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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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Interactions Between Integral Membrane Proteins and the Cytoskeleton in Red Blood Cells Studied by Measured Molecular Lateral Diffusion by  David William Knowles B.Sc.-Hons., University of New South Wales, Australia, 1982 M.Sc., University of British Columbia, Canada, 1986  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY  in THE FACULTY OF GRADUATE STUDIES DEPARTMENT OF PHYSICS  We accept this thesis as conforming to the required standard  THE UNIVERSITY OF  BRITISH COLUMBIA  March 1992 © David William Knowles, 1992  In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission.  (Signature)  Department of  Physics  The University of British Columbia Vancouver, Canada  Date  DE-6 (2/88)  ^le September 1992  Abstract Integral 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 interact with specific agents which control their functional activity. The agents, collectively termed ligands, are commonly other molecules, which vary in size from solubilized ions to large macromolecules. Receptor/ligand interactions can influence lateral organization and conformation of receptor proteins and are the mechanism by which biological signals are passed across the membrane and from cell to cell.  A single receptor protein embedded within a pure lipid bilayer has considerable lateral mobility, which is restricted only by the viscosity of the bilayer, and to a lesser extent, by the viscosity of the surrounding aqueous environment [Saffman & Delbruck 1975]. Cell membranes however, are comprised of many receptor proteins as well as a network of support proteins 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 components. Thus the cell membrane, like many biological systems, is a set of weakly and dynamically coupled components.  To understand the nature of such dynamic coupling, the effect of receptor/ligand interactions 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 well  ii  characterized integral membrane proteins plus a peripheral cytoskeletal network. The major integral proteins are band 3 and several different types of glycophorin. The major cytoskeletal protein is a long (76-200nm) filamentous tetramer known as spectrin. The ligands used were monoclonal antibodies (divalent) and their Fab fragments (monovalent). The antibodies recognize specific epitopes on the extracellular domain of glycophorin A, the major red cell sialoglycoprotein. Ligand 9A3 binds the most distal epitope of the extracellular domain, ligands R10 and 10F7 bind to the mid-region of the extracellular domain and ligand B14 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, membrane deformability (measured by flow ectacytometry) was greatly reduced in normal red cells, but not in red cell variants that lack the cytoplasmic domain of glycophorin A (Miltenberger V red cells). Because the cytoskeletal network (rather than the lipid bilayer) is responsible for membrane rigidity, these results suggested that ligand binding can initiate a transmembrane signal that causes an increased interaction of the cytoplasmic domain of the receptor with the underlying cytoskeletal network.  To test this hypothesis and to investigate the molecular features of such a mechanism, the lateral 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 the amount of ligand (eg. 9A3, R10, 10F7 or B14) bound to the extracellular domain of glycophorin A in both normal and variant red blood cells. Lateral motion (lateral mobility  iii  and mobile fraction) of the integral proteins was measured in situ by the technique of fluorescence recovery after photobleaching (FRAP). This technique involves: (1) quantitating the fluorescence intensity from labels conjugated to the ligand or receptor of interest on the membrane; (2) irreversibly bleaching a small region of the membrane and (3) measuring the subsequent fluorescence recovery due to lateral motion of fluorophore on the surface. Red cell membrane rigidity was measured by the method of pipette aspiration where a portion of the red cell membrane was deformed into a pipette by a minute suction pressure. The dependence of aspiration length on applied pressure is linear for elastic deformations and results in an effective rigidity modulus which is a combination of the elastic shear and the area expansion moduli of the membrane cytoskeleton.  The findings are that anti-glycophorin A ligands (R10 & B14), bound to normal red cell membranes, cause the immobilization of both glycophorin A and band 3, and produce a marked increase in membrane rigidity. These effects are due to an increased interaction between the cytoplasmic domain of the integral proteins and the cytoskeletal network. This was evident for several reasons: (1) the effect was independent of ligand valency, indicating that extracellular cross-linking was not responsible for the immobilization; (2) lateral immobilization and increased rigidity were not seen if ligand was bound to Miltenberger V red cells (In these cells the cytoplasmic domain of glycophorin A is effectively absent due to natural mutation); (3) only partial immobilization and a small increase in membrane rigidity was seen when ligand was bound to spherocytic red cells (These cells are characterized by a severe spectrin deficiency which produces gross defects in the normal hexagonal structure of the cytoskeleton). Immobilization of band 3 resulted only for anti-glycophorin A ligands iv  R10 (10F7) and B14. These ligands bind close to the bilayer surface and produce the greatest increase in membrane rigidity. Ligand 9A3 however, had less of an immobilizing effect on 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 of band 3, and because the cytoplasmic domain of glycophorin A is a small peptide, it is proposed that the cytoplasmic domain of ligand bound glycophorin A interacts (directly) with band 3 to cause immobilization of both integral proteins, and increased membrane rigidity. Finally, this proposed link between band 3 immobilization and the increased membrane rigidity was tested in the case of ovalocytic red cells which are characterized by a tolerance of malarial invasion, and an increased membrane rigidity. It has been long assumed that the high rigidity of these cells, which is responsible for their malaria parasite tolerance, is due 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. A parallel collaborative study shows that ovalocytic band 3 contains a mutation, a deletion of 9 amino acids in its cytoplasmic domain [Mohandas et al 1992]. Thus, it is apparent that the interaction of band 3 with the underlying cytoskeleton plays a crucial role in the determination of membrane mechanical properties, and the lateral motion of integral receptor proteins, and serves as a useful model for the understanding of the molecular mechanisms involved in receptor protein function.  v  Contents Abstract^  ii  Contents^  vi  List of Figures^  ix  List of Tables^  x  xii  Acknowledgements^ 1  2  1  Introduction 1.1  Red Cell Membrane Organization ^  1.2  Laterally Motion of Integral Membrane Proteins ^  1 10  Experimental Methods  17  2.1  The FRAP Technique ^  17  2.2  Membrane Rigidity Measurements ^  20  2.3  Membrane Labelling ^  20  2.4  F.R.A.P. Equipment ^  23  2.4.1^Optics ^  23  2.4.2^Intensified Camera, Video Board and PMT ^  28  2.4.3^Analysis of Recovery Kinetics ^  30  2.4.4^Spot Size Determination ^  32  Red Cell Membrane Elastic Rigidity ^  34  2.5  vi  3 Experiments and Results^  36  3.1 Ligand-Induced Lateral Immobilization of Glycophorin A: Implication of Cy36 toskeletal Entanglement ^ 3.1.1 Ligand-Induced Changes of Glycophorin A Lateral Motion in Normal 37 Red Cells ^ 3.1.2 Lateral Motion of Glycophorin in Miltenberger V Red Cells ^ 39 3.1.3 Lateral Motion of Glycophorin A in Hereditary Spherocytic Red Cells 41 3.1.4 Summary ^  42  3.2 Integral Membrane Proteins Play a Key Role in Membrane Rigidity ^ 45 3.2.1 Integral Protein Lateral Motion and Membrane Rigidity in Membranes of Normal and Miltenberger V Red Cells ^ 46 3.2.2 Membrane Rigidity in Hereditary Spherocytic Red Cells ^ 48 3.2.3 Lateral Motion of Glycophorin A and Band 3, and Membrane Rigidity in Hereditary Ovalocytosis Red Cells ^ 49 3.2.4 Summary ^  50  3.3 Ligand-Induced Interaction Between Glycophorin A and the Cytoskeleton in Human Erythrocytes Involve Band 3 Protein ^ 53 3.3.1 The lateral motion of band 3 in normal red cell membranes altered by ligands bound to glycophorin A ^ 54 3.3.2 The lateral motion of glycophorin C in normal red cell membranes altered by ligands to glycophorin A ^ 55 3.3.3 The lateral motion of band 3 in Miltenberger V red cell membranes altered by ligands to glycophorin A ^ 56 3.3.4 Summary ^  vii  57  4 Discussion and Conclusion^  59  4.1 Receptor/Ligand Interactions in Normal Red Cell Membranes ^ 60 4.2 Receptor-Ligand Interactions in Miltenberger V Red Cell Membranes ^63 4.3 Receptor/Ligand Interactions in HS Red Cell Membranes ^ 64 4.4 The Mobility of Untethered Receptors ^  66  4.5 Receptor Motion in Ovalocytic Red Cell Membranes ^ 67 4.6 Conclusion ^  68  References^  69  viii  List of Figures 1  Structural Organization of the Red Blood Cell Membrane ^  4  2  Structural Organization of the Hexagonal Lattice of the Red Blood Cell Membrane ^  6  3  Fluorescence Recovery of Photobleached Glycophorin on the Red Cell Surface  18  4  Photograph of the FRAP Equipment ^  23  5  Laser Based Epi-Fluorescence Microscope and Microprocessor Controlled Photon Counting and Intensified Image Collection ^  24  6  Double Block Beam Splitter & Attenuator ^  26  7  Correct Block Alignment Eliminates Non-Parallelism Caused by Block Wedge Angle ^  26  8  Epi-Fluorescence Optics ^  28  9  Absorption and Emission Bands of Eosin ^  29  10  Fluorescence Recovery of ETSC Labelled Glycophorin ^  32  11  Gaussian Intensity Profile Convolved with the Point Spread Function of the Objective ^  33  12  Pipette Aspiration Measures Membrane Rigidity ^  34  13  Antibody Binding Epitopes on Glycophorin A ^  37  ix  List of Tables 1^The Major Proteins of the Erythrocyte Membrane  ^3  2^Measured Diffusivity of Lipid and Integral Proteins in Bilayer Vesicles and 14 Red Cell Membranes ^ 3^Alteration of Lateral Motion of ETSC-Labelled Glycophorin A in Normal Red 38 Blood Cells: The Effect of Ligand Binding ^ 4^Alteration of Unlabelled Glycophorin A in Normal Red Blood Cells: The 39 Effect of Binding Labelled Ligand ^ 5^Alteration of Lateral Motion of Hybrid Glycophorin in Miltenberger V Red 40 Cells: The Effect of Ligand Binding ^ 6^Alteration of Lateral Motion of ETSC-Labelled Glycophorin A in Hereditary Spherocytosis Red Cells: The Effect of Spectrin Deficiency ^ 42 7^Alterations of Lateral Motion of Glycophorin A in Hereditary Spherocytic Red Cells Produced by Saturation Binding of Labelled Ligands ^ 42 8^Alteration of Rigidity of Normal Red Cell Membranes by Ligands to Glycophorin A ^ 47 9^Alteration of Rigidity of Normal Red Cell Membranes by Ligands to Glycophorin C ^ 48 10 Alteration of Membrane Rigidity in Miltenberger V Red Cells by Ligands to Glycophorin ^ 48 11 Alteration of Membrane Rigidity in Hereditary Spherocytic Red Cells by Ligands to Glycophorin A ^ 49 12 Lateral Motion of ETSC-labelled Glycophorin A and EMA-Labelled Band 3 in, and Membrane Rigidity of Ovalocytic Red Cell Membranes ^ 50 13 Alteration of EMA-Labelled Band 3 Motion in Normal Red Cells by Ligands to Glycophorin A ^ 55 14 Lateral Motion of Glycophorin C Bound with Fluorescently labelled Bric-10 in Normal Red Cells Altered by Unlabelled Ligands to Glycophorin A . . . . 56  x  15 Lateral Motion of EMA-labelled Band 3 in Miltenberger V Red Cells Altered by Unlabelled Ligands to Glycophorin A ^  xi  57  Acknowledgements  With great pleasure I thank:  Dr. Evan A. Evans, research director, Dr Mohandas Narla for his constant enthusiasm, many helpful discussions and who, along with his colleague Dr. Joel Chasis, supplied the antibodies, the variant red cells and without whom this work would not have been done, Andrew Leung for his experimental and technical excellence, The rest of our (extended) lab: David Needham, who has followed my progress and given so much sound advice; David Berk, Dan Klingenberg and John Ipsen for many helpful discussions; Tony Yeung for many supportive discussions and lamb chops; Wieslawa Rawicz for many a wonderful coffee; Barbara Kukan for showing me, faced with almost certain doom, how to "have it all"; and Andreas Zilker whose positive attitude helped me keep it all in perspective and for help with the diagrams. Thanks also to the groups of Myer Bloom (Physics), Pieter Cullis (Pharmacology) and Mike Wortis (Physics, SFU) whose participation made the summer seminars of 1991 such a success and from which I learnt a great deal, Peter Hass and the Physics machine shop technicians, whoes ability to turn a rough sketch into beautiful reality is world class, Jim Sibley and Rolf Muelchen in the Pathology workshop, for all their work and borrowed tools, Lore Hoffmann, Physics Graduate Secretary and Secretary to the Head, for much help with bureaucratic matters and keeping us grad students 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 Elsie for so much support, And, of course, 'The Girls of the Five'.  1 Introduction 1.1 Red Cell Membrane Organization  The 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 functionally 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 the ,  red cell membrane include phospholipids, namely phosphatidyl -choline (PC), -ethanolamine (PE), -serine (PS), sphingomyelin and a neutral sterol, cholesterol. Distribution of phospholipid is asymmetric with most of the PC and sphingomyelin residing in the outer layer and most of the PE and PS in the inner layer [Marchesi Furthmayr 1976] of the red cell membrane.  Extrinsic proteins in the red cell membrane are adjacent to the bilayer and form the supporting cytoskeletal network. These proteins are so named because of their ease of extraction by the use of low ionic strength buffers [Yu et al 1973]. Once extracted, the proteins are classified by differences in their electrophoretic migration through polymer gels' [Fairbanks 'The major method of red cell membrane protein classification is sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) [Fairbanks et al 1971]. Proteins are dissociated by the binding of the anioic detergent, SDS. The amount of SDS bound and the resulting charge depends on the mass of the protein, thus electrophoretic 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, sialoglycoproteins are stained using Periodic acid and Schiff reagent (PAS), or the proteins can be specifically labelled prior to electrophoresis.  1  et al 1971] which separates the proteins into bands. The major red cell cytoskeletal proteins are bands 1 & 2, which are the a & 13 subunits of spectrin; actin (band 5), band 4.1 and ankyrin (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 are so named because they are not extracted by low ionic strength buffers. Instead, strong detergents are required which solubilize the bilayer and free the integral protein. The major integral 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 PAS 4? or sialoglycoprotein ,3) [Furthmayr et al 1975]. These proteins are glycosylated on their extracellular domain and the resulting sugar/protein matrix is called the glycocalyx.  2  Table 1. THE MAJOR PROTEINS OF THE HUMAN ERYTHROCYTE MEMBRANE Protein  Subunit MW  Native Assembly  Copies/Cell  Extrinsic Proteins Band 1 Spectrin Band 2 Band 2.1 Ankyrin Band 4.1 Band 5 Actin Tropomyosin Adducin  260,000 c, 225, 000/3 215,000 78,000 43,000 29, 000 a ; 27, 0000 103, 000 a ; 97, 000 0  (a, /3)2 tetramer  105 tetramers  monomer  105 2 x 10 5 5 x 10 5 oligomers 7 x 10 4 dimers 3 x 10 4  Integral Proteins Band 3 Glycophorin A GP Glycophorin B GPS Glycophorin C GP /3  oligomer,12-17units hetero-dimer hetero-dimer dimer/tetramer dimer  89,000 31,000 23,000 29,000  10 6 monomers 6 x 10 5 monomers 8 x 10 4 5 x 10 4  The 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 protein having an average span of 76nm in the membrane skeleton [Sheetz 1983] but spanning some 200nm when fully extended [Shen et al 1986]. In normal red cell skeletons 95% of spectrin is in the tetrameric form but the tetramer/dimer equilibrium can be altered by adverse conditions [Palek & Lux 1983]. Electron micrographs of membrane shells show that between 5 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 network is a hexagonal lattice with many structural defects (Fig.2). The junctional complexes are stabilized 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 junctional 3  The Red Blood Cell Membrane Glycoptu3rin B  Gtycoph.orin A^Glycophorin. C  ;#1  Band, 3 GLycocalyx  11 11111111111 3pectrin Tetramer)  Bilayer Band  A nizYrin^6 • junctional Actin (Oligomer) Complex  CytoSkeletal Network  Figure 1 Schematic Cartoon of the Structural Organization of the Red Blood Cell Membrane The red cell membrane consists of a lipid bilayer spanned by integral proteins which are supported by a hexagonal skeletal network. The major integral proteins are band 3 and glycophorin and the major skeletal protein is spectrin.  4  complex and have a low affinity association with several of the junctional components (for a current review see [Bennett 1990]). Evidence of this stabilization is seen in in vitro solubilized spectrin 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 increased fragility, are stabilized and regain normal strength when purified band 4.1 is bound back into their cytoskeletons [Takakuwa et al 1986].  Ankyrin is another spectrin-binding protein [Bennett & Stenbuck 1979a]. The binding domain for ankyrin on spectrin is away from the junctional complex. Ankyrin and band 4.1 play important roles because they bind to the integral proteins band 3 [Bennett & Stenbuck 1979b, 1980] and glycophorin C [Mueller & Morrison 1981] respectively. Such linkages are thought to exist because removal of the lipid bilayer by Triton 2 extraction leaves the majority of the integral proteins band 3 [Bennett & Stenbuck 1979b; Sheetz 1979; Bennett 1982] and glycophorin C [Mueller & Morrison 1981; Anstee et a/ 1984] attached to 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 cytoskeletal network is tethered to the bilayer and could explain the reduced lateral mobilities [Jacobson et 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 chain 2 Triton X is a nonionic detergent which totally disrupts the bilayer by solubilizing the lipid. It leaves the red cell cytoskeletal network (shell) completely intact and in its characteristic discoid shape.  5  Hexagonal Lattice of the Cytoskeletal Network  Figure 2: Schematic Cartoon of the Structural Organization of the Red Blood Cell Cytoskeleton The red cell membrane cytoskeletal network consists mainly of a long (76-200nm) filamentous tetrameric protein, spectrin. Five to seven spectrin tetramers associate with other stabilizing globular proteins, actin and band 4.1, into junctional complexes, thereby forming a hexagonal lattice underneath the lipid bilayer.  6  connected 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] shows that band 3 has a large (hydrophobic) membrane spanning domain and a considerable  —NH3 terminal cytoplasmic domain. A current model suggests that the membrane-spanning domain traverses the bilayer 14 times [Lux et al 1989] resulting in several extracellular loops which extend above the bilayer 4-5nm [Low 1986]. The extracellular domain contains the antigenic determinants involved in blood group specificity (A, B, 0 and possibly Rh and Kell) and is the probable antigen targeted by the auto-immune system at the end of the 120 day average life span of a red cell. The cytoplasmic domain of band 3 is of particular interest because interactions between it and the cytoskeletal network have a marked effect on membrane rigidity [Mohandas et al 1992; Knowles et al 1992(in preparation)]. This domain consists of the first 403 amino acids of the protein which can be cleaved from insideout red cell membranes by treatment with one of several proteinases. This results in a 43kDa fragment consisting of two domains which are linked by a possible 'hinge' and which extends some 25nm beneath the bilayer [Low 1986]. In situ band 3 is thought to exist in a dimer/tetramer equilibrium which favours the dimeric form [Nigg & Cherry 1979]. Both forms are recovered by detergent extraction, and although the ratio of dimer to tetramer varies with the methods the majority is found as a stable dimer.  The other major integral proteins are sialoglycoproteins which separate into 4 bands on SDS gels (PAS1,2,3&4) and are selectively stained with PAS reagent due to their sialic acid content [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) 7  of 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 spanning portion of the protein [Furthmayr 1977], and because the majority of extracted glycophorin A forms a stable dimer, it is thought to exist as a dimer or higher aggregate within the membrane. Glycophorin A has a molecular weight of 3lkDa and there are approximately 6 x 10 5 copies/red cell [Merry et al 1986]. It is a transmembrane protein of 131 amino acids [Tomita et al 1978] containing a single 22 amino acid non-polar region separating two polar regions suggesting that the protein traverses the bilayer once. It has 16 oligosaccharide chains connected to the extracellular —NH3 terminal third of the protein which account for 60% of the molecule's mass [Marchesi et al 1976]. This extracellular region carries the M and N blood group antigens, but other functions are unclear especially since some donors completely lack glycophorin A yet are normal and healthy. The C terminal cytoplasmic domain of glycophorin A contains 39 amino acids [Furthmayr 1977] which could extend some 8-10nm below the bilayer.  The minor sialoglycoproteins are glycophorins B (PAS3) and C (PAS4). Glycophorin  B is a 23kDa protein having 8 x 10 4 copies/red cell. It is a transmembrane protein which traverses the bilayer once although its cytoplasmic domain is small, consisting of only 6 amino acids. Its extracellular domain is related to that of glycophorin A because it has the same —NH3 terminal 26 amino acid sequence, but its attached carbohydrate is slightly different. Glycophorins A and B are thought to be encoded by adjacent genes because mutations occur where these proteins are replaced by mutant, hybrid glycophorins which are combinations of the two [Merry et al 1986]. The amino sequence of glycophorin C 8  is not related to glycophorin A although structurally there are similarities. It is a 128 amino acid transmembrane protein with 11 —NH3 terminal-linked oligosaccharide chains on its extracellular domain. Its molecular weight is 35kDa and there are 5 x 10 4 copies/red cell.  9  1.2 Laterally Motion of Integral Membrane Proteins  Integral 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 and active cell processes and are hindered by the viscous drag of the bilayer and soft (order kT, where k the Boltzman's constant multiplied by T the temperature equals the available thermal energy) association between proteins. The dominant driving energy in the case of the erythrocytes is kT simply because the cell is devoid of internal organelles and is much less biologically active than real cells and thus integral proteins move (diffuse) randomly in the plane of the membrane with a mean square displacement proportional to the diffusivity and the time' (Equ.1).  (x2)  =  4Dt  (1)  3 The diffusion process is analyzed in statistical physics as a random walk. In each time step, the diffusing particle makes a random spatial step and its mean square displacement is plotted as a function of time (its mean displacement being zero). Consequently, the plane on which the particle moves can be divided into a 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 undergoes classical diffusion and its mean square displacement increases linearly with time (Eq.1). However, it is possible that all the sites are not equally accessible; for example, some sites may be sterically blocked. One then assigns a probability, p, of a site being accessible. As p decreases from unity (all sites accessible), the diffusive process becomes increasingly hindered and although at long times its mean square displacement is still 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 this is 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 a certain 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 been used to study the motion of single receptor molecules [Slattery et al 1991a] but the results are yet to be published (personal communication with Watt Webb's group, Cornell University, New York).  10  The lateral diffusivity of a membrane protein is an indirect measure of its specific and non-specific interaction with the other membrane components. Such interactions can be modelled in terms of an average drag coefficient (bd rag ) defined as the drag force on the protein (F drag ) divided by its average velocity (v). The inverse of the drag coefficient is the mobility (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 can consider a single integral protein diffusing in the plane of a pure lipid bilayer membrane. In such a case the lateral motion of the integral protein is restricted only by the viscosity of the lipid bilayer and that of the surrounding aqueous medium 4 . Saffman and Deldruck have calculated 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 a weak 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 in a pure lipid bilayer of 2 x 10 -8 cm 2 /sec.  D kTm ,  kT kTv  b dra g  ^drag  (2)  4 A diffusing particle, physically restricted to 2 dimensions, always interacts with the supporting third dimension. This must be accounted for mathematically, otherwise a non-physical solution results where the 2D velocity field generated around the diffusing particle decays too slowly, resulting in a drag coefficient that diverges (Stokes' Paradox).  11  D =  kT  47Mmemt  ^[  log  ilmemt^7 ;  (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), qw,20 C , surrounding aqueous viscosity 10 -2 poise = 10 -3 kg1(m.sec), kT30OK 4 x 10 -21 J, DFree = 2 x 10 -8 cm 2 /sec.  This has important biological consequence, especially in diffusion limited reactions, because integral proteins of any size can move laterally at the same rate (theoretically). In fact glycophorin reconstituted into giant lipid vesicles in their fluid state has the same diffusivity as the lipid [Kapitza et.a11984]. The positive experimental consequence of this is that integral protein diffusivity is almost unaffected by the size of labels linked to them in lateral motion studies. 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 extracellular domain and only slightly reduce their measured diffusivity [Genes et al 1988, Sheetz et al 1989].  Lateral motion of integral proteins is allowed because the lipid bilayer behaves as a fluid in the plane of the membrane. When integral proteins are reconstituted into artificially-made lipid bilayer vesicles, they have nearly the same lateral diffusivity as the free lipid [Vaz et al 1981,1982 & 1984; Jacobson et a/ 1981; Kapitza et a/1984]. This value of  ti  4 x 10 -8 cm 2 /sec  agrees with the value predicted theoretically (Equ. 3) and when it is measured by the technique of fluorescence photobleaching (Chapter 2) one finds that the fluorescence recovers 12  to 100% of its original value. This indicates that all the molecules (lipid & protein) are free to move laterally. Interestingly enough, the lateral diffusion of lipid in red cell membranes is an order of magnitude lower than this free value. Red cell lipids diffuse at ,-- 4 x 10  -9 cm 2 /see  [Bloom & Webb 1983; Golan et al 1984; Rimon et al 1984; Golan 1989]. This agrees with the values measured for fluorescently labelled PE incorporated into the red cell membrane and in both cases the fluorescence recovery is 100%. The reduction in mobility is presumably due to the high density of integral proteins that sterically hinder the lipid's motion, forcing them to diffuse through a percolative network of integral proteins [Saxton 1989]. Hindered lipid diffusion in red cell membranes is not surprising in light of the finding that the lateral diffusion of the integral proteins differ in two ways from the ideal value. First, the measured diffusivity is decreased by some 2-3 orders of magnitude; and second, the fluorescence recovery is considerably less than 100%. The reduction of diffusivity could result from the congestion of integral proteins but the discovery of an immobile fraction clearly indicates the involvement of the underlying cytoskeletal network because the diffusivity of cytoskeletal spectrin is less than 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 free value is varied and far from understood.  13  Table 2. MEASURED DIFFUSIVITY OF LIPID AND INTEGRAL PROTEINS IN BILAYER VESICLES AND RED CELL MEMBRANES Component  System  NBD-PE Glycophorin Glycophorin Fluorescent PE Phospholipid Cholesterol Glycophorin A  PC Vesicle PC Vesicle DMPC Ves. RBC Mem. RBC Mem. RBC Mem. RBC Mem.  Band 3  RBC Mem.  Spectrin  RBC Skel.  D [cm 2 I sec]  Recovery [%]  4 x 10 -8 4 x 10 -8 (1 — 2) x 10 -8 (3 — 5) x 10' (2 — 4) x 10 -9 (2 — 4) x 10 -9 7.8 x 10" (2 — 3) x 10 -11 (2 — 3) x 10' (1.2 — 2.4) x 10 -11 « 2 x 10 -12  100% 100% 100% 100% 100% 100% 61% 66%-90% 41% 45%-78%  [Ref.] [Kapitza 1984] [Kapitza 1984] [Vaz 1980, 1982] [Golan 1989] [Golan 1984] [Golan 1984] [Knowles 1992] [Golan 1989] [Knowles 1992] [Golan 1989] [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 glycophorin C [Mueller & Morrison 1981] to the spectrin cytoskeleton could be responsible for the im-  mobile fractions measured for these integral proteins [Golan & Veatch 1982]. Incubating the cytoplasmic side of red blood cells with the spectrin binding domain of ankyrin dissociates the 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 mutant mouse red cells which have a complete spectrin deficiency [Sheetz et al 1980], and is higher than normal in human red cells with a partial spectrin deficiency [Golan et al 1991 ASH Abstract]. Band 3 diffusivity also increases if red cells are incubated in a low ionic strength buffer 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 form 14  of tethers [Berk et al 1988]. In contrast to these findings there are many examples where the cytoskeletal network is not responsible for the reduced mobility of integral proteins. If membrane attachment sites immobilize a sub-population of integral proteins, then it is not clear what limits the diffusivity of the mobile portion of the population. Furthermore, deleting most of the cytoskeletal domain of: G proteins in vesicular stomatitis virus [Scullion et al 1987]; epidermal growth factor (EGF) [Livneh et al 1986]; or L d antigens on L cells [Edidin & Zuniga 1984] does not alter their lateral diffusivity.  Another possible region of interaction between membrane bound components is within the bilayer itself. Attractive interactions between bilayer spanning domains of glycophorin A result in the formation of stable dimers. [Furthmayr 1977]. The diffusivity of mobile particles interacting by simple hard core repulsion has been shown to decrease their diffusivity by an order of magnitude as the surface density increases in Monte Carlo simulations [Pink 1985; Saxton 1990]. If attractive, cluster-inducing, interactions are included in such simulations the diffusivity decreases markedly [Pink et al 1986]. Thus simple steric interactions, within the bilayer, could reduce the lateral diffusivity of integral proteins and is the probable cause of 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 integral proteins. This is not surprising because often the majority of an integral protein is extracellular. The diffusivity has been shown to increase linearly with loss of glycosylation of the extracellular domain of mouse Ld class I glycoproteins [Wier & Edidin 1988] while the cytoplasmic and bilayer spanning domains have been shown to have little effect on the mobility  15  of GPI-linked proteins [Zhang et al 1991], or G proteins [Scullion et al 1987]. Interactions in each of the domains, cytoplasmic, bilayer spanning and extracellular, can potentially limit the lateral mobility of integral protein. As to which protein domain interaction predominates will depend on the type of integral receptor protein and its state of conformation.  16  2 Experimental Methods 2.1 The FRAP Technique  The lateral motion of fluorescently labelled macromolecular components whose motion is confined to the plane of the erythrocyte membrane, was measured by the technique of fluorescence 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 both the lateral mobility and the mobile fraction of membrane components and involves several steps. First, the membrane component to be studied was fluorescently labelled. This was done by several methods which attach the fluorescent probe covalently to the membrane component, or indirectly by labelling a specific ligand. Band 3 and glycophorin A were labelled directly by fluorescent probes eosin-5-malimide (EMA) [Nigg Si Cherry 1979] and eosin-5-thiosemicarbazide (ETSC) [Golan et al 1986] respectively. Glycophorins A and C were labelled indirectly by binding fluorescently labelled monoclonal antibodies which are specific for their extracellular domains [Edwards 1980; Anstee & Edwards 1982; Bigbee et al 1983; 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) beam of 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 measured intensity profile and its size is controlled by the FRAP optics. Third, the fluorophor in the area of interest was irreversibly photobleached by a short pulse of a high-intensity (--, 1.3mW,  17  Figure 3: Fluorescence Recovery of Photobleached Glycophorin on the Red Cell Surface 1.) Glycophorin on the red cell surface was labelled with ETSC and imaged in a uniform full field of illumination. The fluorescence labelling was uniform and the variations in intensity are due to the curvature of the membrane.  2.) The second image was taken 0.5 seconds after a region of the cell membrane was irreversibly bleached with a pulse of the Gaussian bleach beam. The normal pulse duration was 20msec but these images were overbleached by a pulse of several seconds to clearly show the bleached area.  3.) The third image was taken approximately lhr after the bleach pulse and shows the recovery of fluorescence due to the lateral motion of labelled glycophorin in the plane of the membrane. The characteristic recovery time for most of the glycophorin is tens of seconds. However, even after 1 hr the fluorescence has not recovered to 100%, indicating that a fraction of the glycophorin is either almost immobile or its lateral motion restricted to small domains.  476 nm) beam of light. The photobleaching-associated cell-surface heating and possible membrane photodamage is minimal and does not affect the lateral motion of the membrane components [Axelrod 1977; Jacobson et al 1978]. Fourth, the low-intensity beam was used to measure the fluorescence recovery in the photobleached area. Recovery is produced by the interchange of unbleached and bleached fluorescently-labelled membrane components as they diffuse across the boundary of the photobleached area (Fig. 3). Finally, recovery kinetics were fitted theoretically to determine the diffusivity and mobile fraction of the labelled component [Axelrod et al 1976; Koppel et al 1979; Yguerabide et al 1982].  18  It is important to realize the limitations of the FRAP methodology. Diffusivity and mobile fraction are least square fit parameters, assuming single component diffusion [Axelrod et.al 1976, Yguerabide 1982], and relate to the temporal window of the FRAP experiment. The lower limit of several milliseconds is determined by the response-time of the shutter and the 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 34 recovery values are measured per cell. This repetition reduces the errors arising from interrogative bleaching while maximizing the signal to noise ratio. The largest errors are due to the sensitivity limit of the PMT and the ability to properly focus the membrane with the illumination spot. The mobile fraction is affected by the first of these types of errors with a typical percentage error of 10%. Diffusivity is affected by both errors and is proportional to the square of the beam-waist diameter. When the size of this waist is determined by fitting its measured intensity profile to a Gaussian function, convolved with the point spread function of the objective, the error is 1%. However, during an experiment a red cell, which is 1-2pmthick, is focused with a conservative focus error of 0.1 to 0.2 pm. This equals the beam waist error because the numerical aperture of the objective is 0.75. Thus, the error in diffusivity can be as high as 60%. The important point is that the accuracy of the FRAP technique is limited to changes of several percent in mobile fraction and to changes by factors of 2 in diffusivity.  19  2.2 Membrane Rigidity Measurements  The elastic rigidity modulus of the red cell membrane was determined by pipette aspiration experiments [Berk et al 1989; Evans & Skalak 1980]. In these experiments a portion of the red cell membrane was deformed into a glass pipette under the influence of a minute suction pressure. The rigidity modulus is inversely proportional to the change in aspiration length with the suction pressure and the deformation is considered to be elastic if this modulus is the same for both the loading and unloading phase (Fig. 12). For ease of analysis, the membrane portion is chosen at the dimple of the discoid cell which makes the deformation axially symmetric. There is a maximum useful aspiration length which decreases with increased rigidity because at some point the work done by the applied pressure goes into membrane buckling.  2.3 Membrane Labelling  Normal blood from healthy volunteers was extracted by vene puncture. Whole blood was stored 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). Miltenberger V and normal travel-control blood samples were supplied by Drs. Mohandas & Chasis, of the Lawrence Berkeley Laboratory, Berkeley, California. This blood was stored at 77K and fast-thawed when needed. Three samples of spherocytic blood were shipped at 4° Cby Dr. 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 blood samples and at the same time as the lateral motion experiments.  20  Ovalocytic blood samples from six individuals, resident in Kuala Lumpur, were supplied by Dr. Mohandas. The blood was shipped direct from Kuala Lumpur along with normal travel-controls. Monoclonal anti-glycophorin A antibody and its Fab fragment were supplied by Dr. Anstee of the South Western Regional Blood Transfusion Center, Bristol, UK. The anti-glycophorin A was fluorescently labelled at the Berkeley Laboratory. Eosin5-thiosemicarbazide (ETSC, cat.# E-120) and eosin-5-maleimide (EMA, cat.#E-118) were purchased from Molecular Probes (Eugene, Oregon, USA). The oxidizing agent, sodium mperiodate, 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 suspended and washed 3 times in lml of PBS/HSA buffer at 4° C. Sialic acid was oxidized by, 50plof 10mM NaI0 4 stock added to prewashed red cells to produce a final concentration of 0.5mM in which cells were incubated for 10min at 4° C, in the dark, before being washed 3 times in PBS/BSA. Finally, ETSC was dissolved in PBS/HSA buffer at 1mg/m1 from which 50plwas added to the washed cells producing a final ETSC concentration of 50µg/ml, the cells were incubated for 30min and again washed 3 times in PBS/BSA. It should be noted that, although ETSC is the state-of-the-art covalent tag for glycophorin A, it is only semi-specific. This results because: (1) ETSC binds only to oxidized sialic acid residues; (2) sodium periodiate is 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 only 60% of the ETSC bound to the red cell surface associates with glycophorin A. 21  Band 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 suspended and washed 3 times in lml of PBS/HSA buffer at room temperature. EMA was dissolved in PBS/HSA buffer at lmg/ml from which 50plwas added to the washed cells producing a final ETSC concentration of 50/ig/ml. The cells were incubated for 30min and washed 3 times in PBS/HSA. The association of EMA with band 3 appears to be highly specific. Golan [Golan et al 1986] reports that over 80% of the EMA bound to red cell surfaces is associated with band 3 and less than 5% associates with sialoglycoproteins.  The extracellular domain of glycophorin A was bound with labelled or unlabelled antiglycophorin A by incubating very low numbers of red cells in a PBS/HSA solution of the  required ligand concentration. These concentrations were determined from measured binding isotherms relating the incubation concentration to the amount of ligand bound to the cell surface. For microscopy studies, labelled red cells were sealed in a chamber made by sandwiching a ring of vacuum grease between two glass cover slips. The chamber was sealed to prevent thermal currents produced by evaporation. In cases where the fluorescence background from the incubation medium was measurable, the incubating medium was replaced with unlabelled buffer by pipette aspiration once the cells had settled and before the chamber was sealed. This method prevented agglutination of IgG-bound cells which result from washing by centrifugation. All experiments were done at room temperature (25.0+-0.5° C).  22  2.4 F.R.A.P. Equipment  Figure 4: Photograph of the FRAP equipment showing the laser, optical table, microscope, intensified CCD camera and a fluorescence image of two red cells in contact.  2.4.1 Optics  The 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 matches the 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 split at 90deg (Fig. 5) was used to produce a full field of illumination with even intensity at the microscope object plane. The other was used to produce the coaxial interrogation and bleach beams. These were produced by the next optical element, a partially aluminized double glass 23  ^  Laser Based Epi-Fluorescence Microscope with Microprocessor Controlled Photon Counting and Intensified Image Collection Beam Steerer  Double Block Splitter  diti^ w4 ..41toN • Aciori __ 111 prer^ ^A '■1 1.a.h.k 1 1 0110  Splitter  Shutter  ■  Spatial Filter  Laser 476 ntn  Shutter  Spatial Filtez  Splitter ABC CCD  Objective, 40X, 1‘10.75 Epi—illumination Diohroio Barrier  Shutter  Video Comer  Dichroic Ocular  Beam Steerer Pre— Amp  Fiber  Intensifier  -  Optic Cooled P VIT  f;i1:241!  Counter  arark-uULri  Visual Display  Figure 5: FRAP Schemitic An argon ion laser beam was split in two, one attenuated by 10 -3 . Computer-controlled shutter synchronization allowed independent use of these two beams for illumination. The two beams were recombined and focused at the back image plane of an epi-fluorescence microscope. Conjugate images were produced by the 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. This light was collected by the objective lens and imaged via an ocular onto a photomultiplier tube. The experiment 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 the bleached area due to lateral motion of unbleached molecules back into the area.  24  block beam splitter/attenuator, which includes two shutters for controlling the beams. This optical set up was introduced into FRAP methods by Koppel [Koppel 1979] and represents a significant improvement over previous methods using solenoid driven neutral density filters because of increased alignment precision, speed, and the lack of vibration caused by moving parts.  The simplicity of the optical setup is seen in Figure 6, where a secondary beam is split and subsequentally recombinded with the first on the original optical axis. The two unaluminized internal reflections reduce the intensity of the secondary beam by three orders of magnitude with respect to the first. The separation of the beams between the blocks is determined by the block thickness, its refractive index and the angle of incidence (Equ. 4). This distance is ultimately limited by the thickness and length of the block (Equ.5).  d = 2t  Omax =  sinOcosO  V  n2  g—  20  (4)  sin  sin -1 fngsin [tan -11 /( 3 0]1  (5)  The blocks, supplied by Spindler and Hoyle (Germany), were made from optical quality glass with optically flat surfaces. A significant problem arises if the faces are not parallel. The internal reflections of the attenuated beam result in an angle between the beams which is more than twice the wedge angle of the block. Even 5 arc seconds ( , 1 thousandth of a degree) between the beams results in considerable alignment error of the two spots in the  25  Correct Block Alignment Eliminates Non-Parallelism Caused by the Block Wedge Angle Double Block Beam Splitter & Attenuator  Figure 6: The double glass block beam splitter/attenuator produces two coaxial beams one attenuated 3 orders of magnitude from the other. The beams are used to produce the observation and bleach spots for the FRAP experiment.  Figure 7: A minute block wedge angle causes the two beams to be non-parallel. Even slight non-parallelism causes considerable alignment error of the two spot images.  plane of the objects. This problem was overcome by cutting the two splitter/attenuator blocks from a single piece. The non-parallelism of the beams produced by the first block can then be cancelled by the second block if it is positioned correctly.  The next optical element was a beam steerer supplied by Newport Corporation, Fountain Valley, California. It not only permits the combination of several beams and makes the FRAP table layout compact, but provides the fine lateral adjustment of the beam required for subsequent optical elements (namely the objective lens) which are less easily positioned 6 For  a beam incident at Oi on a block with refractive index n g and wedge angle between its faces of 0  the primary and secondary beams are transmitted at angle of 0;2  = sin  -  1  (n g sin[sin i^ -  -  On = sin -1 (n g sin [sin -1  30]). The difference (89 =Ott  -  ( -31;:1-e-L) - B] ) and  0t2) is the non-parallelism introduced  by the block wedge angle and for small angle approx.. which introduces the smallest angle, 80 = 2n g G. This angle causes a misalignment of the two spot images produced by the 5cm lens (Fig. 5). The image waist diameter produced by this lens is d = sin0 = 16.7pm. For an alignment displacement error of 5% geometric optics gives 50 = 6 05x5e1 m 6 7im which is less than 3 arc seconds!!  96  (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 the beam profile at its focal plane. This lens, which focuses the beam so it can be filtered by a pin hole, was positioned so that the beam image was at the back image plane of the objective lens. Also, its focal length was chosen such that the divergent light from the beam image just fills the back aperture of the objective. The waist diameter, d, produced by this lens is given by diffraction theory, d A/NA, where A is the wavelength and NA stands for the numerical aperture. This focused waist is the Fourier transform of the angular dependence of the light entering the lens. Consequently, any diffractive artifacts produced by the preceding optics were filtered off at this point by masking the beam image with a pin hole (spatial filter), the diameter 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 a second 50/50 non-polarizing beam splitter cube. Beyond this point the beams entered an inverted 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 objective 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 shorter wavelength) was collected by the objective lens. The unwanted scattered light was rejected by the dichroic/barrier filter pair which in combination have a transmittance to the shorter  27  Figure 8: Epi-Illumination & Fluorescence Optics The stimulating radiation is reflected by the dichroic filter to the object using the objective as a condenser. The fluorescence emission is collected by the objective and passed through the dichroic/barrier pair which has a transmittance 10 -6 to the stimulating light. The figure also Fleur eseeace • Emissiaa x Stitaalatlag Realm ilea  indicates the Gaussian beam waist.  Barrier Filter  wavelength of ti 10 -6 but which allow the longer fluorescent wavelength to pass. The fluorescence 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 imaged onto the scanning plane of an optical fiber of 0.025 inches diameter. The fiber was positioned at the image of the observation spot and collects the fluorescence radiation. The light transmitted 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 PMT  Two photosensitive devices were used to quantify the intensity of the fluorescence image produced by the microscope. The first was a second generation intensifier (Genllsys) optically coupled 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 board  28  KITC Absorption & Emission Bands Absorption Emission Laser Line (476nm)  Oichroic & Barrier  ket  ppir  O  Oichroic  .... ........  • ....^• ........  ...  • • • • • .. •••••.• ...............^• ......  Emission X ^ Oichroic & ^ Barrier 0 400^500^600  ^  Wavelength (nm)  ^I  700  Figure 9: Absorption & Emission Bands of Eosin This 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 pass dichroic/barrier filter combination which has an optical density of 10  -6  to the excitation wavelength but is  transparent to the fluorescence emission. Also shown is the emission band multiplied by the dichroic/barrier cutoff resulting in the effective emission intensity collected and the band pass for the PMT dichroic that reflects the fluorescence emission to the PMT fiber but allows the long wavelength tail to be imaged on the intensified CCD camera.  29  (Imager AT/NP Video Board) supplied by Matrox in Dorval, Quebec. The other was a photomultiplier tube (#C31034A), supplied by Burle-RCA, cooled to -30° C by a Peltier cooled housing, supplied by Products for Research. The PMT charge pulses were pre-amplified, discriminated and counted by associated electronics (Ortec) and the gating sequences and final photon count were controlled and read via an A/D board (QuaTech #PXB-721). For FRAP experiments the PMT was used to quantify the fluorescence intensity because of its superior signal to noise resulting from cooling. However, the intensified camera was invaluable for determining the laser spot size and aligning it with the fiber optic and the region of membrane of interest.  2.4.3 Analysis of Recovery Kinetics  The diffusivity and mobile fraction of the labelled macromolecular species were determined by fitting the experimental fluorescence recovery with a theoretically obtained expression. The theoretical model assumes that fluorescence recovery is due to the lateral diffusion of components with a single diffusivity and is derived from the diffusion equation in 2 dimensions for 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' K and a characteristic recovery time TD. TD is linearly related to the half time of fluorescence recovery (t 112 'YDTD) where 7D is very weakly K dependent and ranges from 1 < yD < 1.45 for 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 bleach K > 5and FP"F(0) —F(0) > 80%) distorts the Gaussian concentration profile and effectively increases the area bleached [Axelrod et al 1976] ^ — F2-- ) and it has been shown that excess bl bleaching^(for K  30  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).  ° , Kr r i^2t  N2  " n=0  D  (—  1 -1  nt^TD1  w2  = 7Dw  (6)  2  471)^4t1/2  F ( 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 percentage bleach (Equ.8). This linear solution however, is in good agreement with the exact solution even for a large percentage bleach. For an initial bleach as high as 85% the least squares deviation of the linear solution and exact solution is less than 4% [Yguerabide et al 1982]. This error is well below the resolution of FRAP measurements and thus the linear solution provides a good measure of the half time recovery and the mobile fraction along with the ease of linear least squares fitting of the data. Figure 10 shows the normalized experimental fluorescence recovery of ETSC-labelled glycophorin A on the red cell membrane surface and the corresponding theoretical recovery curve given by equation 8.  31  Figure 10: This figure shows the normalized fluorescence recovery of ETSC-labelled glycophorin A on the red cell surface and the corresponding least squares fit of the linearized theoretical recovery given by Eq. 8.  2.4.4 Spot Size Determination  The 1/(e 2 )radius of the observation spot (Fig. 8) was determined from the profile of its image produced by the objective lens. This was done by assuming that the intensity profile of the spot is Gaussian and that the information lost in the image is a function of the collection aperture of the objective lens only (diffraction limited). In such a case the resulting image is the intensity profile of the object convolved with the point spread function (PSF) of the lens 8 [Goodman 1968]. Thus the measured image intensity profile of the spot was fitted with a Gaussian profile convolved with the point spread function of the lens to determine sThe point spread function of a lens is the intensity profile of the image that it produces from a point source and is obtained by taking the Fourier transform of the lens' optical transfer function PSF(x) = Ft l[OTF(v)1. The OTF represents the lens' ability to collect light diffracted by the characteristic spatial dimensions of the object and to transmit it to the image. For the case of incoherent illumination (which is the case in fluorescence emission) the diffraction limited OTF equals the autocorrelation of the aperture function of the lens and for a circular aperture this function is —  OTF(  COS  —1(  2  v )^v vmas^-uma  1^  2vmar  2  < 2inna.r where the maxium spatial frequency is  defined by the greatest collection angle (given by numerical aperture) of the lens. v m ,  32  Gaussian Intensity Profile Convolved with the PSF of the Objective  Figure 11: To determine the waist diameter of the Gaussian bleach spot its image, produced by the  Omission  objective lens, was fitted with a  Profile  Gaussian profile, with known diameter, convolved with the  Profile Convolved  point spread function of the objective lens. This was required because the bleach spot has a radius of approximately I wavelength and thus its image is broadened because spatial orders are diffracted beyond the collecting angle of the objective lens are lost in the image. In this figure the Gaussian profile, the convolved profile and the PSF are shown.  its 1/(e 2 )radius. A thin (thickness< A) uniform layer of fluorescent material was deposited on to a glass substrate and positioned at the laser spot. The intensity distribution of the image was measured with the intensified CCD camera. A low intensity beam was used to avoid photobleaching which would have altered the relative peak height to width ratio. Next an intensity profile was produced by averaging several video scan lines through the center of the image. This profile was then fitted with a Gaussian profile convolved with the point spread function of the objective. Figure 11 shows the effect of convolving the point spread function with a Gaussian profile. In the absence of focus error the increase in 1/(e 2 )radius of the image due to the lens is 12%. This increases to 62% when the object is 2A out of focus. The measured spot image intensity profile fitted with a convolved Gaussian resulted in a 1/(e 2 )radius of the bleach spot of (0.55±0.05)itm.  33  2.5 Red Cell Membrane Elastic Rigidity  Figure 12: The rigidity of the red cell membrane was determined by aspirating a portion into a glass pipette and measuring the aspiration length (L) as a function of the applied pressure (AP). Pipette aspiration produces a combined deformation of elastic shear and local area dilatation of the skeletal network and the resulting rigidity modulus, which is inversely proportional to the change in aspiration length with applied pressure, is thus a combination of the compressibility and shear moduli of the red cell skeleton.  Red cell membrane rigidity was determined by pipette aspiration experiments [Berk et al 1989 for review] (Fig. 12) done by Andrew Leung, a technician in our laboratory. The measurements were done concurrently with and on the same blood samples used in the lateral motion experiments. In these experiments a portion of the red cell membrane was aspirated into a narrow glass pipette. The elastic rigidity relates to the ability of the membrane to deform into the pipette under an applied pressure. To simplify the analysis the membrane portion is chosen at the cell dimple so that the deformation is axially symmetric. As the pressure in the pipette is decreased the membrane undergoes an in-plane deformation and is drawn into a cylindrical tongue. The maximum in-plane deformation (tongue length) that can be produced depends on the point at which the membrane outside the pipette starts to buckle. The buckling instability results in out-of-plane deformations which complicate the analysis and, in cases where the rigidity is too high, completely restrict the rigidity 34  measurement.  In the traditional analysis of this experiment the membrane deformation produced was assumed to be one of constant area (pure shear) [Evans & Skalak 1980]. This assumption was made because the area of the lipid bilayer is constant due to its large compressibility modulus. 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 which span 76 nm in the unstressed skeleton but span 200 nm when fully extended. Thus it is not surprising that the deformation produced by pipette aspiration results in a combination of elastic shear and local area dilatation of the cytoskeleton. This indicates that the compressibility modulus of the skeleton is much smaller than that of the lipid bilayer and thus the compressibility of the bilayer would dominate in the measured compressibility for the whole membrane. This is confirmed experimentally because, although the compressibility modulus of the cytoskeleton has not been measured directly, the measured compressibility of the red cell membrane equals that of SOPC (1-stearoyl-2-oleoyl phosphatidyl choline) bilayer vesicles 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 length with applied pressure is a good measure of membrane rigidity, the resulting modulus (p) is a combination of the shear and compressibility moduli of the cytoskeleton.  35  3 Experiments and Results 3.1 Ligand-Induced Lateral Immobilization of Glycophorin A: Implication of Cytoskeletal Entanglement  The interaction of glycophorin A with its underlying cytoskeletal network was studied in normal and variant red cell membranes, by measuring the change in its lateral motion produced by ligands bound to its extracellular domain. The ligands used were a series of monoclonal anti-glycophorin A antibodies with specificities for different regions of the extracellular 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 mid region of the exoplasmic domain, distal to the trypsin cleavage site [Anstee 1982; Bigbee 1983; Edwards 1980] and ligand B14 binds adjacent to the lipid bilayer, between residues 56 and 67 [Ridgwell 1983]. The two membrane variants used were Miltenberger V and hereditary spherocytosis red cells. Miltenberger V red cell membranes result from a mutation involving an 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, but instead contain a hybrid glycoprotein consisting of the extracellular domain of glycophorin  A, 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 the monoclonal antibodies (ligands) 9A3, 10F7 and R10. It also has a significiantly truncated cytoplasmic domain because the cytoplasmic domain of glycophorin B consists of only 6 amino acid residues compared to the 39 residues in the cytoplasmic domain of glycophorin  36  Antibody Binding Epitopes on Gly-A  Figure 13: This figure shows the relative binding position of the monoclonal antibodies 9A3, R10, 10F7 and B14 to the extracellular domain of glycophorin A.  A (Fig. 13). Hereditary spherocytosis is characterized by a partial deficiency of the major cytoskeletal protein, spectrin. Red blood cells were studied from three individuals with spectrin deficiencies of 35%, 41% and 66%. In the case of mild spectrin deficiency (<30%), the hexagonal structure of the skeleton is nearly normal, as determined by electron microscopy of 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 Red -  Cells  Binding isotherms were measured for each fluorescently labelled anti glycophorin A anti-  body. The binding affinities were similar for all four antibodies. Threshold binding, determined by PMT sensitivity, was detected at an incubation concentration of 0.5 fig/m1 and binding saturation was reached at 10 pg/ml. The fluorescence signal increased by almost  37  two 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 crn 2 I sec and a mobile fraction of 61 + 3% (Table 3). Increasing the incubation concentration of 10F7 from 0100 pg/m1 resulted in a dose dependent decrease in the mobile fraction to 40 + 5%, and a slight reduction in the diffusivity to (3.0 ± 0.5) x 10 -11 cm 2 /sec. Similar concentrationdependent changes were produced by 9A3 and monovalent Fab fragment binding. For 9A3 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 it reduced the mobile fraction to 35.2 + 4.8% and diffusivity to (4.1 + 1.0) x  10-112  Table 3. ALTERATION OF LATERAL MOTION OF ETSC-LABELLED GLYCOPHORIN A IN NORMAL RED CELLS: THE EFFECT OF LIGAND BINDING  Ligand^Conc^D ( x10 -11 ) Mobile Fraction Cell Sample Lag/m11^[cm2/seci^[%1^Number Native 9A3 10F7 R10-Fab  Membrane 2.5 10 1 2.5 100 25 100  7.8 1 1.2 2.8 + 0.4 2.6 + 0.4 5.0 ± 0.7 5.0 + 0.5 3.0 ± 0.5 2.8 ± 0.4 4.1 + 1.0  38  61 ± 3 50 ± 5 44.2 + 3.5 53 + 5 40 ± 5 40 + 5 46.4 + 5 35.2 ± 4.8  130 12 12 12 10 25 10 10  /sec.  When unlabelled glycophorin A was bound with fluorescently labelled 9A3 at levels just above threshold, there was a 5 to 10 fold decrease in the diffusivity and the mobile fraction reduced to 40 + 5% (Table 4). Saturation binding further reduced the mobile fraction to 19 + 3%. For fluorescently labelled R1o, B14 and R1O-Fab, both threshold and saturation binding resulted in mobile fractions of 10% to 15%.  Table 4. ALTERATION OF LATERAL MOTION OF UNLABELLED GLYCOPHORIN A IN NORMAL RED CELLS: THE EFFECT OF BINDING LABELLED LIGANDS  Ligand  Native 9A3  R10 R10-Fab  Conc [,ug/m1]  D (x10 - ") [cm 2 /sec]  Mobile Fraction Vol  Cell Sample Number  Membrane 1 10 38 2 10 25 100  7.8 ± 1.2 0.5 ± 0.14 1.4 ± 0.4 1.8 ± 0.7  61+3 40+5 45+5 19+3 <10 <10 18+3 12+3  130 15 10 12 10 35 10 30  3.1.2 Lateral Motion of Glycophorin in Miltenberger V Red Cells  To study the role of the cytoplasmic domain in this ligand-induced immobilization, measurements were made of the effect of ligand binding on the lateral motion of a variant glycophorin A, expressed in Miltenberger V red cells. This variant glycophorin has a large deletion in its cytoplasmic domain. Binding isotherms were measured for fluorescently 39  labelled R10 and 10F7 to these cells. Threshold binding was detected at incubation concentrations of 1 ,ug/m1 and binding saturated at 10 pg/ml. The saturation fluorescence intensity was slightly less than half that measured for normal cells. This supports earlier findings [Merry et al 1986] which showed that the copies/cell of variant glycophorin in Miltenberger V cells is half that of glycophorin A in normal cells. Hybrid glycophorin of Miltenberger V membranes labelled with ETSC had a diffusivity of (6.1+1.2) x 10  -11  crn 2 I sec  and a mobile fraction of 79±4% (Table 5). When unlabelled hybrid glycophorin was bound at saturation with labelled 10F7 or R10 the average measured diffusivity decreased 5 to 10 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 that the cytoplasmic domain is critical for ligand-induced immobilization of glycophorin A in normal cells.  Table 5. ALTERATION OF LATERAL MOTION OF HYBRID GLYCOPHORIN IN MILTENBERGER V RED CELLS: THE EFFECT OF BINDING LABELLED LIGAND Ligand  Conc [pg/m1]  D ( x10 -11 ) [cm2/sec]  Mobile Fraction  Cell Sample Number  Native R10  Membrane 20 100 10  6.1 ± 1.2 1.8 ± 0.5 0.78 + 0.2 0.77 + 0.2  79 ± 5 70 ± 5 69.2 + 5 76 + 5  20 10 30 20  10F7  40  3.1.3 Lateral Motion of Glycophorin A in Hereditary Spherocytic Red Cells  Spherocytic red cells from three individuals were studied to determine the role of cytoskeletal network integrity on the lateral motion of fluorescently labelled ligand bound glycophorin A. The spectrin deficiencies of the individuals were 35%, 41% and 66%, expressed as a percentage of the normal spectrin content. In the absence of ligands, glycophorin A labelled with ETSC gave mobile fractions of 80.6%, 80.5% and 87.3% respectively and the diffusivity 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 with no measurable change in the diffusivity. HS cells saturated with fluorescently labelled B14 gave 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 corresponding diffusivity of (3.0 + 1.5) x 10 -11 cm 2 /sec for the most severe spectrin deficiency. This shows that ligand-induced immobilization is less pronounced if the cell's spectrin content is less than normal.  41  Table 6. ALTERATION OF LATERAL MOTION OF ETSC-LABELLED GLYCOPHORIN A IN HEREDITARY SPHEROCYTOSIS RED CELLS: THE EFFECT OF SPECTRIN DEFICIENCY Spectrin Deficiency [%]  D^x10 -11 ) [cm2/sec]  0 35 41 66  7.8 ± 1.2 6.5 ± 1.2 5.4 ± 1.2 6.6 + 1.2  Mobile Fraction Cell Sample Number [%) 61 ± 3 80.6 ± 5 80.5 ± 5 87.3 ± 5  130 10 10 10  Table 7. ALTERATION OF LATERAL MOTION OF GLYCPHORIN A IN HEREDITARY SPHEROCYTOSIS RED CELLS PRODUCED BY SATURATION BINDING OF LABELLED LIGANDS Spectrin^Ligand D ( x10 -11 ) Mobile Fraction^Cell Sample Deficiency [%1^[cm2/sec]^[70]^Number 0^B14^< 10^10 35^ 16.8 ± 3^10 41^-^14.6 ± 3^10 66^3.0 ± 1.5^32.4 ± 4^10  3.1.4 Summary  In this set of experiments, ligands were bound specifically to the extracellular domain of glycophorin A in normal and variant red cell membranes in order to determine their effect  on glycophorin A lateral motion. 42  In 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 glycophorin A. This established the normal parameters of diffusivity and mobile fraction. Subsequent binding of ligands caused a marked decrease in the previously determined mobile fraction with only a slight decrease in the diffusivity. Such changes are characteristic of mobile species becoming 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, completely immobilized the glycophorin A resulting in a mobile fraction of less than 10%. In these two 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 particular interest because this ligand binds to the epitope furthest from the bilayer on the extracellular domain.  An indication of the origin of this ligand-induced immobilization came from two further experiments. First, labelled monovalent R10-Fab was bound, at saturation, to normal cells and resulted in complete immobilization. This suggested that the immobilization did not involve cross-linking of the extracellular domain by divalent ligands. Second, saturation binding of labelled divalent ligands to Miltenberger V red cell membranes had no effect on the glycophorin A mobile fraction although the diffusivity was decreased by 5 to 10 fold. These experiments show that the cytoplasmic domain of glycophorin A is involved in the ligand-induced immobilization because in Miltenberger V glycophorin there is a large  43  deletion in the cytoplasmic domain.  To further investigate the origin of glycophorin A immobilization, labelled ligands were bound at saturation to the surfaces of spherocytic red cells from 3 individuals with different degrees of spectrin deficiency. In the case of 35% and 41% spectrin deficiencies, labelled ligands completely immobilized the bound glycophorin A. This suggested that these percentages of spectrin deficiency were not enough to alter the interaction between the cytoplasmic domain and the cytoskeleton. However, for the 66% spectrin deficiency, immobilization was much less pronounced. This suggested some dependence on cytoskeletal network density in the immobilization of ligand bound glycophorin A.  The conclusion from these experiments is that ligand binding to specific epitopes on the extracellular domain of glycophorin A promotes an increased interaction between the cytoplasmic domain of glycophorin A and the underlying cytoskeletal network and that this interaction immobilizes the bound protein. Such a process suggests the transduction of a signal across the bilayer in response to bound ligands which is probably facilitated by a conformational change in one of the domains of glycophorin A.  44  3.2 Integral Membrane Proteins Play a Key Role in Membrane Rigidity  To further examine the nature of the ligand-induced interaction between glycophorin A and the cytoskeletal network, cytoskeletal rigidity was measured along with the lateral motion of red cell integral proteins. The postulate being tested was that integral protein lateral motion is inversely related to membrane rigidity. To test the hypothesis, lateral motion and rigidity measurements were made on: normal red cells bound with ligands to glycophorin A 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 antiglycophorin A bound to red cell surfaces decreased membrane deformability 9 in normal cells, but did not change the deformability of Miltenberger V cells [Chasis et al 1988]. These results were the first evidence that ligand induced alterations in membrane rigidity are not due to an extracellular interaction but involve the cytoplasmic domain of transmembrane proteins.  If a relation can be established between integral protein immobilization and increased membrane rigidity, then it is a likely explanation for the high membrane rigidity known to exist in some naturally occuring red cell variants. One such case is hereditary ovalocytosis, a red cell disorder which is widespread in South East Asia [Mohandas et al 1984]. The large rigidity of these cells is believed to be responsible for their ability to resist invasion by malarial parasites [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 ekctacytometry where a shear stress is applied to many cells and their ellipticity is measured by changes in the Fraunhofer diffraction patterns that are produced.  45  was genetically selected in areas where malaria is endemic. Since the skeletal network is responsible for membrane rigidity, it was thought that a mutation would be found in one of the cytoskeletal proteins. However, considerable biochemical analysis has failed to detect any such mutation. The problem was elusive until recently when the ovalocytic genotype was linked to a structural change in the integral protein band 3 [Liu et al 1990]. To test the hypothesis that integral protein lateral motion is linked to membrane rigidity the lateral mobilities of band 3 and glycophorin A were measured in ovalocytic red cell membranes from three individuals.  3.2.1 Integral Protein Lateral Motion and Membrane Rigidity in Membranes of Normal and Miltenberger V Red Cells  Red cell membrane rigidity of normal cells was measured as a function of ligand binding to the 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 remains unaltered. 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 rigidity which further increases at saturation to 15-fold. In the case of ligand B14, threshold binding results in a 46-fold increase in rigidity and at saturation the membrane becomes too rigid for micropipette aspiration.  46  Table 8. ALTERATION OF RIGIDITY OF NORMAL RED CELL MEMBRANES BY LIGANDS TO GLYCOPHORIN A Ligand  Conc itg/m1  Rigidity [fiN/m)  Cell Sample Number  Native Mem 9A3  0 5 76 1 100 2 20  8±1 8±3 64 ± 12 50±5 117±50 370±170 too rigid !  20 5 5 5 5 5 10  10F7 B14  For comparison, the lateral motion and membrane rigidity were measured for ligand Bric10 which binds specifically to an extracellular epitope of glycophorin C. As yet, there is  no method to fluorescently label glycophorin C directly and thus no way of measuring its innate mobility. However, binding the ligand Bric-10 to glycophorin C resulted in a diffusivity of (1.6 ± 0.5) x 10 -11 cm' 1 sec and a mobile fraction of (44 ± 6%) (Table 9), and did not change the rigidity of the membrane indicating clearly that: 1) when an integral protein is not immobilized that there is no change in membrane rigidity; and further that 2) extracellular ligand binding alone does not result in increased membrane rigidity. In a similar vein, ligands 10F7 and R10 bound at saturation to glycophorin A have no effect on the rigidity of Miltenberger V membranes (Table 10).  47  Table 9. LATERAL MOTION AND MEMBRANE RIGIDITY OF LIGAND BOUND GLYCOPHORIN C Ligand^Conc itg/m1  D (x10 -11 ) MFrac. Rigidity Cell Sample [cm 2 /sec]^[%] [ttN/m]^Number  ^ ^ 1.6 ± 0.5 44+6^8+2^30 Bric-10^5 (saturation)  Table 10. ALTERATION OF MEMBRANE RIGIDITY OF MILTENBERGER V RED CELLS BY LIGANDS TO GLYCOPHORIN A Ligand Native Mem 10F7 R10  Conc Rigidity pg/m1 [(LN/m] 0 10 100  8+1 8+1 8+1  Cell Sample Number 20 5 5  3.2.2 Membrane Rigidity in Hereditary Spherocytic Red Cells  Ligands B14 & R10 were bound at saturation to glycophorin A of three individuals with hereditary spherocytosis. For ligand B14, the membranes with mild spectrin deficiencies (35% & 41%) became too rigid for measurement. However, a measurement was possible for 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 deficiency 48  and membrane rigidity. At 0% spectrin deficiency (normal cells) the rigidity of cells bound with ligands R10 is (117 ± 28),(iN/m. This value falls to (84 ± 22)0//m, (32 + 13),uNim and finally to (12 + 3)ttNlm for spectrin deficiencies of 35%, 41% and 66% respectively.  Table 11. ALTERATION OF MEMBRANE RIGIDITY OF HEREDITARY SPHEROCYTOSIS RED CELLS BY LIGANDS TO GLYCOPHORIN A Ligand  Conc pg/m1  Spectrin [70] Deficiency  Rigidity [uN/m]  Cell Sample Number  Native Membrane  0  R10  20  B14  20  0 (Ctrl.) 35 41 66 0(Ctrl.) 35 41 66 0 (Ctrl.) 35 41 66  6.2 ± 1.5 4.5 + 1.0 6.6 ± 2.0 5.4+ 1.3 117 + 28 84 ± 22 32 + 13 12 + 3 370 ± 170 too rigid too rigid 174 + 50  5 10 10 10 5 5 5 10 5 5 5 5  3.2.3 Lateral Motion of Glycophorin A and Band 3, and Membrane Rigidity in Hereditary Ovalocytosis Red Cells  As an application of the hypothesis that integral protein motion is related to membrane rigidity, these two parameters were measured for glycophorin A and band 3 in membranes of three individuals with hereditary ovalocytosis. The rigidity of ovalocytic red cells was 49  greater than normal by factors of 3-fold, 5-fold and 9-fold (Table 12). This increase in membrane rigidity had no effect on the diffusivity of native glycophorin A, but there were slight measured reductions in the mobile fractions. However, in each case, the mobile fraction of the band 3 was less than 10%. Table 12. LATERAL MOTION OF ETSC-LABELLED GLYCOPHORIN A AND EMA-LABELLED BAND 3 IN, AND MEMBRANE RIGIDITY OF OVALOCYTIC RED CELL MEMBRANES Donor  Integral Protein  Normal glycophorin A Control band 3 glycophorin A 901 band 3 glycophorin A 904 band 3 glycophorin A 906 band 3  D ( x10 -11 ) [cm2/sec]  MFrac.  Rigidity [itN/m]  Cell Sample Number  5.5 + 2.4 4.2 ± 1.6 5.3 + 1.3  62 ± 6 35 + 15 40 + 10 < 10 37+6 < 10 49 + 7 < 10  8±1  40 60 10 10 10 10 10 10  5.5 + 1.2 4.9 + 1.9  [%]  25 + 6 39+7 72 + 27  3.2.4 Summary  In this set of experiments, ligands were bound to the extracellular domain of glycophorin A in normal and variant red cell membranes in order to determine their effect on membrane rigidity, and to relate these effects to the changes in glycophorin A lateral motion measured in the last section.  50  In an inital set of experiments, the membrane rigidity was measured for normal red cells bound with anti-glycophorin A. Ligand 9A3 had no effect on membrane rigidity when bound at threshold concentrations but increased the rigidity 8 fold when bound at saturation. Both ligands 10F7 and B14 produced marked rigidity increase at threshold and at saturation. For saturation binding the rigidity increase was highest for B14 which binds close to the phospholipid bilayer and was minimal for 9A3 which binds farthest from the bilayer.  To determine the mechanism responsible for this increased rigidity, two further experiments were designed. First, a ligand, (Brie-10), specific for the extracellular domain of glycophorin C, was bound at saturation to normal cell membranes. Glycophorin C is thought to be a membrane attachment site, because it binds to band 4.1 of the cytoskeletal network. This ligand did not increase the rigidity of the membrane and resulted in a diffusivity for glycophorin C which was similar to that of band 3. The result suggests that 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 of Miltenberger V red cells. In these cells, the binding of ligands had no effect on membrane rigidity. Together, these two experiments show that ligand binding promotes an increased interaction between the cytoplasmic domain of glycophorin A (rather than glycophorin C) and the cytoskeleton that promotes increased rigidity in normal cells.  Finally, the contribution of cytoskeletal spectrin to membrane rigidity was determined by ligand saturation of glycophorin A in spherocytic red cells with varying degrees of spectrin  51  deficiency. Ligand B14, which attached close to the bilayer, caused the cell membranes to become too rigid for pipette aspiration, in all but the most spectrin deficient cells. This suggests that only major spectrin deficiency reduces the rigidifying interaction. R10 ligand causes rigidity that is measurable for each HS donor and decreases dose dependently as the spectrin deficiency increases.  These experiments show that binding anti-glycophorin A ligands increase red cell membrane rigidity. These results, taken together with the changes of lateral mobilities, indicate that interactions between the integral membrane protein's cytoplasmic domain and the cytoskeleton can immobilize the integral protein and cause an increased membrane rigidity. This possibility was tested on ovalocytic red cells which are characterized by very rigid membranes which makes them resistant to malaria. Membrane rigidity and the lateral motion of glycophorin A and band 3 were measured on these Ovalocytic cells. For each of the three individuals studied the measured rigidity was well above normal, as expected. The exciting result was that the lateral motion of glycophorin A was near normal but the entire  band 3 population was immobile. This indicated, for the first time, a strong link between band 3 immobilization and increased membrane rigidity.  52  3.3 Ligand-Induced Interaction Between Glycophorin. A and the Cytoskeleton. in Human Erythrocytes Involve Band 3 Protein  The 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 small cytoplasmic domain (39 amino acids, spanning a possible 10 nm), becomes immobilized and produces such large increases in membrane rigidity in response to extracellular ligand binding.  One proposed mechanism suggested that the interaction of the cytoplasmic domain of glycophorin A with the cytoskeleton is an indirect one which involves the cytoplasmic domain of band 3. Band 3 is implied because it is the major red cell integral glycoprotein, has a considerable cytoplasmic domain that spans some 25nm [Low 1986], and is a known cytoskeletal attachment site between the bilayer and ankyrin [Bennett & Stenbuck 1979b; Golan & Veatch 1982]. Interactions between glycophorin A and band 3 have been suggested in several instances [Nigg et al 1980]. And in the light of our recent work, which relates the increased membrane rigidity in ovalocytes to a mutation in band 3 [Mohandas et al 1992], a cooperative action is strongly indicated. This is because the ovalocytic mutation involves a deletion of a sequence of 9 amino acids adjacent to the bilayer on the cytoplasmic side. The deletion creates a proposed conformational change of the cytoplasmic domain of band 3. The particular amino acid sequence, present in normal band 3, is well within the reach of the cytoplasmic domain of glycophorin A [Mohandas et al 1992].  53  To 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 measured in normal and variant red cell membranes as a function of ligands bound to extracellular epitopes of glycophorin A. The variant red cells were the Miltenberger V type in which glycophorin has a large cytoskeletal deletion.  3.3.1 The lateral motion of band 3 in normal red cell membranes altered by ligands bound to glycophorin A  In these experiments band 3 was fluorescently labelled by eosin-5 malamide (EMA) and its lateral 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 mid region of the extracellular domain of glycophorin A, resulted in a dose-dependent decrease in the band 3 mobile fraction and completely immobilizes the band 3 when bound at saturation. There is no measured change in the corresponding diffusivity. In the case of B14, which recognizes an epitope close to the bilayer on glycophorin A, both threshold and saturation binding resulted in complete band 3 immobilization. However, 9A3, which recognizes the most distal epitope on glycophorin A, leaves the lateral motion of band 3 unaffected even at saturation binding.  54  Table 13. ALTERATION OF EMA-LABELLED BAND 3 MOTION IN NORMAL RED CELLS BY LIGANDS TO GLYCOPHORIN A  Ligand to Gly-A  Conc [pg/m1]  Native 9A3 R10  0 100 1 2.5 100 2.5 100  B14  DBand3( X  10 -11 )  [cm2/sec] 2.9 + 1.4 2.2 + 0.6 2.8 + 0.7 2.8 ± 0.7 1.0 + 0.4  Mobile Fraction of Band 3 [%]  Cell Sample  43 ± 6 39+5 34+5 27.6 ± 2 7±3 17+3 7±4  20 10 10 10 25 15 8  Number  3.3.2 The lateral motion of glycophorin C in normal red cell membranes altered by ligands to glycophorin A  In these experiments, the minor sialoglycoprotein, glycophorin C was fluorescently tagged with labelled ligand Bric-10. This ligand is a monoclonal antibody specific for the extracellular domain of this sialoglycoprotein and earlier results (Table 9) showed that glycophorin C was not immobilized by this ligand and has a similar measured lateral mobility to that of native band 3. Thus, the lateral mobility of Bric-10-labelled glycophorin C can be measured as a function of the amount of RIO bound to glycophorin A. This experiment showed that R10 binding reduces the diffusivity of glycophorin C in a dose dependent fashion, but does not change its mobile fraction which suggests that glycophorin C is not immobilized but that its lateral motion was simply hindered by ligands bound to glycophorin A (Table  55  14).  Table 14. LATERAL MOTION OF GLYCOPHORIN C BOUND WITH FLUORESCENTLY LABELLED BRIC-10 IN NORMAL RED CELLS ALTERED BY UNLABELLED LIGANDS TO GLYCOPHORIN A  Ligand to Gly-A  Conc [µg/ml]  DGty _c (x10 -11 ) [cm2/sec]  Mobile Fraction of Gly-C [%]  Cell Sample  R10  0 1 100  1.6 ± 0.5 0.61 ± 0.2 0.18 ± 0.09  43.7 ± 5 40 + 10 44 ± 15  30 15 10  Number  3.3.3 The lateral motion of band 3 in Miltenberger V red cell membranes altered by ligands to glycophorin A  To test the role of the cytoplasmic domain of glycophorin A in the immobilization of band 3, the lateral motion of EMA-labelled band 3 was measured as a function of R10  binding to the hybrid glycophorin of Miltenberger V cells. In the absence of ligands the lateral 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 no effect on the mobile fraction (Table 15). This indicates that band 3 is not immobilized by ligands to glycophorin A when glycophorin A does not have a cytoplasmic domain and again suggests that the cytoplasmic domain is important for immobilization.  56  Table 15. LATERAL MOTION OF EMA-LABELLED BAND 3 IN MILTENBERGER V RED CELLS ALTERED BY UNLABELLED LIGANDS TO GLYCOPHORIN A Ligand to Gly-A R10  Conc D B „„d3 ( x10 -11 ) [cm2/sec] [ttg/m1] 0 100  2.6 + 0.4) 1.4 ± 0.3  Mobile Fraction of Band 3 [Vo]  Cell Sample Number  41 ± 4 42.2 ± 6  10 30  3.3.4 Summary  Ligands to the extracellular domain of glycophorin A have an immobilizing effect on the lateral motion of band 3. This effect is most pronounced when the ligand B14 is bound because it binds to the extracellular domain of glycophorin A, at an epitope closest to the bilayer. The immobilization is still seen when ligand R10 is bound but is less pronounced at threshold binding levels. However, immobilization is not seen when ligand 9A3 is bound because it binds to the most distal extracellular epitope. The second experiment showes that R10 binding does not have an immobilizing effect on glycophorin C. This indicates that glycophorin C, which is a proposed membrane attachment site, is not involved in the ligand-induced immobilization of glycophorin A and band 3. The third experiment showes 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 involves an interaction in the cytoplasmic domain. Thus, ligand binding to the extracellular domain of glycophorin A promotes an increased interaction between glycophorin's cytoplasmic 57  domain and that of band 3, and results in the immobilization of both integral proteins.  58  4 Discussion and Conclusion  This study describes the dynamic coupling between a membrane receptor-protein and its underlying cytoskeletal network, in a model system, by measuring changes of the receptor's lateral motion and membrane's rigidity, produced by binding ligands to the receptor's extracellular domain. The model system was the red blood cell membrane, both normal and variant types, whoes membrane consist of several well characterized integral proteins and a supporting cytoskeletal protein network. The ligands used were monoclonal antibodies and their Fab fragments which recognize specific epitopes on the extracellular domain of gly-  cophorin A, the major sialoglycoprotein of the red cell membrane. Ligand 9A3 recognizes a glycophorin A epitope at the amino terminus, involving amino acid residue 1 [Bigbee 1983]. Ligands R10 10F7 recognize an epitope in the mid region of the exoplasmic domain, next to the tryipsin cleaving site [Anstee 1982; Bigbee 1983; Edwards 1980]. Ligand  B14 recognizes an epitope adjacent to the bilayer between residues 56 and 67 [Ridgwell 1983].  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 deformability (measured by flow ekctacytometry) was greatly reduced for normal red cells, but not for red cell variants that lack the cytoplasmic domain of glycophorin A (Miltenberger V red cells). Since the cytoskeletal network (rather than the lipid bilayer) is responsible for membrane rigidity, these results suggested that ligand binding can initiate a transmembrane signal causing an increased interaction of the cytoplasmic domain of the receptor with the 59  underlying cytoskeletal network.  Integral protein lateral motion was measured in situ by the technique of fluorescence recovery after photobleaching (FRAP). The technique involves fluorescent labelling of the ligand or the receptor of interest, irreversibly bleaching a small region of the membrane and measuring the subsequent fluorescence recovery due to lateral diffusion exchange of fluorophor coupled proteins. Red cell membrane rigidity was measured by a pipette aspiration technique where a portion of membrane is deformed into a pipette using a minute suction pressure. The dependence of the aspirated tongue length on applied pressure is linear for elastic deformations and results in an effective membrane rigidity modulus which is a combination of the elastic shear and area expansion moduli of the membrane cytoskeleton.  4.1 Receptor/Ligand Interactions in Normal Red Cell Membranes  In normal red cell membranes, the binding of ligands to the extracellular domain of glycophorin A, caused a large increase of membrane rigidity and marked reductions of the mobile fractions of glycophorin A and band 3. Two approaches were used to show that binding anti-glycophorin A ligands markedly reduce the lateral motion of the erythrocyte membrane protein glycophorin A.  The first approach used unlabelled anti-glycophorin A antibodies bound at increasing concentrations to red cell membranes labelled with eosin-5-semithiocarbazide (ETSC). Antibodies 9A3, 10F7 and R10-Fab all showed a similar concentration-dependent decrease in 60  mobile fraction from 61% to 38 ± 6% as the amount of bound antibody increases from zero to saturation. This large reduction of the mobile fraction is indicative of a mobile species becoming immobilized rather than just being hindered.  In the second approach the antibodies were labelled directly and bound to red cell membranes. At saturation, the binding of fluorescently labelled 10F7, R10, R10-Fab and B14 caused the mobile fractions to fall to 10 ± 3%, indicative of near complete glycophorin A immobilization. This effect was less pronounced with ligand 9A3 which binds the most distal extracellular portion of glycophorin A. The term immobilization relates to a very low fluorescence recovery within the FRAP temporal window. This places an upper boundry on the diffusivity of ligand-bound glycophorin A of 2 x 10 -13 cm 2 /secw. This value, which is a reduction of 2 orders of magnitude from the diffusivity of native glycophorin A, is the same as that estimated for the diffusivity of spectrin in the cytoskeleton [Peters 1974]. This suggests 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% lies in the specificity of the ETSC labelling of glycophorin A. ETSC which binds covalently to oxidized 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 other labelled species. Assuming that ETSC labelling is 60% glycophorin A-specific, and that the mobile fraction of ligand-bound glycophorin A is 15% (Table 4), then the mobile fraction 1 °This  value for the diffusivity of liganded glycophorin A is an estimate produced by assuming that its mobile fraction is 60% in infinite time, and by fitting the theoretical recovery (Eq.8) through the point of 10% recovery measured at 360sec, the maximum time of the FRAP experiment.  61  of native glycophorin A would be 50%.  In two further experiments the change of lateral motion of band 3, the major red cell glycoprotein, and glycophorin C, a minor sialoglycoprotein, was measured when glycophorin A was immobilized by bound ligands. In these experiments the glycophorin A ligands are unlabelled and band 3 and glycophorin C are labelled with EMA or the labelled ligand Bric-10, respectively. The band 3 mobile fraction decreased dose dependently with the amount of R10 bound at the mid region of the extracellular domain of glycophorin A. At saturation binding, the band 3 mobile fraction falls to 7 + 3%, suggesting complete band 3 immobilization. This effect was more pronounced when B14 was used because band 3 immobilization is achieved even at threshold ligand binding levels. Furthermore, 9A3 does not 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 immobilization of glycophorin A and band 3. The reduction of band 3 mobile fraction to 7% in this experiment suggests that EMA is highly band 3 specific. This is supported by SDS-PAGE results of both Golan and Nigg [Golan et al 1986, Nigg and Cherry 1979} who report that greater than 801ess than 5amounts are associated with lipids.  Membrane rigidity is a property of the skeletal network only because the lipid bilayer is a 2-dimensional fluid. Consequently, changes of membrane rigidity reflect conformational and/or organizational changes within the cytoskeleton. The binding of ligands 9A3, R10 and B14 to normal red cell membranes increases membrane rigidity. This effect is more  62  pronounced the closer the ligand binds to the bilayer. Ligand 9A3, which binds to the most distal epitope on glycophorin A, had no effect on membrane rigidity when bound at threshold levels but increased membrane rigidity 8 fold when bound at saturation. Ligand R10, which binds to the mid region of glycophorin A's extracellular domain, increased rigidity 6 fold at threshold and 15 + 6 fold at saturation. Ligand B14, which of the ligands binds closest to the lipid bilayer on glycophorin A, increased membrane rigidity 46 ± 21 fold at threshold concentrations and increased rigidity immeasurably at saturation. These results are consistent with earlier results in which 9A3 was found to decrease membrane deformability by 5.8 fold, 10F7 by 10.8 fold and B14 by 18 fold [Chasis et al 1988]. The rigidity results show a concordance with the lateral motion results, which also suggests that integral protein lateral immobilization is linked to membrane rigidity. Furthermore, it is interesting to note that although saturation binding with 9A3 has an immobilizing effect on  glycophorin 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 band  3 immobilization than to glycophorin A immobilization. It appears then that membrane rigidity is controlled by band 3, rather than by glycophorin A, and that the interaction between glycophorin A and the cytoskeleton induced by ligand binding is an indirect one involving band 3.  4.2 Receptor-Ligand Interactions in Miltenberger V Red Cell Membranes  In Miltenberger V red cells the dominant sialoglycoprotein is a hybrid consisting of the extracellular domain of glycophorin A and the transmembrane and cytoskeletal domains of 63  glycophorin B. Consequently, this hybrid glycoprotein still has binding epitopes for ligands R10, 10F7 and 9A3 but has almost no cytoplasmic domain. In these cells the binding of glycophorin A ligands R10 or 10F7, even at saturation, did not change the mobile fraction of glycophorin or band 3. This suggests that neither of these proteins was immobilized and that the cytoplasmic domain of glycophorin A, in normal cell membranes, plays a key role in immobilization. Furthermore, rigidity of Miltenberger V cells was not increased by the binding of these ligands, suggesting that increased membrane rigidity is not caused by an extracellular effect but by one involving the cytoplasmic domain. This result is also supported by the finding that monoclonal R10-Fab causes glycophorin A immobilization in normal red cells because, presumably, Fab cannot cross-link the extracellular domain.  4.3  Receptor/Ligand Interactions in HS Red Cell Membranes  In hereditary spherocytosis red cell membranes there is a partial spectrin deficiency. In mild 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 structural defects.  Spectrin deficiency had no effect on the diffusivity of native glycophorin A although there was 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 red cell membranes. In the most severe case, the membrane rigidity (p) divided by the average rigidity of normal cells  (linormal)  was only 0.87. This reduction is far less than expected if, as  64  Waugh and Agre assumed [Waugh and Agre 1988], each spectrin tetramer acts as an entropic spring, and membrane rigidity is simply related to spectrin surface density. However, spherocytosis has complicating factors which could directly affect membrane rigidity, such as depleted levels of the major integral glycoprotein, band 3 and its cytoskeletal attachment site, ankyrin. It is also conceivable that in severe spectrin deficiencies, the spectrin is fully stretched to allow maximal sub-membrane coverage. This would cause a highly dilated network which might have a reduced shear modulus but would be highly incompressible (elastically). This may explain why the composite rigidity modulus, measured in the micropipette aspiration experiment, does not fall proportionately with spectrin content. This explanation is consistent with Waugh & Agre's results because, although they report that membrane rigidity decreases linearly with spectrin surface density, the reduction is less than 40% in red cells with a 69% spectrin deficiency.  Spectrin deficiency had a considerable effect on membrane rigidity and glycophorin A lateral motion particularly when ligands (R10 & B14) were bound to glycophorin A. In the two cases of mild spectrin deficiency, binding of the ligand B14 caused the immobilization of glycophorin A, while, in the severe case the lateral motion of ligand-bound glycophorin A increased substantially. For R10, membrane rigidity decreased with increased spectrin deficiency. Membrane rigidity falls from 15 x normal membrane rigidity at 0% spectrin deficiency to 10 x, 4 x and 1.5 x for spectrin deficiencies of 35%, 41% and 66% respectively. These results indicate that, in normal cells, a loss of hexagonal structure in the cytoskeletal network disrupts the mechanism responsible for the immobilization of ligand bound glycophorin A. 65  4.4 The Mobility of Untethered Receptors  The native diffusivities of glycophorin A were the same in normal cells, mutant glycophorin in Miltenberger V cells and glycophorin A and in HS red cell with severe spectrin deficiency. This indicates that in normal cells neither the cytoplasmic domain of glycophorin A nor the underlying cytoskeletal network account for the native diffusivity of glycophorin A (which is 2 orders of magnitude below the viscous limit [Saffman Si Delbruck 1975] and that of glycophorin reconstituted into lipid vesicles [Vaz et al 1981, Kapitza et 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 cytoplasmic domain 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 diffusion of native glycophorin A is limited by interactions involving either the bilayer-spanning or extracellular portion of the protein. The involvement of the extracellular domain is likely because the bulk of glycophorin A is extrafacial. Furthermore, extracellular involvement is the probable cause of the hindered lateral motion of the ligand bound glycophorin in Miltenberger V cells (Table 5) or glycophorin C in normal cells (Table 9). Such hindering of lateral motion, which decreases the diffusivity without altering the mobile fraction, has been demonstrated in Monte Carlo simulations where the diffusivity of particles mobile in 2 dimensions is reduced by as much as an order of magnitude when the particle surface density increases [Pink 1985, Pink et al 1986, Saxton 1990]. This may occur because ligand binding 66  increases the effective surface density of glycophorin A by increasing the volume that is occupied (excluded volume) by the protein's extracellular domain. Thus, interactions mediated by the glycocalyx amoung the extracellular domains of neighbouring proteins may well be responsible for native glycophorin A diffusivity and the bound-ligand induced reduced diffusivity of mutant glycophorin.  4.5 Receptor Motion in Ovalocytic Red Cell Membranes  Ovalocytic red cells were used to test the correlation between integral protein lateral immobilization and increased membrane rigidity. This is caused by an increased interaction in the cytoplasmic domain between the integral protein and the cytoskeleton. Hereditary ovalocytosis is a naturally occurring mutation characterized by an increased membrane rigidity which makes the cells resistant to malarial invasion. A great deal of work by a number of researchers 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 hereditary ovalocytosis red cells. The cDNA sequence of the band 3 of hereditary ovalocytosis red cells [Mohandas et al 1992] revealed a deletion of a sequence of 9 amino acids in the cytoplasmic domain next to the lipid bilayer. This finding by collaborators at Lawrence Berkeley Laboratory, together with the lateral motion results (Table 12), indicates a possible conformational change produced by the deletion in the cytoplasmic domain which allows increased interaction with the cytoskeleton, and leads to band 3 immobilization and an increase in the membrane rigidity [Mohandas et al 1992]. Finally, it is interesting to note that band 3 immobilization in ovalocytic red cells did not affect the lateral motion of glycophorin A 67  which suggests that unliganded glycophorin A is not involved in the interactions with the cytoskeleton.  4.6 Conclusion  In summary, this study has shown that the lateral motion of native glycophorin A is regulated neither by its cytoplasmic domain nor the underlying cytoskeletal network. However, binding of ligands specific to the extracellular domain promote an interaction between the cytoplasmic domain of the protein and the cytoskeletal network. This interaction involves a second receptor protein, band 3, and causes the complete immobilization of both glycophorin A and band 3. Lateral motion and membrane rigidity data show that an increase of membrane rigidity relates to band 3, rather than to glycophorin A immobilization. The fore going led to the discovery that the band 3 protein of ovalocytic red cells, which has a mutation in it cytoplasmic domain, is completely immobile. This is the probable explanation of their unusual rigidity.  Our understanding of the architectural components of the cytoskeleton has grown through the use of the model system provided by ligand-induced lateral immobilization and the increase of membrane rigidity of red blood cells and may have important applications to the workings of other biological systems.  68  References Anstee D.J., Edwards P.A.W. 1982 Monoclonal antibodies to human erythrocytes Eur. J. Immunol. 12:228-232. 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