction betweenthe dimers.50 The dimer-dimer interaction is not static, but dynamicin the sense that it opens reversibly under tensile forces imposed bydeformation, and indeed flickers continuously between the openand closed states, as reflected by the relatively low associationconstant.51 This association constant is further strengthened by thebinding of ankyrin to the �-chain.64 The principal constituents ofthe junctional complex at the end of spectrin tetramers are spectrin,F-actin, and protein 4.1R and avidity of this complex is anothermajor determinant of membrane stability.16,63 At least 4 otherproteins, tropomyosin, adducin, tropomodulin, and dematin, arepresent at lower copy numbers of 1 to 2 per junction.65 Theseproteins appear to stabilize the short actin filaments in the complex, buttheir influence on membrane stability appears modest compared withthe primary constituents, spectrin, actin and protein 4.1R.

While a major role for protein-protein interactions in regulatingmechanical stability has been well documented, the contribution of

Figure 3. The horizontal linkages between spectrin-spectrin dimers and between spectrin, actin, andprotein 4.1R in the junctional complex in the spectrin-based membrane skeleton. The repeats of �-spectrinare colored gray while those of �-spectrin are coloredlight green. The single helical repeat at N-terminus of�-spectrin of 1 dimer interacts with the 2 helical repeat atC-terminus of �-spectrin of the second dimer to constitutespectrin dimer-dimer interaction indicated in pink. The PSbinding spectrin repeats are colored in dark blue whilerepeats with low thermal stability (Tm � 37°C) are shownin red. The various components of the junctional complexat the end of spectrin dimer are also shown. EF hands atC-terminus of �-spectrin and the actin-binding domain(ABD) at N-terminus of �-spectrin are also indicated.Illustration by Paulette Dennis.





protein-lipid interactions has received much less attention. Recentstudies have determined that certain of the triple-helical repeats ofboth �- and �-spectrin bind PS (Figure 3) and this binding in situincreases membrane mechanical stability.15,27 In contrast, PIP2binds to N-terminus of �-spectrin and decreases the propensityspectrin to form a ternary complex with actin and protein 4.1R andmay thereby decrease membrane mechanical stability.28

Cytoplasmic viscosity and cell volume regulation. The abilityof normal red cells to rapidly change their shape in response to fluidshear stresses is governed by cytoplasmic viscosity, which is determinedby intracellular hemoglobin concentration. Non-nucleated mammalianred cells regulate their mean cell hemoglobin concentration within avery narrow range (30-35 g/dL) while their mean cell volumes rangecan vary widely (20-200 fL). While the mean cell hemoglobin concen-tration of normal human red cells is 33 g/dL, the distribution ofhemoglobin concentrations in individual red cells in whole blood rangesfrom 27 to 37 g/dL. The viscosity of hemoglobin solution increasessteeply starting at 37 g/dL. While hemoglobin viscosity is only 5 centi-poise (cp) [5 times greater than water] at 27 g/dL increasing to 15 cp at37 g/dL, it rises abruptly to 45 cp at 40 g/dL, up to 170 cp at 45 g/dL and650 cp at 50 g/dL.10,66 By tightly regulating hemoglobin concentrationwithin a narrow range, red cells minimize the cytoplasmic viscousdissipation during cell deformation. Increases in hemoglobin concentra-tions above 37 g/dL markedly decrease the rate at which the cellrecovers its initial shape after both extensional and bending deforma-tions.67 Thus the ability of the cell to accommodate rapidly to a narrowcapillary in the microcirculation will be compromised by increasedcytoplasmic viscosity, and with it, its efficacy in tissue oxygen delivery.Of note, however, cell dehydration and the resultant increase incytoplasmic viscosity only minimally affect red cell survival.

The ability of the red cell to regulate its hemoglobin concentra-tion within narrow limits is critically dependent on its ability tocontrol its volume. As red cell volume is primarily determined bytotal cation content, a host of transport proteins, which transportsodium and potassium across the membrane play a role inregulating cytoplasmic viscosity.68-70

Membrane deformability. A unique feature of the normal redcell membrane is its high elasticity, which enables the cell torapidly respond to applied fluid stresses in the circulation. Whileboth theoretical and experimental evidence implicates a criticalrole for the spectrin-based skeletal network in general, and spectrinin particular, in determining membrane elasticity, the precisestructural basis of the effect remains uncertain. An importantstructural feature of the long filamentous spectrin dimer is thesuccession of 36 spectrin repeats, 20 in �-spectrin and 16 in�-spectrin, which behave in part as independently folding units. Arecent study revealed that the thermal stabilities of the 36 indi-vidual repeats, expressed in terms of the mid-point unfoldingtransition, vary widely, ranging from 21 to 72°C.47 It was inferredthat unfolding of the least stable spectrin repeats might affectmembrane elasticity. A recent elegant study, based on labeling byfluorophores of sterically shielded cysteines of spectrin in intactmembranes in conjunction with quantitative mass spectrometry,demonstrated that such cysteines indeed became available to thereagent when the cell was mechanically stressed, and that thesegroups were located in repeats of low stability.71 These findingssupport the concept that the unfolding and refolding of distinctspectrin repeats make a major contribution to the elasticity of thenormal red cell membrane.

During senescence, normal red cells lose surface area andvolume with little loss of hemoglobin and as a consequence celldensity progressively increases during the red cell’s 120-day life

span. Recent studies have documented that after extensive dehydra-tion, normal red cells as well as sickle red cells, lose their ability tomaintain their cation homeostasis and cell volume increases.72 Ithas been suggested that this cell population may represent the“end-stage” normal red cells destined to be eliminated from cellcirculation. Alternate postulated mechanisms for removal of senes-cent normal red cells include phagocytosis of senescent cells bymacrophages either through recognition of clustered band 3 or PSexposure on the outer monolayer of this cell population.73,74 Whileit is difficult to assign the relative contribution of each of thevarious documented cellular changes to removal of senescent redcells from circulation, it is likely that all of them play some role inthe process.

Thus, our understanding of the mechanistic basis for normal redcell deformability appears reasonably well developed and compre-hensive. We may anticipate that ongoing structural work aimed atdetermining the structures of major red cell membrane proteins atatomic resolution will provide more refined insights into normalred cell membrane structure and function.

Mechanistic basis for altered membrane and cell function ininherited red cell disorders

Inherited red cell disorders with altered membrane and cellfunction can be broadly divided into 2 classes. (1) Altered functiondue to mutations in various membrane or skeletal proteins. Suchconditions include hereditary spherocytosis (HS), hereditary ellip-tocytosis (HE), hereditary ovalocytosis, and hereditary stomatocy-tosis. (2) Altered function due to secondary effects on the mem-brane resulting from mutations in globin genes; these conditionsinclude sickle cell disease, Hb SC disease, Hb CC disease, unstablehemoglobins and thalassemias. A consistent feature of red cells inall of these disorders is decreased cell deformability. We willdiscuss briefly these various inherited disorders based on thedominant cellular features responsible for impaired cell deformabil-ity, and on our current understanding of the molecular andmechanistic basis for the documented cellular alterations.

Altered cell geometry. Decreased cell surface area to volumeratio and consequent increased cell sphericity is a distinguishingfeature of red cells in HS, HE, and overhydrated hereditarystomatocytosis (OHS). In the case of HS and HE, increasedsphericity is the result of loss of cell surface area while in the caseof OHS it results from increased cell volume.

HS is a common inherited hemolytic anemia affecting all ethnicgroups, but is particularly common in people of northern Europeanancestry (1 in 3000).75,76 HS is usually associated with dominantinheritance (75%), although nondominant inheritance (25%) is notuncommon. “Typical” HS is characterized by evidence of hemoly-sis with anemia, jaundice, splenomegaly, reticulocytosis, gall-stones, and the presence of spherocytes on peripheral blood smears.However, the clinical manifestations of HS are highly variableranging from mild to very severe anemia. A common feature of allforms of HS is loss of membrane surface area and resultant changein cell shape from discocytes to stomatocytes to spherocytes(Figure 4). As red cells with decreased membrane surface area areunable to effectively traverse the spleen, they are sequestered andremoved from circulation by the spleen. Importantly, the severity ofanemia is related to extent of decrease in membrane surface area.Splenectomy reduces the severity of anemia by increasing thesurvival of spherocytic red cells. The mechanistic basis formembrane loss in HS is decreased membrane cohesion due to a





reduced number of “vertical” linkages between bilayer and mem-brane skeleton.77 Reduced anchoring results from deficiencies oftransmembrane proteins that link the bilayer to the membraneskeleton (band 3 or RhAG), or of anchoring proteins (ankyrin orprotein 4.2) or of spectrin due to too little membrane skeletonavailable for linkage (Figure 5). Thus, HS is the result of defects ingenes encoding any of the protein components involved in verticallinkages between skeletal network and the membrane.75,78-83

HE is a relatively common, clinically and genetically heteroge-neous disorder, characterized by presence of elliptically shaped red

cells on peripheral blood smear.84 HE has a worldwide distribution,but is more common in malaria endemic regions with prevalenceapproaching 2% in West Africa.85 Inheritance of HE is autosomaldominant. The overwhelming majority of HE is asymptomatic butapproximately 10% of patients have moderate to severe anemiaincluding a few reported cases of hydrops fetalis. Typically, peopleheterozygous for an elliptocytic variant are asymptomatic whilepeople with homozygosity or compound heterozygosity for HEvariants experience mild to severe anemia, including the severevariant hereditary pyropoikilocytosis. A common feature of allforms of HE is a mechanically unstable membrane that results inprogressive transformation of cell shape from discocyte to ellipto-cyte with time in the circulation (Figure 4), and in severe cases,membrane fragmentation and cells with reduced membrane surfacearea (Figure 4). Importantly, the severity of the disease is related toextent of decrease in membrane mechanical stability and resultantloss of membrane surface area.86 While very few patients with HEneed splenectomy, as with HS, splenectomy reduces the severity ofanemia in patients with severe anemia by increasing the circulatorylife span of fragmented red cells. The mechanistic basis fordecreased membrane mechanical stability in HE is weakened“horizontal” linkages in membrane skeleton due either to defectivespectrin dimer-dimer interaction or a defective spectrin-actin-protein 4.1R junctional complex. Thus, HE is the result of defectsin genes encoding for �-spectrin, �-spectrin or protein 4.1R, all ofwhich are involved in “horizontal” linkages in the skeletal network(Figure 3).62,63,79,84,87-90

OHS is a rare disorder characterized by presence of largenumbers of stomatocytes on blood smears in association withmoderately severe to severe anemia.91 Inheritance pattern of OHSis autosomal dominant. The distinctive feature of red cells is theirincreased sphericity due to increased cell volume without concomi-tant increase in membrane surface area. Stomatocytes with in-creased sphericity are sequestered by the spleen. However, whilesplenectomy is highly beneficial in the management of HS and HEpatients, it is contraindicated in OHS, because it leads to increasedrisk of venous thromboembolic complications. The mechanisticbasis for increased volume is the cell’s inability to regulate itscation homeostasis. Stomatocytes exhibit a marked increase in totalcation content due to elevation of intracellular sodium, resulting inincreased cell volume. The molecular basis for OHS has not yetbeen defined.92,93

Increased cytoplasmic viscosity. Red cell dehydration andhence increased cytoplasmic viscosity is a feature of the inherited

Figure 4. Red cell morphology. Hereditary spherocytosis (HS; top panel); nonhemo-lytic hereditary elliptocytosis (HE; middle panel); elliptocytes, poikilocytes, andfragmented red cells in hemolytic HE (bottom panel).

Ankyrin

Protein 4.2

α-spectrin

β-spectrin

Band 3 Band 3

Rh

RhAG

Figure 5. Membrane defects in HS affect the “vertical” interactions anchoringthe membrane skeleton to the lipid bilayer. Deficiency in any one of the proteincomponents (band 3, RhAG, ankyrin, protein 4.2, or spectrin) involved in theanchoring process leads to HS. Illustration by Paulette Dennis.





red cell membrane disorder, dehydrated hereditary stomatocytosis(xerocytosis; DHS).91 Inheritance of DHS is autosomal dominant.The distinctive feature of DHS is the increased MCHC of red cellsas a consequence of decreased total cation content and loss of cellwater. However, in contrast to significantly compromised survivalof hydrated cells of OHS, cell dehydration has only a marginaleffect on survival of DHS red cells. DHS is therefore associatedwith well-compensated anemia with only a mild to moderatelyenlarged spleen. The mechanistic basis for decreased volume is thered cell’s inability to regulate cation homeostasis resulting in decreasedtotal cation content due to decreased intracellular potassium. Themolecular basis for DHS is, like OHS, still unknown.

Failure to regulate cell volume with ensuing cell dehydrationhas long been recognized as a feature of red cells in sickle celldisease, Hb SC disease and Hb CC disease.94,95 However, incontrast to DHS, in which cell dehydration is the only dominantfeature, red cells in hemoglobinopathies are not only dehydratedbut also exhibit significant membrane alterations, including in-creased membrane rigidity.67 The membrane disturbances are theresult of oxidation-induced structural reorganization. While theGardos channel and K-Cl cotransporter have been implicated in analtered state of hydration of red cells in hemoglobinopathies, themechanistic understanding of disordered volume regulation inthese disorders is far from complete.

Altered membrane deformability. Decreased membrane deform-ability is a distinguishing feature of red cells in hereditary ovalocytosis.96

Ovalocytosis is very common in SoutheastAsia where in malaria endemicareas its prevalence ranges from 5% to 25%.97 The condition is character-ized by the presence of oval-shaped red cells with 1 or 2 transverse ridgesor a longitudinal slit on blood smears. Inheritance of ovalocytosis isautosomal dominant and to date only heterozygotes have been identifiedin regions of high incidence implying that homozygosity is embryoniclethal.98 Membranes of ovalocytes are 4 to 8 times less elastic than normalmembranes as assessed by ektacytometry and by micropipette aspira-tion.96,99 In terms of clinical manifestations, it is important to note thatdespite a marked increase in membrane rigidity most affected peopleexperience minimal hemolysis. In all cases of ovalocytosis studied to date,only 1 mutation has been identified: a genomic deletion of 27 bp encodingamino acids 400 to 408 of band 3.100-102 Thus hereditary ovalocytosis isunique among red cell membrane disorders in that the same mutation in asingle gene is responsible for the morphologic phenotype. Althoughseveral hypotheses have been proposed regarding how a mutation inband 3 could lead to a marked increase in membrane rigidity, themechanism of the effect has yet to be established.

Our current understanding of molecular basis for inherited redcell membrane disorders, hereditary spherocytosis, hereditaryelliptocytosis and hereditary ovalocytosis is wide-ranging, yetthere are still cases where the molecular and genetic pathobiologyare unknown. In contrast, the molecular and genetic basis for redcell disorders due to membrane transport defects such as thedehydrated and overhydrated hereditary stomatocytosis syndromesare largely unknown, remaining a continuing challenge.

Future

Studies on the red cell membrane have shed much new, oftenunexpected light on structure and function of plasma membranes

generally, and have, we would argue justified the claims, so oftenmade, of its broad paradigmatic value. There can, at the same time,be no doubt as to the advances in hematology that have flowed fromthis work, for now we have a far-reaching understanding of thecauses of membrane defects in genetic diseases. As result of thesemany advances, it might be felt that we have a complete understand-ing of all aspects of red cell membrane structure and function andvery few new and novel insights will be forthcoming from futureendeavors. This narrow point of view ignores the great continuedpotential of red cell research as revealed by reports published in2008 that outlined unexpected new and novel insights into red cellmembrane structure and function. Furthermore, just a few ex-amples of the great unexhausted potential of red cell researchconcern: nature and function of macromolecular complexes in themembrane; dynamic regulation of skeletal protein and membraneprotein complexes by phosphorylation, and other posttranslationalmodifications, and by phosphoinositides; dynamics of assemblyand function of the membrane microdomains, for which there isnow strong evidence; molecular basis for cell volume regulation;detailed clarification of the sequence of events in regulatedassembly of plasma membrane, and its associated protein skeleton;regulation of membrane properties by biodynamic ligands, includ-ing adrenergic agents, nucleotides and hormones; the nature ofmembrane perturbations in diabetes and many other diseases; theproblems presented by membrane damage in sickle cell and relateddiseases; interaction of red cells with endothelial surfaces in healthand disease; and of course the many problems, basic and practical,presented by malaria. While it is clear these or related topics arecurrently under intensive investigation in many other cell types,because of its simplicity and elegance, it is very likely that the red cellwill continue to provide deep insights into these complex issues.

Acknowledgments

We very much regret that many important papers from several verytalented people who made significant contributions to red cellresearch could not be cited or discussed due to strict spacelimitations. We sincerely apologize for this omission. We gratefullyacknowledge the camaraderie and friendship of a very largenumber of investigators associated with red cell research who overthe years have enriched our careers in red cell research. This workis dedicated to the memory of Sally Marchesi, a dear friend and awonderful colleague.

This work was supported by the National Institutes of Health(DK26263, DK32094, and HL31579 to N.M.; DK62039 andHL65488 to P.G.G.).

Authorship

Contribution: N.M. and P.G.G. cowrote the paper.Conflict-of-interest disclosure: The authors declare no compet-

ing financial interests.Correspondence: Narla Mohandas, New York Blood Center,

310 East 67th Street, New York, NY 10065; e-mail: [email protected].

References

1. Van Leeuwenhoek A. Other microscopical obser-vations made by the same, about the texture ofthe blood, the sap of some plants, the figures ofsugar and salt, and the probable cause of the dif-

ference of their tastes. Philos Trans R Soc Lond.1675;10:380-385.

2. Bessis M, Delpech G. Discovery of the red bloodcell with notes on priorities and credits of discov-

eries, past, present and future. Blood Cells. 1981;7:447-480.

3. Gulliver G. Blood of Vertebrata. Medical Times andGazette. London: John Churchill & Sons; 1862.





4. Gorter E, Grendel F. On the bimolecular layers oflipids on the chromocytes of the blood. J ExpMed. 1925;41:439-443.

5. Singer SJ, Nicolson GL. The fluid mosaic modelof the structure of cell membranes. Science.1972;175:720-731.

6. Marchesi VT, Steers E Jr. Selective solubilizationof a protein component of the red cell membrane.Science. 1968;159:203-204.

7. Steck TL, Fairbanks G, Wallach DF. Dispositionof the major proteins in the isolated erythrocytemembrane. Proteolytic dissection. Biochemistry.1971;10:2617-2624.

8. Rand RP, Burton AC. Mechanical properties ofthe red cell membrane. I. membrane stiffness andintracellular pressure. Biophys J. 1964;4:115-135.

9. Mohandas N, Chasis JA, Shohet SB. The influ-ence of membrane skeleton on red cell deform-ability, membrane material properties, and shape.Semin Hematol. 1983;20:225-242.

10. Mohandas N, Chasis JA. Red blood cell deform-ability, membrane material properties and shape:regulation by transmembrane, skeletal and cyto-solic proteins and lipids. Semin Hematol. 1993;30:171-192.

11. Mohandas N, Evans E. Mechanical properties ofthe red cell membrane in relation to molecularstructure and genetic defects. Annu Rev BiophysBiomol Struct. 1994;23:787-818.

12. Discher DE. New insights into erythrocyte mem-brane organization and microelasticity. Curr OpinHematol. 2000;7:117-122.

13. Delaunay J. The molecular basis of hereditary redcell membrane disorders. Blood Rev. 2007;21:1-20.

14. Rybicki AC, Heath R, Lubin B, Schwartz RS. Hu-man erythrocyte protein 4.1 is a phosphatidylser-ine binding protein. J Clin Invest. 1988;81:255-260.

15. An X, Guo X, Sum H, Morrow J, Gratzer W, Mo-handas N. Phosphatidylserine binding sites inerythroid spectrin: location and implications formembrane stability. Biochemistry. 2004;43:310-315.

16. Walensky LD, Mohandas N, Lux SE. In: HandinRI, Lux SE, Stossel TP, eds. Blood, Principlesand Practice of Hematology (2nd edition). Phila-delphia, PA: Lippincott Williams & Wilkins; 2003:1726-1744.

17. Verkleij AJ, Zwaal RF, Roelofsen B, Comfurius P,Kastelijn D, van Deenen LL. The asymmetric dis-tribution of phospholipids in the human red cellmembrane. A combined study using phospho-lipases and freeze-etch electron microscopy. Bio-chim Biophys Acta. 1973;323:178-193.

18. Zwaal RF, Schroit AJ. Pathophysiologic implica-tions of membrane phospholipid asymmetry inblood cells. Blood. 1997;89:1121-1132.

19. Daleke DL. Regulation of phospholipid asymme-try in the erythrocyte membrane. Curr Opin He-matol. 2008;15:191-195.

20. Sims PJ, Wiedmer T. Unraveling the mysteries ofphospholipid scrambling. Thromb Haemost.2001;86:266-275.

21. Salzer U, Prohaska R. Stomatin, flotillin-1, andflotillin-2 are major integral proteins of erythrocytelipid rafts. Blood. 2001;97:1141-1143.

22. Murphy SC, Samuel BU, Harrison T, et al. Eryth-rocyte detergent-resistant membrane proteins:their characterization and selective uptake duringmalarial infection. Blood. 2004;103:1920-1928.

23. Wood BL, Gibson DF, Tait JF. Increased erythro-cyte phosphatidylserine exposure in sickle celldisease: flow-cytometric measurement and clini-cal associations. Blood. 1996;88:1873-1880.

24. Yasin Z, Witting S, Palascak MB, Joiner CH,Rucknagel DL, Franco RS. Phosphatidylserineexternalization in sickle red blood cells: associa-tions with cell age, density, and hemoglobin F.Blood. 2003;102:365-370.

25. Kuypers FA, Yuan J, Lewis RA, et al. Membranephospholipid asymmetry in human thalassemia.Blood. 1998;91:3044-3051.

26. Setty BN, Kulkarni S, Stuart MJ. Role of erythro-cyte phosphatidylserine in sickle red cell-endo-thelial adhesion. Blood. 2002;99:1564-1571.

27. Manno S, Takakuwa Y, Mohandas N. Identifica-tion of a functional role for lipid asymmetry in bio-logical membranes: Phosphatidylserine-skeletalprotein interactions modulate membrane stability.Proc Natl Acad Sci U S A. 2002;99:1943-1948.

28. An X, Debnath G, Guo X, et al. Identification andfunctional characterization of protein 4.1R andactin-binding sites in erythrocyte beta spectrin:regulation of the interactions by phosphatidylino-sitol-4,5-bisphosphate. Biochemistry. 2005;44:10681-10688.

29. An X, Zhang X, Debnath G, Baines AJ, Mohan-das N. Phosphatidylinositol-4,5-biphosphate(PIP2) differentially regulates the interaction ofhuman erythrocyte protein 4.1 (4.1R) with mem-brane proteins. Biochemistry. 2006;45:5725-5732.

30. Harrison T, Samuel BU, Akompong T, et al. Eryth-rocyte G protein-coupled receptor signaling inmalarial infection. Science. 2003;301:1734-1736.

31. Reid ME, Mohandas N. Red blood cell bloodgroup antigens: structure and function. SeminHematol. 2004;41:93-117.

32. Anstee DJ. Blood group antigens defined by theamino acid sequences of red cell surface pro-teins. Transfus Med. 1995;5:1-13.

33. Telen MJ. Erythrocyte blood group antigens: notso simple after all. Blood. 1995;85:299-306.

34. Cartron JP, Colin Y. Structural and functional di-versity of blood group antigens. Transfus ClinBiol. 2001;8:163-199.

35. Bennett V. Proteins involved in membrane–cy-toskeleton association in human erythrocytes:spectrin, ankyrin, and band 3. Methods Enzymol.1983;96:313-324.

36. Nicolas V, Le Van Kim C, Gane P, et al. Rh-RhAG/ankyrin-R, a new interaction site betweenthe membrane bilayer and the red cell skeleton, isimpaired by Rh(null)-associated mutation. J BiolChem. 2003;278:25526-25533.

37. Reid ME, Takakuwa Y, Conboy J, Tchernia G, Mo-handas N. Glycophorin C content of human eryth-rocyte membrane is regulated by protein 4.1.Blood. 1990;75:2229-2234.

38. Marfatia SM, Lue RA, Branton D, Chishti AH. Invitro binding studies suggest a membrane-asso-ciated complex between erythroid p55, protein4.1, and glycophorin C. J Biol Chem. 1994;269:8631-8634.

39. Salomao M, Zhang X, Yang Y, et al. Protein 4.1R-dependent multiprotein complex: new insightsinto the structural organization of the red bloodcell membrane. Proc Natl Acad Sci U S A. 2008;105:8026-8031.

40. Khan AA, Hanada T, Mohseni M, et al. Dematinand adducin provide a novel link between thespectrin cytoskeleton and human erythrocytemembrane by directly interacting with glucosetransporter-1. J Biol Chem. 2008;283:14600-14609.

41. Bruce LJ, Beckmann R, Ribeiro ML, et al. A band3-based macrocomplex of integral and peripheralproteins in the RBC membrane. Blood. 2003;101:4180-4188.

42. Bennett V. The spectrin-actin junction of erythro-cyte membrane skeletons. Biochim Biophys Acta.1989;988:107-121.

43. Bennett V, Baines AJ. Spectrin and ankyrin-basedpathways: metazoan inventions for integratingcells into tissues. Physiol Rev. 2001;81:1353-1392.

44. Mohandas N, An X. New insights into function ofred cell membrane proteins and their interaction

with spectrin-based membrane skeleton. Trans-fus Clin Biol. 2006;13:29-30.

45. Speicher DW, Marchesi VT. Erythrocyte spectrinis comprised of many homologous triple helicalsegments. Nature. 1984;311:177-180.

46. Djinovic-Carugo K, Gautel M, Ylanne J, Young P.The spectrin repeat: a structural platform for cy-toskeletal protein assemblies. FEBS Lett. 2002;513:119-123.

47. An X, Guo X, Zhang X, et al. Conformational sta-bilities of the structural repeats of erythroid spec-trin and their functional implications. J Biol Chem.2006;281:10527-10532.

48. DeSilva TM, Peng KC, Speicher KD, SpeicherDW. Analysis of human red cell spectrin tetramer(head-to-head) assembly using complementaryunivalent peptides. Biochemistry. 1992;31:10872-10878.

49. Speicher DW, DeSilva TM, Speicher KD, UrsittiJA, Hembach P, Weglarz L. Location of the hu-man red cell spectrin tetramer binding site anddetection of a related “closed” hairpin loop dimerusing proteolytic footprinting. J Biol Chem. 1993;268:4227-4235.

50. Lecomte MC, Nicolas G, Dhermy D, Pinder JC,Gratzer WB. Properties of normal and mutantpolypeptide fragments from the dimer self-asso-ciation sites of human red cell spectrin. Eur Bio-phys J. 1999;28:208-215.

51. An X, Lecomte MC, Chasis JA, Mohandas N,Gratzer W. Shear-response of the spectrin dimer-tetramer equilibrium in the red blood cell mem-brane. J Biol Chem. 2002;277:31796-31800.

52. Ungewickell E, Bennett PM, Calvert R, OhanianV, Gratzer WB. In vitro formation of a complexbetween cytoskeletal proteins of the humanerythrocyte. Nature. 1979;280:811-814.

53. Karinch AM, Zimmer WE, Goodman SR. Theidentification and sequence of the actin-bindingdomain of human red blood cell beta-spectrin.J Biol Chem. 1990;265:11833-11840.

54. Ohanian V, Wolfe LC, John KM, Pinder JC, LuxSE, Gratzer WB. Analysis of the ternary interac-tion of the red cell membrane skeletal proteinsspectrin, actin, and 4.1. Biochemistry. 1984;23:4416-4420.

55. Fowler VM. Regulation of actin filament length inerythrocytes and striated muscle. Curr Opin CellBiol. 1996;8:86-96.

56. Kuhlman PA, Hughes CA, Bennett V, Fowler VM.A new function for adducin. Calcium/calmodulin-regulated capping of the barbed ends of actin fila-ments. J Biol Chem. 1996;271:7986-7991.

57. Fischer RS, Fowler VM. Tropomodulins: life at theslow end. Trends Cell Biol. 2003;13:593-601.

58. Siegel DL, Branton D. Partial purification andcharacterization of an actin-bundling protein,band 4.9, from human erythrocytes. J Cell Biol.1985;100:775-785.

59. Pasini EM, Kirkegaard M, Mortensen P, Lutz HU,Thomas AW, Mann M. In-depth analysis of themembrane and cytosolic proteome of red bloodcells. Blood. 2006;108:791-801.

60. Liu SC, Palek J. Spectrin tetramer-dimer equilib-rium and the stability of erythrocyte membraneskeletons. Nature. 1980;285:586-588.

61. Chasis JA, Mohandas N. Erythrocyte membranedeformability and stability: two distinct membraneproperties that are independently regulated byskeletal protein associations. J Cell Biol. 1986;103:343-350.

62. Marchesi SL, Conboy J, Agre P, et al. Molecularanalysis of insertion/deletion mutations in protein4.1 in elliptocytosis. I. Biochemical identificationof rearrangements in the spectrin/actin bindingdomain and functional characterizations. J ClinInvest. 1990;86:516-523.

63. Tchernia G, Mohandas N, Shohet S. Deficiency ofskeletal membrane protein band 4.1 in homozy-gous hereditary elliptocytosis. Implications for





erythrocyte membrane stability. J Clin Invest.1981;68:454-460.

64. Giorgi M, Cianci CD, Gallagher PG, Morrow JS.Spectrin oligomerization is cooperatively coupledto membrane assembly: a linkage targeted bymany hereditary hemolytic anemias? Exp MolPathol. 2001;70:215-230.

65. Bennett V. Spectrin: a structural mediator be-tween diverse plasma membrane proteins andthe cytoplasm. Curr Opin Cell Biol. 1990;2:51-56.

66. Cokelet GR, Meiselman HJ. Rheological compari-son of hemoglobin solutions and erythrocyte sus-pensions. Science. 1968;162:275-277.

67. Evans E, Mohandas N, Leung A. Static and dy-namic rigidities of normal and sickle erythrocytes.Major influence of cell hemoglobin concentration.J Clin Invest. 1984;73:477-488.

68. Brugnara C. Erythrocyte membrane transportphysiology. Curr Opin Hematol. 1997;4:122-127.

69. Milanick MA, Hoffman JF. Ion transport and vol-ume regulation in red blood cells. Ann N Y AcadSci. 1986;488:174-186.

70. Lew VL, Bookchin RM. Ion transport pathology inthe mechanism of sickle cell dehydration. PhysiolRev. 2005;85:179-200.

71. Johnson CP, Tang HY, Carag C, Speicher DW,Discher DE. Forced unfolding of proteins withincells. Science. 2007;317:663-666.

72. Bookchin RM, Etzion Z, Sorette M, Mohandas N,Skepper JN, Lew VL. Identification and character-ization of a newly recognized population of high-Na�, low-K�, low-density sickle and normal redcells. Proc Natl Acad Sci U S A. 2000;97:8045-8050.

73. Low PS, Waugh SM, Zinke K, Drenckhahn D. Therole of hemoglobin denaturation and band 3 clus-tering in red blood cell aging. Science. 1985;227:531-533.

74. Turrini F, Mannu F, Arese P, Yuan J, Low PS.Characterization of the autologous antibodiesthat opsonize erythrocytes with clustered integralmembrane proteins. Blood. 1993;81:3146-3152.

75. Eber S, Lux SE. Hereditary spherocytosis–de-fects in proteins that connect the membrane skel-eton to the lipid bilayer. Semin Hematol. 2004;41:118-141.

76. Gallagher PG, Ferriera JD. Molecular basis oferythrocyte membrane disorders. Curr Opin He-matol. 1997;4:128-135.

77. Butler J, Mohandas N, Waugh RE. Integral pro-tein linkage and the bilayer-skeletal separationenergy in red blood cells. Biophys J. 2008;95:1826-1836.

78. Jarolim P, Murray JL, Rubin HL, et al. Character-ization of 13 novel band 3 gene defects in heredi-tary spherocytosis with band 3 deficiency. Blood.1996;88:4366-4374.

79. Palek J, Lux SE. Red cell membrane skeletal de-fects in hereditary and acquired hemolytic ane-mias. Semin Hematol. 1983;20:189-224.

80. Agre P, Casella JF, Zinkham WH, McMillan C,Bennett V. Partial deficiency of erythrocyte spec-trin in hereditary spherocytosis. Nature. 1985;314:380-383.

81. Lux SE, Tse WT, Menninger JC, et al. Hereditaryspherocytosis associated with deletion of humanerythrocyte ankyrin gene on chromosome 8. Na-ture. 1990;345:736-739.

82. Eber SW, Gonzalez JM, Lux ML, et al. Ankyrin-1mutations are a major cause of dominant and re-cessive hereditary spherocytosis. Nat Genet.1996;13:214-218.

83. Hassoun H, Vassiliadis JN, Murray J, et al. Char-acterization of the underlying molecular defect inhereditary spherocytosis associated with spectrindeficiency. Blood. 1997;90:398-406.

84. Gallagher PG. Hereditary elliptocytosis: spectrinand protein 4.1R. Semin Hematol. 2004;41:142-164.

85. Dhermy D, Schrevel J, Lecomte MC. Spectrin-based skeleton in red blood cells and malaria.Curr Opin Hematol. 2007;14:198-202.

86. Lane PA, Shew RL, Iarocci TA, Mohandas N,Hays T, Mentzer WC. Unique alpha-spectrin mu-tant in a kindred with common hereditary ellipto-cytosis. J Clin Invest. 1987;79:989-996.

87. Delaunay J, Dhermy D. Mutations involving thespectrin heterodimer contact site: clinical expres-sion and alterations in specific function. SeminHematol. 1993;30:21-33.

88. Coetzer TL, Palek J. Partial spectrin deficiency inhereditary pyropoikilocytosis. Blood. 1986;67:919-924.

89. Gaetani M, Mootien S, Harper S, Gallagher PG,Speicher DW. Structural and functional effects ofhereditary hemolytic anemia-associated pointmutations in the alpha spectrin tetramer site.Blood. 2008;111:5712-5720.

90. Johnson CP, Gaetani M, Ortiz V, et al. Pathogenicproline mutation in the linker between spectrinrepeats: disease caused by spectrin unfolding.Blood. 2007;109:3538-3543.

91. Delaunay J. The hereditary stomatocytoses: ge-netic disorders of the red cell membrane perme-ability to monovalent cations. Semin Hematol.2004;41:165-172.

92. Bruce LJ, Robinson HC, Guizouarn H, et al.Monovalent cation leaks in human red cellscaused by single amino-acid substitutions in thetransport domain of the band 3 chloride-bicarbon-ate exchanger, AE1. Nat Genet. 2005;37:1258-1263.

93. Iolascon A, Perrotta S, Stewart GW. Red bloodcell membrane defects. Rev Clin Exp Hematol.2003;7:22-56.

94. Fabry ME, Kaul DK, Raventos C, Baez S, RiederR, Nagel RL. Some aspects of the pathophysiol-ogy of homozygous Hb CC erythrocytes. J ClinInvest. 1981;67:1284-1291.

95. Embury SH, Clark MR, Monroy G, Mohandas N.Concurrent sickle cell anemia and alpha-thalas-semia. Effect on pathological properties of sickleerythrocytes. J Clin Invest. 1984;73:116-123.

96. Mohandas N, Lie-Injo LE, Friedman M, Mak JW.Rigid membranes of Malayan ovalocytes: a likelygenetic barrier against malaria. Blood. 1984;63:1385-1392.

97. Amato D, Booth PB. Hereditary ovalocytosis inMelanesians. P N G Med J. 1977;20:26-32.

98. Liu SC, Jarolim P, Rubin HL, et al. The homozy-gous state for the band 3 protein mutation inSoutheast Asian Ovalocytosis may be lethal.Blood. 1994;84:3590-3591.

99. Mohandas N, Winardi R, Knowles D, et al. Mo-lecular basis for membrane rigidity of hereditaryovalocytosis. A novel mechanism involving thecytoplasmic domain of band 3. J Clin Invest.1992;89:686-692.

100. Jarolim P, Palek J, Amato D, et al. Deletion inerythrocyte band 3 gene in malaria-resistantSoutheast Asian ovalocytosis. Proc Natl Acad SciU S A. 1991;88:11022-11026.

101. Mohandas N. Molecular basis for red cell mem-brane viscoelastic properties. Biochem SocTrans. 1992;20:776-782.

102. Schofield AE, Tanner MJ, Pinder JC, et al. Basisof unique red cell membrane properties in heredi-tary ovalocytosis. J Mol Biol. 1992;223:949-958.





Narla Mohandas

I arrived in the United States in 1968 with a degree in chemical engineering to begin graduate stud-ies at the School of Engineering at Washington University, St Louis. It was there it all started—andnot the way I had envisioned. My thesis supervisor, Robert Hochmuth, introduced me to the fieldof red cell research. It became my first professional love and I have stayed faithful to it ever since.After completing my DSc, I spent 3 formative years in Paris working with Marcel Bessis. Using theektacytometer that we developed together, we found interesting differences in the deformability ofred cells in a variety of disorders. Realizing that my engineering background had not equipped mefor a broad understanding of the pathophysiologic implications of the phenomenon I was study-ing, in 1976 I began to look for collaborators with expertise in hematology, biochemistry, biophys-ics, cell biology, molecular biology, and genetics. This journey, spanning the past 3 decades, hasbeen extraordinarily rewarding both professionally and personally. During these years I have hadthe privilege of working closely with an array of wonderful colleagues who have become closefriends. I would like especially to acknowledge my collaborators of many years, Xiuli An, Joel AnnChasis, Margaret Clark, John Conboy, Ross Coppel, Dennis Discher, Evan Evans, Gil Tchernia,and Yuichi Takakuwa, and my many good friends, always generous with their support and help,including Peter Agre, David Anstee, Anthony Baines, Jane Barker, Vann Bennett, David Bodine,Pat Gallagher, Walter Gratzer, Kasturi Haldar, Bob Hebbel, Joe Hoffman, Phil Low, Sam Lux, JonMorrow, Luanne Peters, and Stan Schrier. A happy feature of the red cell field is its collegiality, andit has been a particular pleasure to associate with so many excellent scientists, whose friendshipand support has meant so much to me. My fascination with red cells is still unabated after devot-ing the past 4 decades of my professional life to it.

Patrick G. Gallagher

The little boy with sickle cell disease in Room 316A was hospitalized with the diagnosis of “im-pending crisis.” “What did his peripheral smear show?” Dr Beatrice Lampkin asked me on myfourth day of internship at Cincinnati Children’s Hospital. “Let’s go look.” Listening to her de-scribe the morphology and its significance and discuss the clinical manifestations and pathobiol-ogy of his disease and what it meant to this boy on a daily basis was a life lesson in hematology.People in the field of hematology care, they are thoughtful, they want to understand how thingswork, and they want to improve people’s lives. I also learned another lesson of hematology: youcan learn a lot more sitting around a microscope with a group of hematologists than just what’s onthe peripheral smear. After residency, I moved to Yale University for fellowship training, where Ibegan training in the laboratory of Dr Bernard Forget. The research training I received from Berniewas priceless, going from experimental conception, design, execution, and interpretation to pre-sentation. Group meetings in the Hematology Division at Yale at that time were exciting, with pre-sentations on hemoglobin switching, gene expression, receptor signaling, gene structure andfunction, membrane biology, and inherited and acquired human disease, which instilled an inter-est in a broad range of topics that continues today. I also greatly benefitted from William Tse’slaboratory tutelage, for which I am most grateful. I think of my experiences in hematology asvignettes. Bernie Forget coaching the art of peer review: “First, always say something nice.”Watching the influence of Edward Benz’s smile and encouraging words on the people around him.Sitting in an airport listening to Peter Agre comparing academics to drug addiction, explainingthat no matter the ups and downs, we just love academics and can’t stop. Introducing myself toSamuel Lux when he was visiting New Haven; he replied, “I know who you are. Are you putting in aK?” Meeting my collaborator and friend David Bodine at a Gordon Conference reception to dis-cuss erythroid-specific promoters, and the Red Sox. David Nathan rescuing me from a toughquestioner at a Children’s Health Research Center meeting. Reading the name badge of the kindman who came over to talk to me about my poster at ASH while I was a fellow: “Mohandas Narla” it

read. Mohan is now a great friend and colleague and I am proud to coauthor this review with him. I have found the field of hematology to be exciting, col-legial, and compassionate. I look forward to many, many more years.





membrana eritrocitaria

Documents