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Interactions between integral membrane proteins and the cytoskeleton in red blood cells studied by measured… Knowles, David W. 1992

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We accept this thesis as conformingto the required standardTHE UNIVERSITY OF BRITISH COLUMBIAMarch 1992Interactions Between Integral Membrane Proteinsand the Cytoskeleton in Red Blood Cells Studied byMeasured Molecular Lateral DiffusionbyDavid William KnowlesB.Sc.-Hons., University of New South Wales, Australia, 1982M.Sc., University of British Columbia, Canada, 1986A THESIS SUBMITTED IN PARTIAL FULFILMENT OFTHE REQUIREMENTS FOR THE DEGREE OFDOCTOR OF PHILOSOPHYinTHE FACULTY OF GRADUATE STUDIESDEPARTMENT OF PHYSICS© David William Knowles, 1992In presenting this thesis in partial fulfilment of the requirements for an advanceddegree at the University of British Columbia, I agree that the Library shall make itfreely available for reference and study. I further agree that permission for extensivecopying of this thesis for scholarly purposes may be granted by the head of mydepartment or by his or her representatives. It is understood that copying orpublication of this thesis for financial gain shall not be allowed without my writtenpermission.(Signature)Department of Physics The University of British ColumbiaVancouver, CanadaDate ^le September 1992DE-6 (2/88)AbstractIntegral membrane receptor proteins are involved in many aspects of biological function.These proteins are restricted to the lipid bilayer but can move laterally within it and in-teract with specific agents which control their functional activity. The agents, collectivelytermed ligands, are commonly other molecules, which vary in size from solubilized ions tolarge macromolecules. Receptor/ligand interactions can influence lateral organization andconformation of receptor proteins and are the mechanism by which biological signals arepassed across the membrane and from cell to cell.A single receptor protein embedded within a pure lipid bilayer has considerable lateral mo-bility, which is restricted only by the viscosity of the bilayer, and to a lesser extent, by theviscosity of the surrounding aqueous environment [Saffman & Delbruck 1975]. Cell mem-branes however, are comprised of many receptor proteins as well as a network of supportproteins which closely associate with the bilayer on its cytoplasmic side. Consequently,receptor-protein lateral mobility is considerably lower in membranes than in lipid bilayers,presumably because of weak non-covalent interactions with peripheral membrane compo-nents. Thus the cell membrane, like many biological systems, is a set of weakly and dynam-ically coupled components.To understand the nature of such dynamic coupling, the effect of receptor/ligand interac-tions on receptor lateral motion and membrane rigidity was studied in a model-cell system.The model system chosen was the red blood cell membrane because it contains several welli icharacterized integral membrane proteins plus a peripheral cytoskeletal network. The majorintegral proteins are band 3 and several different types of glycophorin. The major cy-toskeletal protein is a long (76-200nm) filamentous tetramer known as spectrin. The ligandsused were monoclonal antibodies (divalent) and their Fab fragments (monovalent). The anti-bodies recognize specific epitopes on the extracellular domain of glycophorin A, the majorred cell sialoglycoprotein. Ligand 9A3 binds the most distal epitope of the extracellular do-main, ligands R10 and 10F7 bind to the mid-region of the extracellular domain and ligandB14 binds close to the lipid bilayer on the extracellular side of glycophorin A.Results from earlier studies [Chasis et al 1988] demonstrated that when these ligands (9A3,R10, 10F7 & B14) are bound to the extracellular domain of glycophorin A, membranedeformability (measured by flow ectacytometry) was greatly reduced in normal red cells, butnot in red cell variants that lack the cytoplasmic domain of glycophorin A (Miltenberger Vred cells). Because the cytoskeletal network (rather than the lipid bilayer) is responsible formembrane rigidity, these results suggested that ligand binding can initiate a transmembranesignal that causes an increased interaction of the cytoplasmic domain of the receptor withthe underlying cytoskeletal network.To test this hypothesis and to investigate the molecular features of such a mechanism, thelateral motion of red cell integral receptor proteins (glycophorin A, glycophorin C &band 3) and the mechanical rigidity of the membrane were measured in relation to theamount of ligand (eg. 9A3, R10, 10F7 or B14) bound to the extracellular domain of gly-cophorin A in both normal and variant red blood cells. Lateral motion (lateral mobilityi i iand mobile fraction) of the integral proteins was measured in situ by the technique of flu-orescence recovery after photobleaching (FRAP). This technique involves: (1) quantitatingthe fluorescence intensity from labels conjugated to the ligand or receptor of interest on themembrane; (2) irreversibly bleaching a small region of the membrane and (3) measuring thesubsequent fluorescence recovery due to lateral motion of fluorophore on the surface. Redcell membrane rigidity was measured by the method of pipette aspiration where a portionof the red cell membrane was deformed into a pipette by a minute suction pressure. Thedependence of aspiration length on applied pressure is linear for elastic deformations andresults in an effective rigidity modulus which is a combination of the elastic shear and thearea expansion moduli of the membrane cytoskeleton.The findings are that anti-glycophorin A ligands (R10 & B14), bound to normal red cellmembranes, cause the immobilization of both glycophorin A and band 3, and producea marked increase in membrane rigidity. These effects are due to an increased interactionbetween the cytoplasmic domain of the integral proteins and the cytoskeletal network. Thiswas evident for several reasons: (1) the effect was independent of ligand valency, indicatingthat extracellular cross-linking was not responsible for the immobilization; (2) lateral immo-bilization and increased rigidity were not seen if ligand was bound to Miltenberger V redcells (In these cells the cytoplasmic domain of glycophorin A is effectively absent due tonatural mutation); (3) only partial immobilization and a small increase in membrane rigiditywas seen when ligand was bound to spherocytic red cells (These cells are characterized by asevere spectrin deficiency which produces gross defects in the normal hexagonal structure ofthe cytoskeleton). Immobilization of band 3 resulted only for anti-glycophorin A ligandsivR10 (10F7) and B14. These ligands bind close to the bilayer surface and produce the great-est increase in membrane rigidity. Ligand 9A3 however, had less of an immobilizing effecton glycophorin A, had little effect on membrane rigidity and did not immobilize band 3.In light of the strong correlation between increased membrane rigidity and immobilization ofband 3, and because the cytoplasmic domain of glycophorin A is a small peptide, it is pro-posed that the cytoplasmic domain of ligand bound glycophorin A interacts (directly) withband 3 to cause immobilization of both integral proteins, and increased membrane rigidity.Finally, this proposed link between band 3 immobilization and the increased membranerigidity was tested in the case of ovalocytic red cells which are characterized by a toleranceof malarial invasion, and an increased membrane rigidity. It has been long assumed thatthe high rigidity of these cells, which is responsible for their malaria parasite tolerance, isdue to a mutation in one of the skeletal proteins. No such mutation has ever been found.However, this study shows that the entire band 3 population is immobile in these cells. Aparallel collaborative study shows that ovalocytic band 3 contains a mutation, a deletionof 9 amino acids in its cytoplasmic domain [Mohandas et al 1992]. Thus, it is apparent thatthe interaction of band 3 with the underlying cytoskeleton plays a crucial role in the de-termination of membrane mechanical properties, and the lateral motion of integral receptorproteins, and serves as a useful model for the understanding of the molecular mechanismsinvolved in receptor protein function.vContentsAbstract^ iiContents^ viList of Figures^ ixList of Tables^ xAcknowledgements^ xii1 Introduction 11.1 Red Cell Membrane Organization ^ 11.2 Laterally Motion of Integral Membrane Proteins ^ 102 Experimental Methods 172.1 The FRAP Technique ^ 172.2 Membrane Rigidity Measurements^ 202.3 Membrane Labelling ^ 202.4 F.R.A.P. Equipment 232.4.1^Optics ^ 232.4.2^Intensified Camera, Video Board and PMT^ 282.4.3^Analysis of Recovery Kinetics ^ 302.4.4^Spot Size Determination 322.5 Red Cell Membrane Elastic Rigidity ^ 34vi3 Experiments and Results^ 363.1 Ligand-Induced Lateral Immobilization of Glycophorin A: Implication of Cy-toskeletal Entanglement ^  363.1.1 Ligand-Induced Changes of Glycophorin A Lateral Motion in NormalRed Cells ^  373.1.2 Lateral Motion of Glycophorin in Miltenberger V Red Cells ^ 393.1.3 Lateral Motion of Glycophorin A in Hereditary Spherocytic Red Cells 413.1.4 Summary ^  423.2 Integral Membrane Proteins Play a Key Role in Membrane Rigidity ^ 453.2.1 Integral Protein Lateral Motion and Membrane Rigidity in Membranesof Normal and Miltenberger V Red Cells ^  463.2.2 Membrane Rigidity in Hereditary Spherocytic Red Cells ^ 483.2.3 Lateral Motion of Glycophorin A and Band 3, and Membrane Rigidityin Hereditary Ovalocytosis Red Cells ^  493.2.4 Summary ^  503.3 Ligand-Induced Interaction Between Glycophorin A and the Cytoskeleton inHuman Erythrocytes Involve Band 3 Protein ^  533.3.1 The lateral motion of band 3 in normal red cell membranes altered byligands bound to glycophorin A ^  543.3.2 The lateral motion of glycophorin C in normal red cell membranesaltered by ligands to glycophorin A ^  553.3.3 The lateral motion of band 3 in Miltenberger V red cell membranesaltered by ligands to glycophorin A ^  563.3.4 Summary ^  57vii4 Discussion and Conclusion^ 594.1 Receptor/Ligand Interactions in Normal Red Cell Membranes ^ 604.2 Receptor-Ligand Interactions in Miltenberger V Red Cell Membranes^634.3 Receptor/Ligand Interactions in HS Red Cell Membranes ^ 644.4 The Mobility of Untethered Receptors ^  664.5 Receptor Motion in Ovalocytic Red Cell Membranes^  674.6 Conclusion ^  68References^ 69viiiList of Figures1 Structural Organization of the Red Blood Cell Membrane^ 42 Structural Organization of the Hexagonal Lattice of the Red Blood Cell Mem-brane ^ 63 Fluorescence Recovery of Photobleached Glycophorin on the Red Cell Surface 184 Photograph of the FRAP Equipment ^ 235 Laser Based Epi-Fluorescence Microscope and Microprocessor Controlled Pho-ton Counting and Intensified Image Collection ^ 246 Double Block Beam Splitter & Attenuator 267 Correct Block Alignment Eliminates Non-Parallelism Caused by Block WedgeAngle ^ 268 Epi-Fluorescence Optics ^ 289 Absorption and Emission Bands of Eosin ^ 2910 Fluorescence Recovery of ETSC Labelled Glycophorin^ 3211 Gaussian Intensity Profile Convolved with the Point Spread Function of theObjective ^ 3312 Pipette Aspiration Measures Membrane Rigidity ^ 3413 Antibody Binding Epitopes on Glycophorin A 37ixList of Tables1^The Major Proteins of the Erythrocyte Membrane ^32^Measured Diffusivity of Lipid and Integral Proteins in Bilayer Vesicles andRed Cell Membranes ^  143^Alteration of Lateral Motion of ETSC-Labelled Glycophorin A in Normal RedBlood Cells: The Effect of Ligand Binding ^  384^Alteration of Unlabelled Glycophorin A in Normal Red Blood Cells: TheEffect of Binding Labelled Ligand ^  395^Alteration of Lateral Motion of Hybrid Glycophorin in Miltenberger V RedCells: The Effect of Ligand Binding ^  406^Alteration of Lateral Motion of ETSC-Labelled Glycophorin A in HereditarySpherocytosis Red Cells: The Effect of Spectrin Deficiency ^ 427^Alterations of Lateral Motion of Glycophorin A in Hereditary SpherocyticRed Cells Produced by Saturation Binding of Labelled Ligands ^ 428^Alteration of Rigidity of Normal Red Cell Membranes by Ligands to Gly-cophorin A^  479^Alteration of Rigidity of Normal Red Cell Membranes by Ligands to Gly-cophorin C ^  4810 Alteration of Membrane Rigidity in Miltenberger V Red Cells by Ligands toGlycophorin ^  4811 Alteration of Membrane Rigidity in Hereditary Spherocytic Red Cells by Lig-ands to Glycophorin A ^  4912 Lateral Motion of ETSC-labelled Glycophorin A and EMA-Labelled Band 3in, and Membrane Rigidity of Ovalocytic Red Cell Membranes ^ 5013 Alteration of EMA-Labelled Band 3 Motion in Normal Red Cells by Ligandsto Glycophorin A ^  5514 Lateral Motion of Glycophorin C Bound with Fluorescently labelled Bric-10in Normal Red Cells Altered by Unlabelled Ligands to Glycophorin A . . . . 56x15 Lateral Motion of EMA-labelled Band 3 in Miltenberger V Red Cells Alteredby Unlabelled Ligands to Glycophorin A ^  57xiAcknowledgementsWith great pleasure I thank:Dr. Evan A. Evans, research director,Dr Mohandas Narla for his constant enthusiasm, many helpfuldiscussions and who, along with his colleague Dr. Joel Chasis,supplied the antibodies, the variant red cells and without whomthis work would not have been done,Andrew Leung for his experimental and technical excellence,The rest of our (extended) lab: David Needham, who has followedmy progress and given so much sound advice; David Berk, DanKlingenberg and John Ipsen for many helpful discussions; TonyYeung for many supportive discussions and lamb chops; WieslawaRawicz for many a wonderful coffee; Barbara Kukan for showingme, faced with almost certain doom, how to "have it all"; andAndreas Zilker whose positive attitude helped me keep it all inperspective and for help with the diagrams. Thanks also to thegroups of Myer Bloom (Physics), Pieter Cullis (Pharmacology) andMike Wortis (Physics, SFU) whose participation made the summerseminars of 1991 such a success and from which I learnt a greatdeal,Peter Hass and the Physics machine shop technicians, whoes abilityto turn a rough sketch into beautiful reality is world class,Jim Sibley and Rolf Muelchen in the Pathology workshop, for alltheir work and borrowed tools,Lore Hoffmann, Physics Graduate Secretary and Secretary to theHead, for much help with bureaucratic matters and keeping us gradstudents in line,Frances, for her love and encouragement,Wayne, for his gentle nature and all his hugs,Lee, Lou, Justy, Carol, Lucie, Jacky, Claire, Kim, May and Elsiefor so much support,And, of course, 'The Girls of the Five'.1 Introduction1.1 Red Cell Membrane OrganizationThe red cell membrane can be divided based on structure into 3 regions: the glycocalyx,the bilayer and the cytoskeletal network (Fig.1). The membrane is also composed of 3 func-tionally different components: lipids, integral (or intrinsic) proteins and extrinsic proteins.Lipids are the smaller of the amphiphilic macromolecules which assemble into a planar bilayer( ,--5nm thick), in which they are free to move rotationally and laterally. The lipids of thered cell membrane include phospholipids, namely phosphatidyl -choline (PC), -ethanolamine(PE), -serine (PS), sphingomyelin and a neutral sterol, cholesterol. Distribution of phospho-lipid is asymmetric with most of the PC and sphingomyelin residing in the outer layer andmost of the PE and PS in the inner layer [Marchesi Furthmayr 1976] of the red cellmembrane.Extrinsic proteins in the red cell membrane are adjacent to the bilayer and form the support-ing cytoskeletal network. These proteins are so named because of their ease of extractionby the use of low ionic strength buffers [Yu et al 1973]. Once extracted, the proteins areclassified by differences in their electrophoretic migration through polymer gels' [Fairbanks'The major method of red cell membrane protein classification is sodium dodecyl sulphate polyacrylamidegel electrophoresis (SDS-PAGE) [Fairbanks et al 1971]. Proteins are dissociated by the binding of the anioicdetergent, SDS. The amount of SDS bound and the resulting charge depends on the mass of the protein, thuselectrophoretic migration through polyacrylamide gels separates proteins into bands according to their mass.Several techniques can then be used for protein analysis. The proteins can be stained with coomassie blue,known molecular weight markers can be added for relative molecular weight measures, sialoglycoproteinsare stained using Periodic acid and Schiff reagent (PAS), or the proteins can be specifically labelled prior toelectrophoresis.1et al 1971] which separates the proteins into bands. The major red cell cytoskeletal proteinsare bands 1 & 2, which are the a & 13 subunits of spectrin; actin (band 5), band 4.1 andankyrin (band 2.1). Minor proteins are band 4.2, adducin and tropomyosin.The integral membrane proteins are those that exist within the lipid bilayer. They areso named because they are not extracted by low ionic strength buffers. Instead, strongdetergents are required which solubilize the bilayer and free the integral protein. The majorintegral proteins of the red cell membrane are the transmembrane glycoproteins band 3[Furthmayr et al 1976; Steck 1978] and glycophorin A (PAS1 & 2 or sialoglycoprotein a),glycophorin B (PAS3 or sialoglycoprotein 8) & glycophorin C (glycoconnectin or PAS4? or sialoglycoprotein ,3) [Furthmayr et al 1975]. These proteins are glycosylated on theirextracellular domain and the resulting sugar/protein matrix is called the glycocalyx.2Table 1.THE MAJOR PROTEINS OF THE HUMAN ERYTHROCYTE MEMBRANEProtein Subunit MW Native Assembly Copies/CellExtrinsic ProteinsSpectrin Band 1 260,000 c, (a, /3)2 tetramer 105 tetramersBand 2 225, 000/3Ankyrin Band 2.1 215,000 monomer 105Band 4.1 78,000 2 x 105Actin Band 5 43,000 oligomer,12-17units 5 x 105 oligomersTropomyosin 29, 000a ; 27, 0000 hetero-dimer 7 x 104 dimersAdducin 103, 000a ; 97, 0000 hetero-dimer 3 x 104Integral ProteinsBand 3 89,000 dimer/tetramer 106 monomersGlycophorin A GP 31,000 dimer 6 x 105 monomersGlycophorin B GPS 23,000 8 x 104Glycophorin C GP /3 29,000 5 x 104The current view of the structural organization of the red cell membrane is shown in Figure 1.Spectrin associates into heterodimers which head link into a tetrameric filamentous proteinhaving an average span of 76nm in the membrane skeleton [Sheetz 1983] but spanning some200nm when fully extended [Shen et al 1986]. In normal red cell skeletons 95% of spectrinis in the tetrameric form but the tetramer/dimer equilibrium can be altered by adverseconditions [Palek & Lux 1983]. Electron micrographs of membrane shells show that between5 and 7 spectrin tetramer tails associate at junctional complexes [Byers & Branton 1985;Shen et al 1986; Liu et al 1987]. Thus the overall structure of the cytoskeletal networkis a hexagonal lattice with many structural defects (Fig.2). The junctional complexes arestabilized by other extrinsic proteins namely actin, band 4.1, adducin and tropomyosin.These proteins either bind spectrin directly (actin and 4.1), or reside at the junctional3Glycoptu3rin B Gtycoph.orin A^Glycophorin. C;#1 Band, 311 111111111113pectrinTetramer)GLycocalyxBilayerCyto-SkeletalNetworkBandA nizYrin^6 •Actin junctional(Oligomer) ComplexThe Red Blood Cell MembraneFigure 1Schematic Cartoon of the Structural Organization of the Red Blood Cell MembraneThe red cell membrane consists of a lipid bilayer spanned by integral proteins which are supported by ahexagonal skeletal network. The major integral proteins are band 3 and glycophorin and the majorskeletal protein is spectrin.4complex and have a low affinity association with several of the junctional components (for acurrent review see [Bennett 1990]). Evidence of this stabilization is seen in in vitro solubilizedspectrin which only forms a gel in the presence of actin [Fowler & Taylor 1980; Morrow& Marchesi 1981] and in band 4.1-deficient red cells, which are characterized by increasedfragility, are stabilized and regain normal strength when purified band 4.1 is bound backinto their cytoskeletons [Takakuwa et al 1986].Ankyrin is another spectrin-binding protein [Bennett & Stenbuck 1979a]. The bindingdomain for ankyrin on spectrin is away from the junctional complex. Ankyrin and band4.1 play important roles because they bind to the integral proteins band 3 [Bennett &Stenbuck 1979b, 1980] and glycophorin C [Mueller & Morrison 1981] respectively. Suchlinkages are thought to exist because removal of the lipid bilayer by Triton 2 extraction leavesthe majority of the integral proteins band 3 [Bennett & Stenbuck 1979b; Sheetz 1979;Bennett 1982] and glycophorin C [Mueller & Morrison 1981; Anstee et a/ 1984] attachedto the cytoskeletal shell, whereas the majority of glycophorin A is extracted with the lipid[Chasis et a/ 1985]. Such linkages would also provide a mechanism by which the cytoskeletalnetwork is tethered to the bilayer and could explain the reduced lateral mobilities [Jacobsonet al 1987] of the integral proteins as compared to their predicted and measured free values[Saffman & Delbruck 1975; Golan 1989].Band 3 is a transmembrane integral glycoprotein with a molecular weight of 90-100kD[Steck 1978]. It is a known anion exchanger and has a single branched carbohydrate chain2Triton X is a nonionic detergent which totally disrupts the bilayer by solubilizing the lipid. It leaves thered cell cytoskeletal network (shell) completely intact and in its characteristic discoid shape.5Hexagonal Lattice ofthe Cytoskeletal NetworkFigure 2:Schematic Cartoon of the Structural Organization of the Red Blood Cell CytoskeletonThe red cell membrane cytoskeletal network consists mainly of a long (76-200nm) filamentous tetramericprotein, spectrin. Five to seven spectrin tetramers associate with other stabilizing globular proteins, actinand band 4.1, into junctional complexes, thereby forming a hexagonal lattice underneath the lipid bilayer.6connected to its extracellular domain. Band 3 associated carbohydrate accounts for 5-8%of the total membrane carbohydrate. The amino acid sequence [Tanner et al 1988] showsthat band 3 has a large (hydrophobic) membrane spanning domain and a considerable—NH3 terminal cytoplasmic domain. A current model suggests that the membrane-spanningdomain traverses the bilayer 14 times [Lux et al 1989] resulting in several extracellular loopswhich extend above the bilayer 4-5nm [Low 1986]. The extracellular domain contains theantigenic determinants involved in blood group specificity (A, B, 0 and possibly Rh andKell) and is the probable antigen targeted by the auto-immune system at the end of the120 day average life span of a red cell. The cytoplasmic domain of band 3 is of particularinterest because interactions between it and the cytoskeletal network have a marked effecton membrane rigidity [Mohandas et al 1992; Knowles et al 1992(in preparation)]. Thisdomain consists of the first 403 amino acids of the protein which can be cleaved from inside-out red cell membranes by treatment with one of several proteinases. This results in a43kDa fragment consisting of two domains which are linked by a possible 'hinge' and whichextends some 25nm beneath the bilayer [Low 1986]. In situ band 3 is thought to exist ina dimer/tetramer equilibrium which favours the dimeric form [Nigg & Cherry 1979]. Bothforms are recovered by detergent extraction, and although the ratio of dimer to tetramervaries with the methods the majority is found as a stable dimer.The other major integral proteins are sialoglycoproteins which separate into 4 bands onSDS gels (PAS1,2,3&4) and are selectively stained with PAS reagent due to their sialic acidcontent [Fairbanks et al 1975]. Bands PAS1&2 comprise 75% to 80% of the sialoglycoprotein[Furthmayr et al 1975] and are shown to be dimeric and monomeric forms (respectively)7of glycophorin A because PAS1 readily converts to PAS2 when heated in SDS [Marchesi& Furthmayr 1976]. The interaction site for self-aggregation is within the bilayer spanningportion of the protein [Furthmayr 1977], and because the majority of extracted glycophorinA forms a stable dimer, it is thought to exist as a dimer or higher aggregate within themembrane. Glycophorin A has a molecular weight of 3lkDa and there are approximately6 x 10 5 copies/red cell [Merry et al 1986]. It is a transmembrane protein of 131 aminoacids [Tomita et al 1978] containing a single 22 amino acid non-polar region separating twopolar regions suggesting that the protein traverses the bilayer once. It has 16 oligosaccharidechains connected to the extracellular —NH3 terminal third of the protein which account for60% of the molecule's mass [Marchesi et al 1976]. This extracellular region carries the Mand N blood group antigens, but other functions are unclear especially since some donorscompletely lack glycophorin A yet are normal and healthy. The C terminal cytoplasmicdomain of glycophorin A contains 39 amino acids [Furthmayr 1977] which could extendsome 8-10nm below the bilayer.The minor sialoglycoproteins are glycophorins B (PAS3) and C (PAS4). GlycophorinB is a 23kDa protein having 8 x 10 4 copies/red cell. It is a transmembrane protein whichtraverses the bilayer once although its cytoplasmic domain is small, consisting of only 6amino acids. Its extracellular domain is related to that of glycophorin A because it hasthe same —NH3 terminal 26 amino acid sequence, but its attached carbohydrate is slightlydifferent. Glycophorins A and B are thought to be encoded by adjacent genes becausemutations occur where these proteins are replaced by mutant, hybrid glycophorins whichare combinations of the two [Merry et al 1986]. The amino sequence of glycophorin C8is not related to glycophorin A although structurally there are similarities. It is a 128amino acid transmembrane protein with 11 —NH3 terminal-linked oligosaccharide chains onits extracellular domain. Its molecular weight is 35kDa and there are 5 x 10 4 copies/red cell.91.2 Laterally Motion of Integral Membrane ProteinsIntegral proteins are able to undergo rotational and lateral motion in the plane of the bilayer.These motions, an essential part of cellular function, are driven by thermal energy andactive cell processes and are hindered by the viscous drag of the bilayer and soft (orderkT, where k the Boltzman's constant multiplied by T the temperature equals the availablethermal energy) association between proteins. The dominant driving energy in the case ofthe erythrocytes is kT simply because the cell is devoid of internal organelles and is muchless biologically active than real cells and thus integral proteins move (diffuse) randomly inthe plane of the membrane with a mean square displacement proportional to the diffusivityand the time' (Equ.1).(x2) = 4Dt (1)3The diffusion process is analyzed in statistical physics as a random walk. In each time step, the diffusingparticle makes a random spatial step and its mean square displacement is plotted as a function of time (itsmean displacement being zero). Consequently, the plane on which the particle moves can be divided intoa network of spatial steps (bonds) or particle sites (Bond or Site Percolation [Mitescu & Roussenq 1983,Stauffer 1985, Saxton 1990]). If all the sites on the plane are equally accessible, then the particle undergoesclassical diffusion and its mean square displacement increases linearly with time (Eq.1). However, it ispossible that all the sites are not equally accessible; for example, some sites may be sterically blocked. Onethen assigns a probability, p, of a site being accessible. As p decreases from unity (all sites accessible), thediffusive process becomes increasingly hindered and although at long times its mean square displacement isstill linear with time, its effective diffusivity (D) decreases. The random walk is thus confined to a 'cluster'of nearest neighbour accessible sites. For large p (p c < p < 1) such a cluster has infinite size, however thisis not the case at some critical probability (p,) known as the percolation threshold. At this probability,the cluster size becomes finite, the diffusivity goes to zero and the mean square displacement saturates in acertain characteristic time. As p decreases further, both this characteristic time and the cluster size decrease.For the analysis in this study, receptor lateral motion is assumed to be due to a single constant diffusivity.Thus, the percolative network on which they move is infinitly connected. Percolation type analysis has beenused to study the motion of single receptor molecules [Slattery et al 1991a] but the results are yet to bepublished (personal communication with Watt Webb's group, Cornell University, New York).10The lateral diffusivity of a membrane protein is an indirect measure of its specific andnon-specific interaction with the other membrane components. Such interactions can bemodelled in terms of an average drag coefficient (bd rag ) defined as the drag force on theprotein (Fdrag ) divided by its average velocity (v). The inverse of the drag coefficient is themobility (m) and this relates directly to the diffusivity (D) via the Stokes-Einstein relation(Equ. 2). For an upper theoretical limit of the diffusivity of an integral protein one canconsider a single integral protein diffusing in the plane of a pure lipid bilayer membrane. Insuch a case the lateral motion of the integral protein is restricted only by the viscosity ofthe lipid bilayer and that of the surrounding aqueous medium 4 . Saffman and Deldruck havecalculated an expression for the diffusion of a particle in an ideal fluid layer (Equ. 3) [Saffman& Delbruck 1975]. The expression shows that the diffusivity in 2 dimensions varies with aweak logarithmic dependence on particle size and with the substitution of appropriate values(as indicated) results in a theoretical upper limit for the diffusivity of an integral protein ina pure lipid bilayer of 2 x 10 -8 cm2 /sec.D kTm , kT kTvbdrag ^drag(2)4A diffusing particle, physically restricted to 2 dimensions, always interacts with the supporting thirddimension. This must be accounted for mathematically, otherwise a non-physical solution results where the2D velocity field generated around the diffusing particle decays too slowly, resulting in a drag coefficient thatdiverges (Stokes' Paradox).11D = kT ^[log ilmemt^7 ;47Mmemt(3 )a— particle radius ti 1nm,t— interaction thickness^2x bilayer thickness ) ti lOnm,= 0.5772, Euler's constant,7/mem , membrane viscosity ti 1poise=0.1kg1(m.sec),surrounding aqueous viscosity 10-2poise = 10 -3kg1(m.sec),qw,20 C ,kT30OK 4 x 10 -21 J,DFree = 2 x 10-8 cm2 /sec.This has important biological consequence, especially in diffusion limited reactions, becauseintegral proteins of any size can move laterally at the same rate (theoretically). In factglycophorin reconstituted into giant lipid vesicles in their fluid state has the same diffusivityas the lipid [Kapitza et.a11984]. The positive experimental consequence of this is that integralprotein diffusivity is almost unaffected by the size of labels linked to them in lateral motionstudies. This is even the case in nanometer resolved optical receptor tracking where (huge)30-50nm gold particles or low density lipoprotein are bound to the receptor's extracellulardomain and only slightly reduce their measured diffusivity [Genes et al 1988, Sheetz et al1989].Lateral motion of integral proteins is allowed because the lipid bilayer behaves as a fluid inthe plane of the membrane. When integral proteins are reconstituted into artificially-madelipid bilayer vesicles, they have nearly the same lateral diffusivity as the free lipid [Vaz et al1981,1982 & 1984; Jacobson et a/ 1981; Kapitza et a/1984]. This value of ti 4 x 10 -8cm2/secagrees with the value predicted theoretically (Equ. 3) and when it is measured by thetechnique of fluorescence photobleaching (Chapter 2) one finds that the fluorescence recovers12to 100% of its original value. This indicates that all the molecules (lipid & protein) are freeto move laterally. Interestingly enough, the lateral diffusion of lipid in red cell membranes isan order of magnitude lower than this free value. Red cell lipids diffuse at ,-- 4 x 10 -9cm2 /see[Bloom & Webb 1983; Golan et al 1984; Rimon et al 1984; Golan 1989]. This agrees with thevalues measured for fluorescently labelled PE incorporated into the red cell membrane andin both cases the fluorescence recovery is 100%. The reduction in mobility is presumably dueto the high density of integral proteins that sterically hinder the lipid's motion, forcing themto diffuse through a percolative network of integral proteins [Saxton 1989]. Hindered lipiddiffusion in red cell membranes is not surprising in light of the finding that the lateral diffusionof the integral proteins differ in two ways from the ideal value. First, the measured diffusivityis decreased by some 2-3 orders of magnitude; and second, the fluorescence recovery isconsiderably less than 100%. The reduction of diffusivity could result from the congestion ofintegral proteins but the discovery of an immobile fraction clearly indicates the involvementof the underlying cytoskeletal network because the diffusivity of cytoskeletal spectrin is lessthan 4 orders of magnitude below the level of the free diffusivity [Peters et al 1974]. However,the nature of the interactions which reduce the motion of integral proteins from their freevalue is varied and far from understood.13Table 2.MEASURED DIFFUSIVITY OF LIPID AND INTEGRAL PROTEINSIN BILAYER VESICLES AND RED CELL MEMBRANESComponent System D [cm 2 I sec] Recovery [%] [Ref.]NBD-PE PC Vesicle 4 x 10-8 100% [Kapitza 1984]Glycophorin PC Vesicle 4 x 10-8 100% [Kapitza 1984]Glycophorin DMPC Ves. (1 — 2) x 10 -8 100% [Vaz 1980, 1982]Fluorescent PE RBC Mem. (3 — 5) x 10' 100% [Golan 1989]Phospholipid RBC Mem. (2 — 4) x 10 -9 100% [Golan 1984]Cholesterol RBC Mem. (2 — 4) x 10 -9 100% [Golan 1984]Glycophorin A RBC Mem. 7.8 x 10" 61% [Knowles 1992](2 — 3) x 10 -11 66%-90% [Golan 1989]Band 3 RBC Mem. (2 — 3) x 10' 41% [Knowles 1992](1.2 — 2.4) x 10 -11 45%-78% [Golan 1989]Spectrin RBC Skel. « 2 x 10 -12 [Peters 1974]The role of the cytoskeleton in reducing integral protein motion is implied by several findings.The high-affinity attachment sites of band 3 [Bennett & Stenbuck 1979b] and glycophorinC [Mueller & Morrison 1981] to the spectrin cytoskeleton could be responsible for the im-mobile fractions measured for these integral proteins [Golan & Veatch 1982]. Incubating thecytoplasmic side of red blood cells with the spectrin binding domain of ankyrin dissociatesthe spectrin from the bilayer resulting in an increased lateral mobility of band 3 [Fowler& Bennett 1978; Tsuji & Ohnishi 1986]. Band 3 diffusivity is increased 50x in mutantmouse red cells which have a complete spectrin deficiency [Sheetz et al 1980], and is higherthan normal in human red cells with a partial spectrin deficiency [Golan et al 1991 ASHAbstract]. Band 3 diffusivity also increases if red cells are incubated in a low ionic strengthbuffer which partially disrupts the cytoskeletal protein associations [Golan & Veatch 1980],or when the lipid bilayer and its integral proteins are pulled off the cytoskeleton in the form14of tethers [Berk et al 1988]. In contrast to these findings there are many examples where thecytoskeletal network is not responsible for the reduced mobility of integral proteins. If mem-brane attachment sites immobilize a sub-population of integral proteins, then it is not clearwhat limits the diffusivity of the mobile portion of the population. Furthermore, deletingmost of the cytoskeletal domain of: G proteins in vesicular stomatitis virus [Scullion et al1987]; epidermal growth factor (EGF) [Livneh et al 1986]; or Ld antigens on L cells [Edidin& Zuniga 1984] does not alter their lateral diffusivity.Another possible region of interaction between membrane bound components is within thebilayer itself. Attractive interactions between bilayer spanning domains of glycophorin Aresult in the formation of stable dimers. [Furthmayr 1977]. The diffusivity of mobile particlesinteracting by simple hard core repulsion has been shown to decrease their diffusivity by anorder of magnitude as the surface density increases in Monte Carlo simulations [Pink 1985;Saxton 1990]. If attractive, cluster-inducing, interactions are included in such simulationsthe diffusivity decreases markedly [Pink et al 1986]. Thus simple steric interactions, withinthe bilayer, could reduce the lateral diffusivity of integral proteins and is the probable causeof the reduced diffusivity of lipids in red cell membranes from their ideal value (Equ.3).Finally, the extracellular domain of the membrane can hinder the lateral motion of integralproteins. This is not surprising because often the majority of an integral protein is extra-cellular. The diffusivity has been shown to increase linearly with loss of glycosylation of theextracellular domain of mouse Ld class I glycoproteins [Wier & Edidin 1988] while the cyto-plasmic and bilayer spanning domains have been shown to have little effect on the mobility15of GPI-linked proteins [Zhang et al 1991], or G proteins [Scullion et al 1987]. Interactions ineach of the domains, cytoplasmic, bilayer spanning and extracellular, can potentially limitthe lateral mobility of integral protein. As to which protein domain interaction predominateswill depend on the type of integral receptor protein and its state of conformation.162 Experimental Methods2.1 The FRAP TechniqueThe lateral motion of fluorescently labelled macromolecular components whose motion isconfined to the plane of the erythrocyte membrane, was measured by the technique of fluo-rescence recovery after photobleaching (FRAP) [Jacobson et al 1976; McGregor et al 1984](for a review of FRAP equipment design see [Wolf 1989]). The technique determines boththe lateral mobility and the mobile fraction of membrane components and involves severalsteps. First, the membrane component to be studied was fluorescently labelled. This wasdone by several methods which attach the fluorescent probe covalently to the membranecomponent, or indirectly by labelling a specific ligand. Band 3 and glycophorin A werelabelled directly by fluorescent probes eosin-5-malimide (EMA) [Nigg Si Cherry 1979] andeosin-5-thiosemicarbazide (ETSC) [Golan et al 1986] respectively. Glycophorins A and Cwere labelled indirectly by binding fluorescently labelled monoclonal antibodies which arespecific for their extracellular domains [Edwards 1980; Anstee & Edwards 1982; Bigbee et al1983; Anstee et a/ 1984; Merry et al 1984; Dahr et a/ 1989].Second, an area of the membrane was excited by a low-intensity (,-,, 12.81CW, 476 nm) beamof light and the intensity of the emitted fluorescence was measured by a photomultiplier tube(PMT). The area of the beam profile at the membrane, was determined from its measuredintensity profile and its size is controlled by the FRAP optics. Third, the fluorophor in thearea of interest was irreversibly photobleached by a short pulse of a high-intensity (--, 1.3mW,17Figure 3:Fluorescence Recovery of Photobleached Glycophorin on the Red Cell Surface1.) Glycophorin on the red cellsurface was labelled with ETSCand imaged in a uniform fullfield of illumination. Thefluorescence labelling wasuniform and the variations inintensity are due to thecurvature of the membrane.2.) The second image was taken0.5 seconds after a region of thecell membrane was irreversiblybleached with a pulse of theGaussian bleach beam. Thenormal pulse duration was20msec but these images wereoverbleached by a pulse ofseveral seconds to clearly showthe bleached area.3.) The third image was takenapproximately lhr after thebleach pulse and shows therecovery of fluorescence due tothe lateral motion of labelledglycophorin in the plane of themembrane. The characteristicrecovery time for most of theglycophorin is tens of seconds.However, even after 1 hr thefluorescence has not recovered to100%, indicating that a fractionof the glycophorin is eitheralmost immobile or its lateralmotion restricted to smalldomains.476 nm) beam of light. The photobleaching-associated cell-surface heating and possiblemembrane photodamage is minimal and does not affect the lateral motion of the membranecomponents [Axelrod 1977; Jacobson et al 1978]. Fourth, the low-intensity beam was usedto measure the fluorescence recovery in the photobleached area. Recovery is produced by theinterchange of unbleached and bleached fluorescently-labelled membrane components as theydiffuse across the boundary of the photobleached area (Fig. 3). Finally, recovery kineticswere fitted theoretically to determine the diffusivity and mobile fraction of the labelledcomponent [Axelrod et al 1976; Koppel et al 1979; Yguerabide et al 1982].18It is important to realize the limitations of the FRAP methodology. Diffusivity and mobilefraction are least square fit parameters, assuming single component diffusion [Axelrod et.al1976, Yguerabide 1982], and relate to the temporal window of the FRAP experiment. Thelower limit of several milliseconds is determined by the response-time of the shutter andthe upper limit of minutes by the characteristic time for gross lateral membrane motion.Fluorescence recovery kinetics are composed of the data of several (10-20) cells, where 3-4 recovery values are measured per cell. This repetition reduces the errors arising frominterrogative bleaching while maximizing the signal to noise ratio. The largest errors are dueto the sensitivity limit of the PMT and the ability to properly focus the membrane with theillumination spot. The mobile fraction is affected by the first of these types of errors witha typical percentage error of 10%. Diffusivity is affected by both errors and is proportionalto the square of the beam-waist diameter. When the size of this waist is determined byfitting its measured intensity profile to a Gaussian function, convolved with the point spreadfunction of the objective, the error is 1%. However, during an experiment a red cell, whichis 1-2pmthick, is focused with a conservative focus error of 0.1 to 0.2 pm. This equals thebeam waist error because the numerical aperture of the objective is 0.75. Thus, the error indiffusivity can be as high as 60%. The important point is that the accuracy of the FRAPtechnique is limited to changes of several percent in mobile fraction and to changes by factorsof 2 in diffusivity.192.2 Membrane Rigidity MeasurementsThe elastic rigidity modulus of the red cell membrane was determined by pipette aspirationexperiments [Berk et al 1989; Evans & Skalak 1980]. In these experiments a portion of thered cell membrane was deformed into a glass pipette under the influence of a minute suctionpressure. The rigidity modulus is inversely proportional to the change in aspiration lengthwith the suction pressure and the deformation is considered to be elastic if this modulus is thesame for both the loading and unloading phase (Fig. 12). For ease of analysis, the membraneportion is chosen at the dimple of the discoid cell which makes the deformation axiallysymmetric. There is a maximum useful aspiration length which decreases with increasedrigidity because at some point the work done by the applied pressure goes into membranebuckling.2.3 Membrane LabellingNormal blood from healthy volunteers was extracted by vene puncture. Whole blood wasstored at 4° C, washed and diluted for experiments in a phosphate buffered saline solution(290 mOsm, pH7.4) containing 0.05g% human serum albumin (PBS /HSA). MiltenbergerV and normal travel-control blood samples were supplied by Drs. Mohandas & Chasis,of the Lawrence Berkeley Laboratory, Berkeley, California. This blood was stored at 77Kand fast-thawed when needed. Three samples of spherocytic blood were shipped at 4° CbyDr. Peter Agre of the John Hopkins University, School of Medicine, Baltimore, Maryland.5 These experiments were done by Andrew Leung, a technician in this laboratory, on the same bloodsamples and at the same time as the lateral motion experiments.20Ovalocytic blood samples from six individuals, resident in Kuala Lumpur, were suppliedby Dr. Mohandas. The blood was shipped direct from Kuala Lumpur along with normaltravel-controls. Monoclonal anti-glycophorin A antibody and its Fab fragment were sup-plied by Dr. Anstee of the South Western Regional Blood Transfusion Center, Bristol, UK.The anti-glycophorin A was fluorescently labelled at the Berkeley Laboratory. Eosin-5-thiosemicarbazide (ETSC, cat.# E-120) and eosin-5-maleimide (EMA, cat.#E-118) werepurchased from Molecular Probes (Eugene, Oregon, USA). The oxidizing agent, sodium m-periodate, used in the ETSC labelling procedure, was purchased from Sigma, St. Louis,Missouri, U.S.A. (cat.# S-1878).Glycophorin A was labelled in situ with ETSC using the technique modified from Cherry[Cherry et al 1980] and Golan [Golan et al 1986]. Briefly, 10,alof whole blood was suspendedand washed 3 times in lml of PBS/HSA buffer at 4° C. Sialic acid was oxidized by, 50plof10mM NaI04 stock added to prewashed red cells to produce a final concentration of 0.5mMin which cells were incubated for 10min at 4° C, in the dark, before being washed 3 times inPBS/BSA. Finally, ETSC was dissolved in PBS/HSA buffer at 1mg/m1 from which 50plwasadded to the washed cells producing a final ETSC concentration of 50µg/ml, the cells were in-cubated for 30min and again washed 3 times in PBS/BSA. It should be noted that, althoughETSC is the state-of-the-art covalent tag for glycophorin A, it is only semi-specific. Thisresults because: (1) ETSC binds only to oxidized sialic acid residues; (2) sodium periodiateis a non-specific oxidizing agent; and (3) although glycophorin A is the major glycophorin,all the red cell glycophorins contain sialic acid. Golan [Golan et al 1986] reports that only60% of the ETSC bound to the red cell surface associates with glycophorin A.21Band 3 was labelled in situ with EMA using the technique modified from Nigg & Cherry[Nigg & Cherry 1979] and Golan [Golan et al 1986]. Briefly, 10,alof whole blood was sus-pended and washed 3 times in lml of PBS/HSA buffer at room temperature. EMA wasdissolved in PBS/HSA buffer at lmg/ml from which 50plwas added to the washed cellsproducing a final ETSC concentration of 50/ig/ml. The cells were incubated for 30min andwashed 3 times in PBS/HSA. The association of EMA with band 3 appears to be highlyspecific. Golan [Golan et al 1986] reports that over 80% of the EMA bound to red cellsurfaces is associated with band 3 and less than 5% associates with sialoglycoproteins.The extracellular domain of glycophorin A was bound with labelled or unlabelled anti-glycophorin A by incubating very low numbers of red cells in a PBS/HSA solution of therequired ligand concentration. These concentrations were determined from measured bind-ing isotherms relating the incubation concentration to the amount of ligand bound to thecell surface. For microscopy studies, labelled red cells were sealed in a chamber made bysandwiching a ring of vacuum grease between two glass cover slips. The chamber was sealedto prevent thermal currents produced by evaporation. In cases where the fluorescence back-ground from the incubation medium was measurable, the incubating medium was replacedwith unlabelled buffer by pipette aspiration once the cells had settled and before the cham-ber was sealed. This method prevented agglutination of IgG-bound cells which result fromwashing by centrifugation. All experiments were done at room temperature (25.0+-0.5° C).222.4 F.R.A.P. EquipmentFigure 4:Photograph of the FRAPequipment showing the laser,optical table, microscope,intensified CCD camera and afluorescence image of two redcells in contact.2.4.1 OpticsThe source of illumination was an argon ion laser supplied by Coherent Co. in Palo Alto,California. The laser produced a linearily polarized beam with a Gaussian profile (TEM00)at 350mW when tuned to 476nm. This wavelength was chosen because it closely matchesthe absorption peak of eosin and fluorsine as determined by spectrophotometry (Fig. 9).The first optical element was a 50/50 non-polarizing beam splitting cube. The beam splitat 90deg (Fig. 5) was used to produce a full field of illumination with even intensity at themicroscope object plane. The other was used to produce the coaxial interrogation and bleachbeams. These were produced by the next optical element, a partially aluminized double glass23DichroicEpi—illuminationDiohroioBarrierf;i1:241!OcularVisualDisplayarark-uULriLaser Based Epi-FluorescenceMicroscope with MicroprocessorControlled Photon Counting andIntensified Image CollectionBeam SteererSpatialFilterSplitterObjective, 40X,1‘10.75Double Block Splitter.^A diti^.41toN^• Aciori.a.h.k__ ■111 prer 0110'■1111111w4ShutterABC CCDVideoComerIntensifierShutter SplitterShutterCounterSpatial Filtez-BeamSteererPre— AmpFiberPOptic CooledVITLaser476 ntnFigure 5:FRAP SchemiticAn argon ion laser beam was split in two, one attenuated by 10 -3 . Computer-controlled shutter synchro-nization allowed independent use of these two beams for illumination. The two beams were recombined andfocused at the back image plane of an epi-fluorescence microscope. Conjugate images were produced bythe objective at the object. These images are diffraction limited in size with a Gaussian intensity profile[l/(e 2)radius = 0.55pm]. The attenuated beam was used to excite fluorescence light from the object. Thislight was collected by the objective lens and imaged via an ocular onto a photomultiplier tube. The exper-iment consisted of measuring the fluorescence intensity in an area of interest using the attenuated beam,bleaching the area with the unattenuated beam and measuring the subsequent fluorescence recovery in thebleached area due to lateral motion of unbleached molecules back into the area.24block beam splitter/attenuator, which includes two shutters for controlling the beams. Thisoptical set up was introduced into FRAP methods by Koppel [Koppel 1979] and representsa significant improvement over previous methods using solenoid driven neutral density filtersbecause of increased alignment precision, speed, and the lack of vibration caused by movingparts.The simplicity of the optical setup is seen in Figure 6, where a secondary beam is split andsubsequentally recombinded with the first on the original optical axis. The two unaluminizedinternal reflections reduce the intensity of the secondary beam by three orders of magnitudewith respect to the first. The separation of the beams between the blocks is determined bythe block thickness, its refractive index and the angle of incidence (Equ. 4). This distanceis ultimately limited by the thickness and length of the block (Equ.5).sinOcosOd = 2tOmax = sin -1 fngsin [tan -11/(30]1The blocks, supplied by Spindler and Hoyle (Germany), were made from optical quality glasswith optically flat surfaces. A significant problem arises if the faces are not parallel. Theinternal reflections of the attenuated beam result in an angle between the beams which ismore than twice the wedge angle of the block. Even 5 arc seconds ( ,1 thousandth of adegree) between the beams results in considerable alignment error of the two spots in theVn2 20g — sin (4)(5)25Correct Block AlignmentEliminates Non-ParallelismCaused by the Block Wedge AngleDouble Block Beam Splitter & AttenuatorFigure 6:The double glass block beamsplitter/attenuator produces two coaxialbeams one attenuated 3 orders ofmagnitude from the other. The beamsare used to produce the observation andbleach spots for the FRAP experiment.Figure 7:A minute block wedge angle causes the two beams to benon-parallel. Even slight non-parallelism causesconsiderable alignment error of the two spot images.plane of the objects. This problem was overcome by cutting the two splitter/attenuatorblocks from a single piece. The non-parallelism of the beams produced by the first block canthen be cancelled by the second block if it is positioned correctly.The next optical element was a beam steerer supplied by Newport Corporation, FountainValley, California. It not only permits the combination of several beams and makes theFRAP table layout compact, but provides the fine lateral adjustment of the beam requiredfor subsequent optical elements (namely the objective lens) which are less easily positioned6 For a beam incident at Oi on a block with refractive index ng and wedge angle between its faces of 0the primary and secondary beams are transmitted at angle of On = sin-1 (ng sin [sin-1 ( 31;:--1-e-L) - B] ) and0;2 = sin - 1 (n gsin[sin - i^- 30]). The difference (89 =Ott - 0t2) is the non-parallelism introducedby the block wedge angle and for small angle approx.. which introduces the smallest angle, 80 = 2ng G.This angle causes a misalignment of the two spot images produced by the 5cm lens (Fig. 5). The imagewaist diameter produced by this lens is d = sin0 = 16.7pm. For an alignment displacement error of 5%geometric optics gives 50 = 6 05x5e1 m6 7im which is less than 3 arc seconds!!9 6(by the Boy's method etc.) relative to the optical axis.The next element was a planoconvex lens (Newport, cm) which makes an image of thebeam profile at its focal plane. This lens, which focuses the beam so it can be filtered by apin hole, was positioned so that the beam image was at the back image plane of the objectivelens. Also, its focal length was chosen such that the divergent light from the beam image justfills the back aperture of the objective. The waist diameter, d, produced by this lens is givenby diffraction theory, d A/NA, where A is the wavelength and NA stands for the numericalaperture. This focused waist is the Fourier transform of the angular dependence of the lightentering the lens. Consequently, any diffractive artifacts produced by the preceding opticswere filtered off at this point by masking the beam image with a pin hole (spatial filter), thediameter of which slightly exceeds that of the waist (Newport, 100ympin hole).Next, the beam is combined with the full field shutter-controlled illumination beam by asecond 50/50 non-polarizing beam splitter cube. Beyond this point the beams entered aninverted epi-illuminated fluorescence microscope (Leitz Diavert) equipped with a dichroic /barrier filter set, a 40 x piano objective with a 0.75 numerical aperture and a 16 x ocular.Once in the microscope, the laser beam reflects off the dichroic mirror and enters the ob-jective lens which mades a diffraction-limited image of the beam profile in the image plane.The excited fluorescence emission, along with some back scattered laser radiation (of shorterwavelength) was collected by the objective lens. The unwanted scattered light was rejectedby the dichroic/barrier filter pair which in combination have a transmittance to the shorter27Fleur eseeace• Emissiaax StitaalatlagRealm ileaBarrierFilterFigure 8:Epi-Illumination & Fluorescence OpticsThe stimulating radiation is reflected by thedichroic filter to the object using the objective asa condenser. The fluorescence emission iscollected by the objective and passed through thedichroic/barrier pair which has a transmittance10 -6 to the stimulating light. The figure alsoindicates the Gaussian beam waist.wavelength of ti 10-6 but which allow the longer fluorescent wavelength to pass. The fluo-rescence light was imaged either onto the phosphor of a second generation image intensifier,or reflected off a second dichroic mirror (Newport, 595 nm long pass at 45°) and imagedonto the scanning plane of an optical fiber of 0.025 inches diameter. The fiber was posi-tioned at the image of the observation spot and collects the fluorescence radiation. The lighttransmitted by the fiber, was collected at its far end by a biconvex lens (Newport, fr.:19mm,#KBX043AR.14) and projected onto a photomultiplier tube.2.4.2 Intensified Camera, Video Board and PMTTwo photosensitive devices were used to quantify the intensity of the fluorescence image pro-duced by the microscope. The first was a second generation intensifier (Genllsys) opticallycoupled to a CCD video camera (CCD72). both supplied by Dage-MTI in Michigan City,Indiana. The video signal was digitized and analyzed in real time by a video imaging board28^IketO0ppirOichroic............• ....^• ........Emission X^ Oichroic &^ Barrier...• • • • • .. •••••.• ...............^• ......AbsorptionEmissionOichroic &BarrierLaserLine(476nm)KITC Absorption & Emission Bands400^500^600 700Wavelength (nm)Figure 9:Absorption & Emission Bands of EosinThis figure shows the absorption and emission bands of eosin (data taken from [Chen^Scott 1985]),the fluorescent probe used in the experiments, in relation to the excitation line (476nm) and the long passdichroic/barrier filter combination which has an optical density of 10 -6 to the excitation wavelength but istransparent to the fluorescence emission. Also shown is the emission band multiplied by the dichroic/barriercutoff resulting in the effective emission intensity collected and the band pass for the PMT dichroic thatreflects the fluorescence emission to the PMT fiber but allows the long wavelength tail to be imaged on theintensified CCD camera.29(Imager AT/NP Video Board) supplied by Matrox in Dorval, Quebec. The other was a pho-tomultiplier tube (#C31034A), supplied by Burle-RCA, cooled to -30° C by a Peltier cooledhousing, supplied by Products for Research. The PMT charge pulses were pre-amplified,discriminated and counted by associated electronics (Ortec) and the gating sequences andfinal photon count were controlled and read via an A/D board (QuaTech #PXB-721). ForFRAP experiments the PMT was used to quantify the fluorescence intensity because of itssuperior signal to noise resulting from cooling. However, the intensified camera was invalu-able for determining the laser spot size and aligning it with the fiber optic and the region ofmembrane of interest.2.4.3 Analysis of Recovery KineticsThe diffusivity and mobile fraction of the labelled macromolecular species were determinedby fitting the experimental fluorescence recovery with a theoretically obtained expression.The theoretical model assumes that fluorescence recovery is due to the lateral diffusion ofcomponents with a single diffusivity and is derived from the diffusion equation in 2 dimensionsfor an initial axially symmetric (Gaussian) concentration gradient of bleached fluorophor[Axelrod et al 1976]. The series solution (Equ.6) is given in terms of a bleach parameter' Kand a characteristic recovery time TD. TD is linearly related to the half time of fluorescencerecovery (t 112 'YDTD) where 7D is very weakly K dependent and ranges from 1 < yD < 1.45for a bleach percentage ranging from 0% to 80%. The relation of TD to diffusivity is given'The bleach parameter is given in terms of the fluorescence intensity ratio before and after the bleachK^  — 2--F bleaching) and it has been shown that excess ^(for K > 5and FP" —F(0) > 80%) distorts theF(0)Gaussian concentration profile and effectively increases the area bleached [Axelrod et al 1976]30(6)by Eq.1 where the mean square displacement is the bleach area given by the beam waist (w)squared(Equ.7) (w is the 1/(e 2 )radius).°N2, (—Kr ri^2t 1 -1nt^TD1" n=0w2 = 7Dw2D471)^4t1/2F (0 ) + F(00) (01 /2 ) F(t) =^1 + (t/t 1 /2 )The series solution for fluorescence recovery is easily linearized for the case of small per-centage bleach (Equ.8). This linear solution however, is in good agreement with the exactsolution even for a large percentage bleach. For an initial bleach as high as 85% the leastsquares deviation of the linear solution and exact solution is less than 4% [Yguerabide et al1982]. This error is well below the resolution of FRAP measurements and thus the linearsolution provides a good measure of the half time recovery and the mobile fraction along withthe ease of linear least squares fitting of the data. Figure 10 shows the normalized exper-imental fluorescence recovery of ETSC-labelled glycophorin A on the red cell membranesurface and the corresponding theoretical recovery curve given by equation 8.31Figure 10:This figure shows the normalizedfluorescence recovery ofETSC-labelled glycophorin Aon the red cell surface and thecorresponding least squares fit ofthe linearized theoreticalrecovery given by Eq. 8.2.4.4 Spot Size DeterminationThe 1/(e2 )radius of the observation spot (Fig. 8) was determined from the profile of its imageproduced by the objective lens. This was done by assuming that the intensity profile of thespot is Gaussian and that the information lost in the image is a function of the collectionaperture of the objective lens only (diffraction limited). In such a case the resulting imageis the intensity profile of the object convolved with the point spread function (PSF) of thelens8 [Goodman 1968]. Thus the measured image intensity profile of the spot was fittedwith a Gaussian profile convolved with the point spread function of the lens to determinesThe point spread function of a lens is the intensity profile of the image that it produces froma point source and is obtained by taking the Fourier transform of the lens' optical transfer functionPSF(x) = Ft — l[OTF(v)1. The OTF represents the lens' ability to collect light diffracted by thecharacteristic spatial dimensions of the object and to transmit it to the image. For the case of in-coherent illumination (which is the case in fluorescence emission) the diffraction limited OTF equalsthe autocorrelation of the aperture function of the lens and for a circular aperture this function isCOS —1(  v  )^v 2vmas^-uma < 2inna.r where the maxium spatial frequency isOTF( 1^2vmar 2defined by the greatest collection angle (given by numerical aperture) of the lens. v m,32Gaussian Intensity ProfileConvolved with the PSFof the ObjectiveOmissionProfileConvolvedProfileFigure 11:To determine the waist diameterof the Gaussian bleach spot itsimage, produced by theobjective lens, was fitted with aGaussian profile, with knowndiameter, convolved with thepoint spread function of theobjective lens. This wasrequired because the bleach spothas a radius of approximately Iwavelength and thus its image isbroadened because spatial ordersare diffracted beyond thecollecting angle of the objectivelens are lost in the image. Inthis figure the Gaussian profile,the convolved profile and thePSF are shown.its 1/(e2 )radius. A thin (thickness< A) uniform layer of fluorescent material was depositedon to a glass substrate and positioned at the laser spot. The intensity distribution of theimage was measured with the intensified CCD camera. A low intensity beam was used toavoid photobleaching which would have altered the relative peak height to width ratio. Nextan intensity profile was produced by averaging several video scan lines through the centerof the image. This profile was then fitted with a Gaussian profile convolved with the pointspread function of the objective. Figure 11 shows the effect of convolving the point spreadfunction with a Gaussian profile. In the absence of focus error the increase in 1/(e 2 )radiusof the image due to the lens is 12%. This increases to 62% when the object is 2A out offocus. The measured spot image intensity profile fitted with a convolved Gaussian resultedin a 1/(e 2 )radius of the bleach spot of (0.55±0.05)itm.332.5 Red Cell Membrane Elastic RigidityFigure 12:The rigidity of the red cell membrane wasdetermined by aspirating a portion into aglass pipette and measuring the aspirationlength (L) as a function of the appliedpressure (AP). Pipette aspiration producesa combined deformation of elastic shear andlocal area dilatation of the skeletal networkand the resulting rigidity modulus, which isinversely proportional to the change inaspiration length with applied pressure, isthus a combination of the compressibilityand shear moduli of the red cell skeleton.Red cell membrane rigidity was determined by pipette aspiration experiments [Berk et al1989 for review] (Fig. 12) done by Andrew Leung, a technician in our laboratory. Themeasurements were done concurrently with and on the same blood samples used in the lateralmotion experiments. In these experiments a portion of the red cell membrane was aspiratedinto a narrow glass pipette. The elastic rigidity relates to the ability of the membrane todeform into the pipette under an applied pressure. To simplify the analysis the membraneportion is chosen at the cell dimple so that the deformation is axially symmetric. As thepressure in the pipette is decreased the membrane undergoes an in-plane deformation and isdrawn into a cylindrical tongue. The maximum in-plane deformation (tongue length) thatcan be produced depends on the point at which the membrane outside the pipette startsto buckle. The buckling instability results in out-of-plane deformations which complicatethe analysis and, in cases where the rigidity is too high, completely restrict the rigidity34measurement.In the traditional analysis of this experiment the membrane deformation produced was as-sumed to be one of constant area (pure shear) [Evans & Skalak 1980]. This assumption wasmade because the area of the lipid bilayer is constant due to its large compressibility mod-ulus. The linearized solution from this analysis showed that the membrane shear modulus(j) was proportional to the change in tongue length (L) with applied pressure (AP) (Fig.12). However, it is the skeletal network not the bilayer which undergoes elastic deformation.The red cell skeletal network is a hexagonal lattice of filamentous spectrin tetramers whichspan 76 nm in the unstressed skeleton but span 200 nm when fully extended. Thus it isnot surprising that the deformation produced by pipette aspiration results in a combinationof elastic shear and local area dilatation of the cytoskeleton. This indicates that the com-pressibility modulus of the skeleton is much smaller than that of the lipid bilayer and thusthe compressibility of the bilayer would dominate in the measured compressibility for thewhole membrane. This is confirmed experimentally because, although the compressibilitymodulus of the cytoskeleton has not been measured directly, the measured compressibility ofthe red cell membrane equals that of SOPC (1-stearoyl-2-oleoyl phosphatidyl choline) bilayervesicles which contain the same mole fraction of cholesterol (40%) as the red cell membrane[Needham et al 1990]. Thus it should be noted that although the change in tongue lengthwith applied pressure is a good measure of membrane rigidity, the resulting modulus (p) isa combination of the shear and compressibility moduli of the cytoskeleton.353 Experiments and Results3.1 Ligand-Induced Lateral Immobilization of Glycophorin A:Implication of Cytoskeletal EntanglementThe interaction of glycophorin A with its underlying cytoskeletal network was studiedin normal and variant red cell membranes, by measuring the change in its lateral motionproduced by ligands bound to its extracellular domain. The ligands used were a series ofmonoclonal anti-glycophorin A antibodies with specificities for different regions of theextracellular domain (Fig. 13). Ligand 9A3 recognizes an epitope at the amino terminus,involving amino acid residue 1 [Bigbee 1983]. Ligands R10 and 10F7 bind in the midregion of the exoplasmic domain, distal to the trypsin cleavage site [Anstee 1982; Bigbee1983; Edwards 1980] and ligand B14 binds adjacent to the lipid bilayer, between residues 56and 67 [Ridgwell 1983]. The two membrane variants used were Miltenberger V and hereditaryspherocytosis red cells. Miltenberger V red cell membranes result from a mutation involvingan overlap in adjacent genes which code for the integral proteins glycophorin A and B.Consequently, Miltenberger V membranes are totally deficient in both these proteins, butinstead contain a hybrid glycoprotein consisting of the extracellular domain of glycophorinA, and the transmembrane and cytoplasmic domains of glycophorin B [Vignal et al 1989,Huang & Blumenfeld 1991]. Thus, this hybrid glycoprotein contains the binding sites for themonoclonal antibodies (ligands) 9A3, 10F7 and R10. It also has a significiantly truncatedcytoplasmic domain because the cytoplasmic domain of glycophorin B consists of only 6amino acid residues compared to the 39 residues in the cytoplasmic domain of glycophorin36Antibody Binding Epitopes on Gly-A Figure 13:This figure shows the relative binding po-sition of the monoclonal antibodies 9A3,R10, 10F7 and B14 to the extracellulardomain of glycophorin A.A (Fig. 13). Hereditary spherocytosis is characterized by a partial deficiency of the majorcytoskeletal protein, spectrin. Red blood cells were studied from three individuals withspectrin deficiencies of 35%, 41% and 66%. In the case of mild spectrin deficiency (<30%), thehexagonal structure of the skeleton is nearly normal, as determined by electron microscopyof spread membrane shells [Liu et a/ 1990]. In contrast, cytoskeletons with severe deficiency(>45%) show gross structural defects and a complete loss of normal hexagonal structure.3.1.1 Ligand -Induced Changes of Glycophorin A Lateral Motion in Normal RedCellsBinding isotherms were measured for each fluorescently labelled anti -glycophorin A anti-body. The binding affinities were similar for all four antibodies. Threshold binding, deter-mined by PMT sensitivity, was detected at an incubation concentration of 0.5 fig/m1 andbinding saturation was reached at 10 pg/ml. The fluorescence signal increased by almost37ALTERATION OF LATERAL MOTIONOF ETSC-LABELLED GLYCOPHORIN AIN NORMAL RED CELLS:THE EFFECT OF LIGAND BINDINGLigand^Conc^D ( x10-11 ) Mobile Fraction Cell SampleLag/m11^[cm2/seci^[%1^NumberNative9A310F7R10-Fab1 1.2+ 0.4+ 0.4± 0.7+ 0.5± 0.5± 0.4+ 1.0Membrane 7.82.5 2.810 2.61 5.02.5 5.0100 3.025 2.8100 4.161 ± 3 13050 ± 5 1244.2 + 3.5 1253 + 5 1240 ± 5 1040 + 5 2546.4 + 5 1035.2 ± 4.8 10two orders of magnitude over this range.Analysis of the fluorescence recovery of native glycophorin A in normal red cell membranes,labelled in situ with ETSC, produced a diffusivity of (7.8+ 1.2) x 10 -11 crn2 I sec and a mobilefraction of 61 + 3% (Table 3). Increasing the incubation concentration of 10F7 from 0-100 pg/m1 resulted in a dose dependent decrease in the mobile fraction to 40 + 5%, anda slight reduction in the diffusivity to (3.0 ± 0.5) x 10 -11 cm 2 /sec. Similar concentration-dependent changes were produced by 9A3 and monovalent Fab fragment binding. For9A3 bound at saturation the mobile fraction fell to 44.2 + 3.5% and the diffusivity to(2.6 + 0.4) x 10 -11 cm 2 /sec. The Fab fragment was produced from R10 and at saturation itreduced the mobile fraction to 35.2 + 4.8% and diffusivity to (4.1 + 1.0) x 10-112/sec.Table 3.38ALTERATION OF LATERAL MOTIONOF UNLABELLED GLYCOPHORIN AIN NORMAL RED CELLS:THE EFFECT OF BINDING LABELLED LIGANDSLigand Conc D (x10-") Mobile Fraction Cell Sample[,ug/m1] [cm 2/sec] Vol NumberNative Membrane 7.8 ± 1.2 61+3 1309A3 1 0.5 ± 0.14 40+5 1510 1.4 ± 0.4 45+5 1038 1.8 ± 0.7 19+3 12R10 2 <10 1010 <10 35R10-Fab 25 18+3 10100 12+3 30When unlabelled glycophorin A was bound with fluorescently labelled 9A3 at levels justabove threshold, there was a 5 to 10 fold decrease in the diffusivity and the mobile fractionreduced to 40 + 5% (Table 4). Saturation binding further reduced the mobile fraction to19 + 3%. For fluorescently labelled R1o, B14 and R1O-Fab, both threshold and saturationbinding resulted in mobile fractions of 10% to 15%.Table 4.3.1.2 Lateral Motion of Glycophorin in Miltenberger V Red CellsTo study the role of the cytoplasmic domain in this ligand-induced immobilization, mea-surements were made of the effect of ligand binding on the lateral motion of a variantglycophorin A, expressed in Miltenberger V red cells. This variant glycophorin has alarge deletion in its cytoplasmic domain. Binding isotherms were measured for fluorescently39ALTERATION OF LATERAL MOTIONOF HYBRID GLYCOPHORININ MILTENBERGER V RED CELLS:THE EFFECT OF BINDING LABELLED LIGANDLigand Conc D ( x10-11 ) Mobile Fraction Cell Sample[pg/m1] [cm2/sec] NumberNative Membrane 6.1 ± 1.2 79 ± 5 20R10 20 1.8 ± 0.5 70 ± 5 10100 0.78 + 0.2 69.2 + 5 3010F7 10 0.77 + 0.2 76 + 5 20labelled R10 and 10F7 to these cells. Threshold binding was detected at incubation con-centrations of 1 ,ug/m1 and binding saturated at 10 pg/ml. The saturation fluorescenceintensity was slightly less than half that measured for normal cells. This supports earlierfindings [Merry et al 1986] which showed that the copies/cell of variant glycophorin inMiltenberger V cells is half that of glycophorin A in normal cells. Hybrid glycophorin ofMiltenberger V membranes labelled with ETSC had a diffusivity of (6.1+1.2) x 10 -11 crn 2 I secand a mobile fraction of 79±4% (Table 5). When unlabelled hybrid glycophorin was boundat saturation with labelled 10F7 or R10 the average measured diffusivity decreased 5 to10 fold to (1.1 ± 0.4) x 10 -11 cm 2 /sec and the mobile fraction reduced slightly to 72 ± 5%.This data shows that the labelled hybrid glycophorin was not immobilized suggesting thatthe cytoplasmic domain is critical for ligand-induced immobilization of glycophorin A innormal cells.Table 5.403.1.3 Lateral Motion of Glycophorin A in Hereditary Spherocytic Red CellsSpherocytic red cells from three individuals were studied to determine the role of cytoskeletalnetwork integrity on the lateral motion of fluorescently labelled ligand bound glycophorinA. The spectrin deficiencies of the individuals were 35%, 41% and 66%, expressed as apercentage of the normal spectrin content. In the absence of ligands, glycophorin Alabelled with ETSC gave mobile fractions of 80.6%, 80.5% and 87.3% respectively and thediffusivity was constant at (6.2 + 1.2) x 10-11 cm 2 isec (Table 6). Compared to normal cells,the reduction of spectrin content resulted in a large increase in the mobile fraction withno measurable change in the diffusivity. HS cells saturated with fluorescently labelled B14gave mobile fractions of 16.8 ± 3% and 14.6 ± 3% for spectrin deficiencies of 35% and 41%respectively (Table 7). However, the mobile fraction was 32.4 + 4% with a correspondingdiffusivity of (3.0 + 1.5) x 10 -11 cm2 /sec for the most severe spectrin deficiency. This showsthat ligand-induced immobilization is less pronounced if the cell's spectrin content is lessthan normal.41ALTERATION OF LATERAL MOTION OFETSC-LABELLED GLYCOPHORIN AIN HEREDITARY SPHEROCYTOSIS RED CELLS:THE EFFECT OF SPECTRIN DEFICIENCYSpectrin D^x10 -11 ) Mobile Fraction Cell SampleDeficiency [%] [cm2/sec] [%) Number0 7.8 ± 1.2 61 ± 3 13035 6.5 ± 1.2 80.6 ± 5 1041 5.4 ± 1.2 80.5 ± 5 1066 6.6 + 1.2 87.3 ± 5 10Table 6.Table 7.ALTERATION OF LATERAL MOTION OF GLYCPHORIN AIN HEREDITARY SPHEROCYTOSIS RED CELLSPRODUCED BY SATURATION BINDING OF LABELLED LIGANDSSpectrin^Ligand D ( x10 -11 ) Mobile Fraction^Cell SampleDeficiency [%1 [cm2/sec]^[70]^Number0^B14^< 10^1035 16.8 ± 3 1041 -^14.6 ± 3^1066^3.0 ± 1.5^32.4 ± 4 103.1.4 SummaryIn this set of experiments, ligands were bound specifically to the extracellular domain ofglycophorin A in normal and variant red cell membranes in order to determine their effecton glycophorin A lateral motion.42In the first experiment, unlabelled ligands were bound to red cell surfaces labelled with ETSC,a fluorescent probe which covalently links to oxidized sialic acid residues on glycophorinA. This established the normal parameters of diffusivity and mobile fraction. Subsequentbinding of ligands caused a marked decrease in the previously determined mobile fractionwith only a slight decrease in the diffusivity. Such changes are characteristic of mobile speciesbecoming immobilized.In a second experiment, the ligands were labelled directly and bound to normal, unlabelled,red cell surfaces. In this case, ligands R10 and B14, bound at saturation, completelyimmobilized the glycophorin A resulting in a mobile fraction of less than 10%. In thesetwo cases a measurement of the diffusivity cannot be made (an upper limit is discussed later).The immobilization was less pronounced in the case of ligand 9A3 which is of particularinterest because this ligand binds to the epitope furthest from the bilayer on the extracellulardomain.An indication of the origin of this ligand-induced immobilization came from two furtherexperiments. First, labelled monovalent R10-Fab was bound, at saturation, to normal cellsand resulted in complete immobilization. This suggested that the immobilization did notinvolve cross-linking of the extracellular domain by divalent ligands. Second, saturationbinding of labelled divalent ligands to Miltenberger V red cell membranes had no effecton the glycophorin A mobile fraction although the diffusivity was decreased by 5 to 10fold. These experiments show that the cytoplasmic domain of glycophorin A is involved inthe ligand-induced immobilization because in Miltenberger V glycophorin there is a large43deletion in the cytoplasmic domain.To further investigate the origin of glycophorin A immobilization, labelled ligands werebound at saturation to the surfaces of spherocytic red cells from 3 individuals with differentdegrees of spectrin deficiency. In the case of 35% and 41% spectrin deficiencies, labelled lig-ands completely immobilized the bound glycophorin A. This suggested that these percent-ages of spectrin deficiency were not enough to alter the interaction between the cytoplasmicdomain and the cytoskeleton. However, for the 66% spectrin deficiency, immobilization wasmuch less pronounced. This suggested some dependence on cytoskeletal network density inthe immobilization of ligand bound glycophorin A.The conclusion from these experiments is that ligand binding to specific epitopes on theextracellular domain of glycophorin A promotes an increased interaction between the cy-toplasmic domain of glycophorin A and the underlying cytoskeletal network and that thisinteraction immobilizes the bound protein. Such a process suggests the transduction of asignal across the bilayer in response to bound ligands which is probably facilitated by aconformational change in one of the domains of glycophorin A.443.2 Integral Membrane Proteins Play a Key Role in MembraneRigidityTo further examine the nature of the ligand-induced interaction between glycophorin A andthe cytoskeletal network, cytoskeletal rigidity was measured along with the lateral motionof red cell integral proteins. The postulate being tested was that integral protein lateralmotion is inversely related to membrane rigidity. To test the hypothesis, lateral motion andrigidity measurements were made on: normal red cells bound with ligands to glycophorinA and glycophorin C; Miltenberger V cells with ligands bound to glycophorin A and;spherocytic cells with ligands bound to glycophorin A. Earlier studies showed that anti-glycophorin A bound to red cell surfaces decreased membrane deformability 9 in normalcells, but did not change the deformability of Miltenberger V cells [Chasis et al 1988]. Theseresults were the first evidence that ligand induced alterations in membrane rigidity are notdue to an extracellular interaction but involve the cytoplasmic domain of transmembraneproteins.If a relation can be established between integral protein immobilization and increased mem-brane rigidity, then it is a likely explanation for the high membrane rigidity known to existin some naturally occuring red cell variants. One such case is hereditary ovalocytosis, a redcell disorder which is widespread in South East Asia [Mohandas et al 1984]. The large rigid-ity of these cells is believed to be responsible for their ability to resist invasion by malarialparasites [Kidson et al 1981; Hadley et al 1983]. Thus it is likely that this red cell variant'Membrane deformability which relates inversely to membrane rigidity can be measured by flow ekcta-cytometry where a shear stress is applied to many cells and their ellipticity is measured by changes in theFraunhofer diffraction patterns that are produced.45was genetically selected in areas where malaria is endemic. Since the skeletal network isresponsible for membrane rigidity, it was thought that a mutation would be found in oneof the cytoskeletal proteins. However, considerable biochemical analysis has failed to detectany such mutation. The problem was elusive until recently when the ovalocytic genotypewas linked to a structural change in the integral protein band 3 [Liu et al 1990]. To testthe hypothesis that integral protein lateral motion is linked to membrane rigidity the lateralmobilities of band 3 and glycophorin A were measured in ovalocytic red cell membranesfrom three individuals.3.2.1 Integral Protein Lateral Motion and Membrane Rigidity in Membranesof Normal and Miltenberger V Red CellsRed cell membrane rigidity of normal cells was measured as a function of ligand binding tothe extracellular domain of glycophorin A. The rigidity of ligand-free normal cells was (8 ±1)/iNim (Table 8). When the ligand 9A3 is bound at threshold levels, the rigidity remainsunaltered. However, at saturation there is an 8-fold increase in rigidity to (64 + 12)//N/m.When ligand 10F7 is bound at threshold levels there is a marked 6-fold increase in rigiditywhich further increases at saturation to 15-fold. In the case of ligand B14, threshold bindingresults in a 46-fold increase in rigidity and at saturation the membrane becomes too rigidfor micropipette aspiration.46ALTERATION OF RIGIDITYOF NORMAL RED CELL MEMBRANESBY LIGANDS TO GLYCOPHORIN ALigand Conc Rigidity Cell Sampleitg/m1 [fiN/m) NumberNative Mem 0 8±1 209A3 5 8±3 576 64 ± 12 510F7 1 50±5 5100 117±50 5B14 2 370±170 520 too rigid ! 10Table 8.For comparison, the lateral motion and membrane rigidity were measured for ligand Bric-10 which binds specifically to an extracellular epitope of glycophorin C. As yet, there isno method to fluorescently label glycophorin C directly and thus no way of measuringits innate mobility. However, binding the ligand Bric-10 to glycophorin C resulted in adiffusivity of (1.6 ± 0.5) x 10 -11 cm' 1 sec and a mobile fraction of (44 ± 6%) (Table 9), anddid not change the rigidity of the membrane indicating clearly that: 1) when an integralprotein is not immobilized that there is no change in membrane rigidity; and further that2) extracellular ligand binding alone does not result in increased membrane rigidity. In asimilar vein, ligands 10F7 and R10 bound at saturation to glycophorin A have no effecton the rigidity of Miltenberger V membranes (Table 10).47LATERAL MOTION AND MEMBRANE RIGIDITYOF LIGAND BOUND GLYCOPHORIN CLigand^Concitg/m1D (x10-11 ) MFrac.[cm2/sec]^[%]Rigidity Cell Sample[ttN/m]^Number Bric-10^5^1.6 ± 0.5^44+6^8+2^30(saturation)ALTERATION OF MEMBRANE RIGIDITYOF MILTENBERGER V RED CELLSBY LIGANDS TO GLYCOPHORIN ALigand Conc Rigidity Cell Samplepg/m1 [(LN/m] NumberNative Mem 0 8+1 2010F7 10 8+1 5R10 100 8+1 5Table 9.Table 10.3.2.2 Membrane Rigidity in Hereditary Spherocytic Red CellsLigands B14 & R10 were bound at saturation to glycophorin A of three individuals withhereditary spherocytosis. For ligand B14, the membranes with mild spectrin deficiencies(35% & 41%) became too rigid for measurement. However, a measurement was possiblefor the cells with severe spectrin deficiency (66%), and these cells had a rigidity of (174 ±50),aN/m (Table 11). Ligand R10 shows an inverse relationship between spectrin deficiency48ALTERATION OF MEMBRANE RIGIDITYOF HEREDITARY SPHEROCYTOSIS RED CELLSBY LIGANDS TO GLYCOPHORIN ALigand Conc Spectrin [70] Rigidity Cell Samplepg/m1 Deficiency [uN/m] NumberNative 0 0 (Ctrl.) 6.2 ± 1.5 5Membrane 35 4.5 + 1.0 1041 6.6 ± 2.0 1066 5.4+ 1.3 10R10 20 0(Ctrl.) 117 + 28 535 84 ± 22 541 32 + 13 566 12 + 3 10B14 20 0 (Ctrl.) 370 ± 170 535 too rigid 541 too rigid 566 174 + 50 5and membrane rigidity. At 0% spectrin deficiency (normal cells) the rigidity of cells boundwith ligands R10 is (117 ± 28),(iN/m. This value falls to (84 ± 22)0//m, (32 + 13),uNimand finally to (12 + 3)ttNlm for spectrin deficiencies of 35%, 41% and 66% respectively.Table 11.3.2.3 Lateral Motion of Glycophorin A and Band 3, and Membrane Rigidity inHereditary Ovalocytosis Red CellsAs an application of the hypothesis that integral protein motion is related to membranerigidity, these two parameters were measured for glycophorin A and band 3 in membranesof three individuals with hereditary ovalocytosis. The rigidity of ovalocytic red cells was49LATERAL MOTION OFETSC-LABELLED GLYCOPHORIN A AND EMA-LABELLED BAND 3 IN,AND MEMBRANE RIGIDITY OFOVALOCYTIC RED CELL MEMBRANESDonor Integral D ( x10 -11 ) MFrac. Rigidity Cell SampleProtein [cm2/sec] [%] [itN/m] NumberNormal glycophorin A 5.5 + 2.4 62 ± 6 8 ± 1 40Control band 3 4.2 ± 1.6 35 + 15 60901 glycophorin A 5.3 + 1.3 40 + 10 25 + 6 10band 3 < 10 10904 glycophorin A 5.5 + 1.2 37+6 39+7 10band 3 < 10 10906 glycophorin A 4.9 + 1.9 49 + 7 72 + 27 10band 3 < 10 10greater than normal by factors of 3-fold, 5-fold and 9-fold (Table 12). This increase inmembrane rigidity had no effect on the diffusivity of native glycophorin A, but there wereslight measured reductions in the mobile fractions. However, in each case, the mobile fractionof the band 3 was less than 10%.Table 12.3.2.4 SummaryIn this set of experiments, ligands were bound to the extracellular domain of glycophorinA in normal and variant red cell membranes in order to determine their effect on membranerigidity, and to relate these effects to the changes in glycophorin A lateral motion measuredin the last section.50In an inital set of experiments, the membrane rigidity was measured for normal red cellsbound with anti-glycophorin A. Ligand 9A3 had no effect on membrane rigidity whenbound at threshold concentrations but increased the rigidity 8 fold when bound at satura-tion. Both ligands 10F7 and B14 produced marked rigidity increase at threshold and atsaturation. For saturation binding the rigidity increase was highest for B14 which bindsclose to the phospholipid bilayer and was minimal for 9A3 which binds farthest from thebilayer.To determine the mechanism responsible for this increased rigidity, two further experimentswere designed. First, a ligand, (Brie-10), specific for the extracellular domain of gly-cophorin C, was bound at saturation to normal cell membranes. Glycophorin C isthought to be a membrane attachment site, because it binds to band 4.1 of the cytoskele-tal network. This ligand did not increase the rigidity of the membrane and resulted in adiffusivity for glycophorin C which was similar to that of band 3. The result suggeststhat glycophorin C is not immobilized by a ligand specific for its extracellular domain.Second, ligands R10 and 10F7 were bound at saturation to the hybrid glycophorin ofMiltenberger V red cells. In these cells, the binding of ligands had no effect on membranerigidity. Together, these two experiments show that ligand binding promotes an increasedinteraction between the cytoplasmic domain of glycophorin A (rather than glycophorinC) and the cytoskeleton that promotes increased rigidity in normal cells.Finally, the contribution of cytoskeletal spectrin to membrane rigidity was determined byligand saturation of glycophorin A in spherocytic red cells with varying degrees of spectrin51deficiency. Ligand B14, which attached close to the bilayer, caused the cell membranes tobecome too rigid for pipette aspiration, in all but the most spectrin deficient cells. Thissuggests that only major spectrin deficiency reduces the rigidifying interaction. R10 ligandcauses rigidity that is measurable for each HS donor and decreases dose dependently as thespectrin deficiency increases.These experiments show that binding anti-glycophorin A ligands increase red cell mem-brane rigidity. These results, taken together with the changes of lateral mobilities, indicatethat interactions between the integral membrane protein's cytoplasmic domain and the cy-toskeleton can immobilize the integral protein and cause an increased membrane rigidity.This possibility was tested on ovalocytic red cells which are characterized by very rigidmembranes which makes them resistant to malaria. Membrane rigidity and the lateral mo-tion of glycophorin A and band 3 were measured on these Ovalocytic cells. For each ofthe three individuals studied the measured rigidity was well above normal, as expected. Theexciting result was that the lateral motion of glycophorin A was near normal but the entireband 3 population was immobile. This indicated, for the first time, a strong link betweenband 3 immobilization and increased membrane rigidity.523.3 Ligand-Induced Interaction Between Glycophorin. A and theCytoskeleton. in Human Erythrocytes Involve Band 3 Pro-teinThe final set of experiments was inspired by the questions of how the integral protein,glycophorin A, with only a single helical transmembrane domain and a relatively smallcytoplasmic domain (39 amino acids, spanning a possible 10 nm), becomes immobilizedand produces such large increases in membrane rigidity in response to extracellular ligandbinding.One proposed mechanism suggested that the interaction of the cytoplasmic domain of gly-cophorin A with the cytoskeleton is an indirect one which involves the cytoplasmic domainof band 3. Band 3 is implied because it is the major red cell integral glycoprotein, hasa considerable cytoplasmic domain that spans some 25nm [Low 1986], and is a known cy-toskeletal attachment site between the bilayer and ankyrin [Bennett & Stenbuck 1979b;Golan & Veatch 1982]. Interactions between glycophorin A and band 3 have been sug-gested in several instances [Nigg et al 1980]. And in the light of our recent work, whichrelates the increased membrane rigidity in ovalocytes to a mutation in band 3 [Mohandas etal 1992], a cooperative action is strongly indicated. This is because the ovalocytic mutationinvolves a deletion of a sequence of 9 amino acids adjacent to the bilayer on the cytoplasmicside. The deletion creates a proposed conformational change of the cytoplasmic domain ofband 3. The particular amino acid sequence, present in normal band 3, is well within thereach of the cytoplasmic domain of glycophorin A [Mohandas et al 1992].53To test the involvement of band 3 in the ligand-induced immobilization of glycophorin A,the lateral motion of band 3 and a minor sialoglycoprotein, glycophorin C, were measuredin normal and variant red cell membranes as a function of ligands bound to extracellularepitopes of glycophorin A. The variant red cells were the Miltenberger V type in whichglycophorin has a large cytoskeletal deletion.3.3.1 The lateral motion of band 3 in normal red cell membranes altered byligands bound to glycophorin AIn these experiments band 3 was fluorescently labelled by eosin-5 malamide (EMA) and itslateral motion measured in normal red cell membranes as a function of binding ligands 9A3,R10 and B14 to extracellular epitopes of glycophorin A.In the absence of ligands, EMA labelled band 3 has a diffusivity of (2.9+1.4) x 10-11 crn 2 lsec,and a mobile fraction of 43 ± 6% (Table 13). Binding ligand R10, which recognizes the midregion of the extracellular domain of glycophorin A, resulted in a dose-dependent decreasein the band 3 mobile fraction and completely immobilizes the band 3 when bound atsaturation. There is no measured change in the corresponding diffusivity. In the case ofB14, which recognizes an epitope close to the bilayer on glycophorin A, both thresholdand saturation binding resulted in complete band 3 immobilization. However, 9A3, whichrecognizes the most distal epitope on glycophorin A, leaves the lateral motion of band 3unaffected even at saturation binding.54ALTERATION OF EMA-LABELLED BAND 3 MOTIONIN NORMAL RED CELLSBY LIGANDS TO GLYCOPHORIN ALigand Conc DBand3( X 10-11 ) Mobile Fraction Cell Sampleto Gly-A [pg/m1] [cm2/sec] of Band 3 [%] NumberNative 0 2.9 + 1.4 43 ± 6 209A3 100 2.2 + 0.6 39+5 10R10 1 2.8 + 0.7 34+5 102.5 2.8 ± 0.7 27.6 ± 2 10100 7 ± 3 25B14 2.5 1.0 + 0.4 17+3 15100 7 ± 4 8Table 13.3.3.2 The lateral motion of glycophorin C in normal red cell membranes alteredby ligands to glycophorin AIn these experiments, the minor sialoglycoprotein, glycophorin C was fluorescently taggedwith labelled ligand Bric-10. This ligand is a monoclonal antibody specific for the extracel-lular domain of this sialoglycoprotein and earlier results (Table 9) showed that glycophorinC was not immobilized by this ligand and has a similar measured lateral mobility to that ofnative band 3. Thus, the lateral mobility of Bric-10-labelled glycophorin C can be mea-sured as a function of the amount of RIO bound to glycophorin A. This experiment showedthat R10 binding reduces the diffusivity of glycophorin C in a dose dependent fashion, butdoes not change its mobile fraction which suggests that glycophorin C is not immobilizedbut that its lateral motion was simply hindered by ligands bound to glycophorin A (Table55LATERAL MOTION OF GLYCOPHORIN CBOUND WITH FLUORESCENTLY LABELLED BRIC-10IN NORMAL RED CELLSALTERED BY UNLABELLED LIGANDS TO GLYCOPHORIN ALigand Conc DGty_c (x10 -11 ) Mobile Fraction Cell Sampleto Gly-A [µg/ml] [cm2/sec] of Gly-C [%] NumberR10 0 1.6 ± 0.5 43.7 ± 5 301 0.61 ± 0.2 40 + 10 15100 0.18 ± 0.09 44 ± 15 1014).Table 14.3.3.3 The lateral motion of band 3 in Miltenberger V red cell membranes al-tered by ligands to glycophorin ATo test the role of the cytoplasmic domain of glycophorin A in the immobilization ofband 3, the lateral motion of EMA-labelled band 3 was measured as a function of R10binding to the hybrid glycophorin of Miltenberger V cells. In the absence of ligands thelateral motion of EMA-labelled band 3 was the same as that for band 3 in normal cells.Saturation binding of R10 caused a slight, 2-fold reduction in the diffusivity but had noeffect on the mobile fraction (Table 15). This indicates that band 3 is not immobilized byligands to glycophorin A when glycophorin A does not have a cytoplasmic domain andagain suggests that the cytoplasmic domain is important for immobilization.56LATERAL MOTION OF EMA-LABELLED BAND 3IN MILTENBERGER V RED CELLSALTERED BY UNLABELLED LIGANDS TO GLYCOPHORIN ALigand Conc DB„„d3 ( x10-11 ) Mobile Fraction Cell Sampleto Gly-A [ttg/m1] [cm2/sec] of Band 3 [Vo] NumberR10 0 2.6 + 0.4) 41 ± 4 10100 1.4 ± 0.3 42.2 ± 6 30Table 15.3.3.4 SummaryLigands to the extracellular domain of glycophorin A have an immobilizing effect on thelateral motion of band 3. This effect is most pronounced when the ligand B14 is boundbecause it binds to the extracellular domain of glycophorin A, at an epitope closest to thebilayer. The immobilization is still seen when ligand R10 is bound but is less pronouncedat threshold binding levels. However, immobilization is not seen when ligand 9A3 is boundbecause it binds to the most distal extracellular epitope. The second experiment showesthat R10 binding does not have an immobilizing effect on glycophorin C. This indicatesthat glycophorin C, which is a proposed membrane attachment site, is not involved inthe ligand-induced immobilization of glycophorin A and band 3. The third experimentshowes that ligands bound to a variant glycophorin which has a large cytoskeletal deletion,do not immobilize band 3, suggesting that band 3 immobilization in normal cells involvesan interaction in the cytoplasmic domain. Thus, ligand binding to the extracellular domainof glycophorin A promotes an increased interaction between glycophorin's cytoplasmic57domain and that of band 3, and results in the immobilization of both integral proteins.584 Discussion and ConclusionThis study describes the dynamic coupling between a membrane receptor-protein and itsunderlying cytoskeletal network, in a model system, by measuring changes of the receptor'slateral motion and membrane's rigidity, produced by binding ligands to the receptor's ex-tracellular domain. The model system was the red blood cell membrane, both normal andvariant types, whoes membrane consist of several well characterized integral proteins and asupporting cytoskeletal protein network. The ligands used were monoclonal antibodies andtheir Fab fragments which recognize specific epitopes on the extracellular domain of gly-cophorin A, the major sialoglycoprotein of the red cell membrane. Ligand 9A3 recognizesa glycophorin A epitope at the amino terminus, involving amino acid residue 1 [Bigbee1983]. Ligands R10 10F7 recognize an epitope in the mid region of the exoplasmic do-main, next to the tryipsin cleaving site [Anstee 1982; Bigbee 1983; Edwards 1980]. LigandB14 recognizes an epitope adjacent to the bilayer between residues 56 and 67 [Ridgwell1983].Results from earlier studies [Chasis et al 1988] demonstrated that when these ligands (9A3,R10, 10F7 & B14) are bound to the extracellular domain of glycophorin A, membrane de-formability (measured by flow ekctacytometry) was greatly reduced for normal red cells, butnot for red cell variants that lack the cytoplasmic domain of glycophorin A (MiltenbergerV red cells). Since the cytoskeletal network (rather than the lipid bilayer) is responsible formembrane rigidity, these results suggested that ligand binding can initiate a transmembranesignal causing an increased interaction of the cytoplasmic domain of the receptor with the59underlying cytoskeletal network.Integral protein lateral motion was measured in situ by the technique of fluorescence recoveryafter photobleaching (FRAP). The technique involves fluorescent labelling of the ligand orthe receptor of interest, irreversibly bleaching a small region of the membrane and measuringthe subsequent fluorescence recovery due to lateral diffusion exchange of fluorophor coupledproteins. Red cell membrane rigidity was measured by a pipette aspiration technique wherea portion of membrane is deformed into a pipette using a minute suction pressure. The de-pendence of the aspirated tongue length on applied pressure is linear for elastic deformationsand results in an effective membrane rigidity modulus which is a combination of the elasticshear and area expansion moduli of the membrane cytoskeleton.4.1 Receptor/Ligand Interactions in Normal Red CellMembranesIn normal red cell membranes, the binding of ligands to the extracellular domain of gly-cophorin A, caused a large increase of membrane rigidity and marked reductions of themobile fractions of glycophorin A and band 3. Two approaches were used to show thatbinding anti-glycophorin A ligands markedly reduce the lateral motion of the erythrocytemembrane protein glycophorin A.The first approach used unlabelled anti-glycophorin A antibodies bound at increasingconcentrations to red cell membranes labelled with eosin-5-semithiocarbazide (ETSC). An-tibodies 9A3, 10F7 and R10-Fab all showed a similar concentration-dependent decrease in60mobile fraction from 61% to 38 ± 6% as the amount of bound antibody increases from zeroto saturation. This large reduction of the mobile fraction is indicative of a mobile speciesbecoming immobilized rather than just being hindered.In the second approach the antibodies were labelled directly and bound to red cell mem-branes. At saturation, the binding of fluorescently labelled 10F7, R10, R10-Fab and B14caused the mobile fractions to fall to 10 ± 3%, indicative of near complete glycophorinA immobilization. This effect was less pronounced with ligand 9A3 which binds the mostdistal extracellular portion of glycophorin A. The term immobilization relates to a verylow fluorescence recovery within the FRAP temporal window. This places an upper boundryon the diffusivity of ligand-bound glycophorin A of 2 x 10-13 cm 2 /secw. This value, whichis a reduction of 2 orders of magnitude from the diffusivity of native glycophorin A, is thesame as that estimated for the diffusivity of spectrin in the cytoskeleton [Peters 1974]. Thissuggests that immobilization is due to an interaction with the cytoskeletal network.The answer to why the mobile fraction in the first approach does not fall below 38 + 6% liesin the specificity of the ETSC labelling of glycophorin A. ETSC which binds covalently tooxidized sialic acid residues on the membrane surface is only 60% glycophorin A specific[Golan 1986]. This lack of specificity means that the mobile fraction of native, ETSC labelled,glycophorin A lies between 33% and 100% depending on the mobile fraction of the otherlabelled species. Assuming that ETSC labelling is 60% glycophorin A-specific, and that themobile fraction of ligand-bound glycophorin A is 15% (Table 4), then the mobile fraction1 °This value for the diffusivity of liganded glycophorin A is an estimate produced by assuming that itsmobile fraction is 60% in infinite time, and by fitting the theoretical recovery (Eq.8) through the point of10% recovery measured at 360sec, the maximum time of the FRAP experiment.61of native glycophorin A would be 50%.In two further experiments the change of lateral motion of band 3, the major red cell glyco-protein, and glycophorin C, a minor sialoglycoprotein, was measured when glycophorinA was immobilized by bound ligands. In these experiments the glycophorin A ligands areunlabelled and band 3 and glycophorin C are labelled with EMA or the labelled ligandBric-10, respectively. The band 3 mobile fraction decreased dose dependently with theamount of R10 bound at the mid region of the extracellular domain of glycophorin A. Atsaturation binding, the band 3 mobile fraction falls to 7 + 3%, suggesting complete band3 immobilization. This effect was more pronounced when B14 was used because band 3immobilization is achieved even at threshold ligand binding levels. Furthermore, 9A3 doesnot alter the band 3 mobile fraction and thus has no immobilizing effect even at saturation.In contrast, the mobile fraction of glycophorin C was not affected by the ligand (R10)bound to glycophorin A. This suggests that glycophorin C is not involved in the im-mobilization of glycophorin A and band 3. The reduction of band 3 mobile fraction to7% in this experiment suggests that EMA is highly band 3 specific. This is supported bySDS-PAGE results of both Golan and Nigg [Golan et al 1986, Nigg and Cherry 1979} whoreport that greater than 801ess than 5amounts are associated with lipids.Membrane rigidity is a property of the skeletal network only because the lipid bilayer isa 2-dimensional fluid. Consequently, changes of membrane rigidity reflect conformationaland/or organizational changes within the cytoskeleton. The binding of ligands 9A3, R10and B14 to normal red cell membranes increases membrane rigidity. This effect is more62pronounced the closer the ligand binds to the bilayer. Ligand 9A3, which binds to themost distal epitope on glycophorin A, had no effect on membrane rigidity when bound atthreshold levels but increased membrane rigidity 8 fold when bound at saturation. LigandR10, which binds to the mid region of glycophorin A's extracellular domain, increasedrigidity 6 fold at threshold and 15 + 6 fold at saturation. Ligand B14, which of the ligandsbinds closest to the lipid bilayer on glycophorin A, increased membrane rigidity 46 ± 21fold at threshold concentrations and increased rigidity immeasurably at saturation. Theseresults are consistent with earlier results in which 9A3 was found to decrease membranedeformability by 5.8 fold, 10F7 by 10.8 fold and B14 by 18 fold [Chasis et al 1988]. Therigidity results show a concordance with the lateral motion results, which also suggests thatintegral protein lateral immobilization is linked to membrane rigidity. Furthermore, it isinteresting to note that although saturation binding with 9A3 has an immobilizing effect onglycophorin A, that it did not increase membrane rigidity and did not immobilize band 3.In the light of this information, increased membrane rigidity correlates more closely to band3 immobilization than to glycophorin A immobilization. It appears then that membranerigidity is controlled by band 3, rather than by glycophorin A, and that the interactionbetween glycophorin A and the cytoskeleton induced by ligand binding is an indirect oneinvolving band 3.4.2 Receptor-Ligand Interactions in Miltenberger V Red CellMembranesIn Miltenberger V red cells the dominant sialoglycoprotein is a hybrid consisting of theextracellular domain of glycophorin A and the transmembrane and cytoskeletal domains of63glycophorin B. Consequently, this hybrid glycoprotein still has binding epitopes for ligandsR10, 10F7 and 9A3 but has almost no cytoplasmic domain. In these cells the binding ofglycophorin A ligands R10 or 10F7, even at saturation, did not change the mobile fractionof glycophorin or band 3. This suggests that neither of these proteins was immobilizedand that the cytoplasmic domain of glycophorin A, in normal cell membranes, plays akey role in immobilization. Furthermore, rigidity of Miltenberger V cells was not increasedby the binding of these ligands, suggesting that increased membrane rigidity is not causedby an extracellular effect but by one involving the cytoplasmic domain. This result is alsosupported by the finding that monoclonal R10-Fab causes glycophorin A immobilizationin normal red cells because, presumably, Fab cannot cross-link the extracellular domain.4.3 Receptor/Ligand Interactions in HS Red Cell MembranesIn hereditary spherocytosis red cell membranes there is a partial spectrin deficiency. Inmild cases (< 30%) this leaves the hexagonal structure of the cytoskeletal network intact,but in severe cases (> 50%) totally disrupts this hexagonal structure to cause gross structuraldefects.Spectrin deficiency had no effect on the diffusivity of native glycophorin A although therewas a higher mobile fraction than in normal red cells. This result is discussed further below.The native membrane rigidity of these HS cells was only slightly less than that of normal redcell membranes. In the most severe case, the membrane rigidity (p) divided by the averagerigidity of normal cells (linormal) was only 0.87. This reduction is far less than expected if, as64Waugh and Agre assumed [Waugh and Agre 1988], each spectrin tetramer acts as an en-tropic spring, and membrane rigidity is simply related to spectrin surface density. However,spherocytosis has complicating factors which could directly affect membrane rigidity, such asdepleted levels of the major integral glycoprotein, band 3 and its cytoskeletal attachmentsite, ankyrin. It is also conceivable that in severe spectrin deficiencies, the spectrin isfully stretched to allow maximal sub-membrane coverage. This would cause a highly dilatednetwork which might have a reduced shear modulus but would be highly incompressible(elastically). This may explain why the composite rigidity modulus, measured in the mi-cropipette aspiration experiment, does not fall proportionately with spectrin content. Thisexplanation is consistent with Waugh & Agre's results because, although they report thatmembrane rigidity decreases linearly with spectrin surface density, the reduction is lessthan 40% in red cells with a 69% spectrin deficiency.Spectrin deficiency had a considerable effect on membrane rigidity and glycophorin Alateral motion particularly when ligands (R10 & B14) were bound to glycophorin A. Inthe two cases of mild spectrin deficiency, binding of the ligand B14 caused the immobi-lization of glycophorin A, while, in the severe case the lateral motion of ligand-boundglycophorin A increased substantially. For R10, membrane rigidity decreased with in-creased spectrin deficiency. Membrane rigidity falls from 15 x normal membrane rigidityat 0% spectrin deficiency to 10 x, 4 x and 1.5 x for spectrin deficiencies of 35%, 41% and66% respectively. These results indicate that, in normal cells, a loss of hexagonal structurein the cytoskeletal network disrupts the mechanism responsible for the immobilization ofligand bound glycophorin A.654.4 The Mobility of Untethered ReceptorsThe native diffusivities of glycophorin A were the same in normal cells, mutant gly-cophorin in Miltenberger V cells and glycophorin A and in HS red cell with severespectrin deficiency. This indicates that in normal cells neither the cytoplasmic domainof glycophorin A nor the underlying cytoskeletal network account for the native diffusivityof glycophorin A (which is 2 orders of magnitude below the viscous limit [Saffman Si Del-bruck 1975] and that of glycophorin reconstituted into lipid vesicles [Vaz et al 1981, Kapitzaet al 1984]). This conflicts with reports of increased diffusivities of the membrane proteins,band 3, in spectrin-deficient spherocytic membranes of mouse red cells [Sheetz et al 1980]and, band 3 and glycophorin, in spectrin-deficient spherocytic membranes of human[Golan et al 1990] red cells, but is consistent with reports that integral protein cytoplasmicdomain does not regulate protein lateral diffusion [Edidin Zuniga 1984, Livneh et al 1986,Scullion et al 1987, Zhang et al 1991]. Consequently, this data suggests that the diffusionof native glycophorin A is limited by interactions involving either the bilayer-spanning orextracellular portion of the protein. The involvement of the extracellular domain is likelybecause the bulk of glycophorin A is extrafacial. Furthermore, extracellular involvementis the probable cause of the hindered lateral motion of the ligand bound glycophorin inMiltenberger V cells (Table 5) or glycophorin C in normal cells (Table 9). Such hinderingof lateral motion, which decreases the diffusivity without altering the mobile fraction, hasbeen demonstrated in Monte Carlo simulations where the diffusivity of particles mobile in 2dimensions is reduced by as much as an order of magnitude when the particle surface densityincreases [Pink 1985, Pink et al 1986, Saxton 1990]. This may occur because ligand binding66increases the effective surface density of glycophorin A by increasing the volume that isoccupied (excluded volume) by the protein's extracellular domain. Thus, interactions medi-ated by the glycocalyx amoung the extracellular domains of neighbouring proteins may wellbe responsible for native glycophorin A diffusivity and the bound-ligand induced reduceddiffusivity of mutant glycophorin.4.5 Receptor Motion in Ovalocytic Red Cell MembranesOvalocytic red cells were used to test the correlation between integral protein lateral immo-bilization and increased membrane rigidity. This is caused by an increased interaction inthe cytoplasmic domain between the integral protein and the cytoskeleton. Hereditary oval-ocytosis is a naturally occurring mutation characterized by an increased membrane rigiditywhich makes the cells resistant to malarial invasion. A great deal of work by a number of re-searchers has failed to identify mutations of any of the cytoskeletal proteins, spectrin, actin,band 4.1 and ankyrin. However, this study has revealed that band 3 is immobile in heredi-tary ovalocytosis red cells. The cDNA sequence of the band 3 of hereditary ovalocytosis redcells [Mohandas et al 1992] revealed a deletion of a sequence of 9 amino acids in the cyto-plasmic domain next to the lipid bilayer. This finding by collaborators at Lawrence BerkeleyLaboratory, together with the lateral motion results (Table 12), indicates a possible confor-mational change produced by the deletion in the cytoplasmic domain which allows increasedinteraction with the cytoskeleton, and leads to band 3 immobilization and an increase inthe membrane rigidity [Mohandas et al 1992]. Finally, it is interesting to note that band 3immobilization in ovalocytic red cells did not affect the lateral motion of glycophorin A67which suggests that unliganded glycophorin A is not involved in the interactions with thecytoskeleton.4.6 ConclusionIn summary, this study has shown that the lateral motion of native glycophorin A is reg-ulated neither by its cytoplasmic domain nor the underlying cytoskeletal network. However,binding of ligands specific to the extracellular domain promote an interaction between thecytoplasmic domain of the protein and the cytoskeletal network. This interaction involvesa second receptor protein, band 3, and causes the complete immobilization of both gly-cophorin A and band 3. Lateral motion and membrane rigidity data show that an increaseof membrane rigidity relates to band 3, rather than to glycophorin A immobilization. Thefore going led to the discovery that the band 3 protein of ovalocytic red cells, which has amutation in it cytoplasmic domain, is completely immobile. This is the probable explanationof their unusual rigidity.Our understanding of the architectural components of the cytoskeleton has grown throughthe use of the model system provided by ligand-induced lateral immobilization and theincrease of membrane rigidity of red blood cells and may have important applications to theworkings of other biological systems.68ReferencesAnstee D.J., Edwards P.A.W. 1982Monoclonal antibodies to human erythrocytesEur. J. 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