Lipid diffusibility in the intact erythrocyte membrane

The lateral diffusion of fluorescent lipid analogues in the plasma membrane of intact erythrocytes from man, mouse, rabbit, and frog has been measured by fluorescence photobleaching recovery (FPR). Intact cells from dystrophic, normoblastic, hemolytic, and spherocytotic mouse mutants; from hypercholesterolemic rabbits and humans; and from prenatal, neonatal, and juvenile mice have been compared with corresponding normals. The lateral diffusion coefficient (D) for 3,3'-dioctadecylindodicarbocyanine iodide (DiI[5]) in intact normal human erythrocytes is D = 8.2 +/- 1.2 X 10(-9) cm2/s at 25 degrees C and D = 2.1 +/- 0.4 X 10(-8) cm2/s at 37 degrees C, and varies approximately 50-fold between 1 degree and 42 degrees C. The diffusion constants of lipid analogue rhodamine-B phosphatidylethanolamine (RBPE) are about twice those of DiI[5]. The temperature dependence and magnitude of D vary by up to a factor of 3 between species and are only influenced by donor age in prenatals. DiI[5] diffusibility is not perturbed by the presence of calcium or local anesthetics or by spectrin depletion (via mutation). However, lipid-analogue diffusibility in erythrocyte ghosts may differ from intact cells. Dietary hypercholesterolemia in rabbits reduces the diffusion coefficient and eliminates the characteristic break in Arrhenius plots of D found in all other cells studied except frog.     

Characterization of electric field-induced fusion in erythrocyte ghost membranes

Fusion has been reported to occur in a variety of membrane systems in response to the application of certain electric currents to the medium (Zimmermann, U., 1982, Biochim. Biophys. Acta., 694:227-277). The application of a weak but continuous alternating current causes the membranes in suspension to become rearranged into the "pearl-chain" formation. Fusion can then be induced by one or more strong direct current pulses that cause pore formation. This results in the conversion of individual membranes in the "pearl-chain" formation to a single membrane with one or more hourglass constrictions that form lumens which connect the cytoplasmic compartments. As the diameter of the lumens increases, the overall membrane shape grows to one large sphere. To further characterize electric field-induced fusion, experiments were conducted using the erythrocyte ghost as a model membrane, and a new combination of electrical circuit and fusion chamber that is simpler and improved over previous systems. All odd- shaped ghosts (collapsed or partly collapsed spherical shapes, echinocytes, discocytes, and stomatocytes) in 30 mM phosphate buffer was first converted to spherocytes and then fused with increasing yields by increasing the number of pulses. After fusion, the lateral diffusion of a fluorescent lipid soluble label (Dil) from labeled to unlabeled membranes was observed to occur both with and without the appearance in phase-contrast optics of distinct communication (lumens) between cytoplasmic compartments of the fused membranes. Connections between cytoplasmic compartments, however, were unmistakable with the instant transfer of a fluorescent water-soluble label (fluorescein isothiocyanate-dextran) from labeled to unlabeled cytoplasmic compartments upon fusion. Although pulses still resulted in the lateral diffusion of Dil to unlabeled membranes, the presence of glycerol in the medium strongly reduced the yield of lumens observable by phase- contrast optics in fusion events. The presence of glycerol also inhibited the conversion of membranes to spherocytes, but did not inhibit the lateral diffusion of Dil from labeled to unlabeled membranes.     

Lateral mobility of a lipid analog in the membrane of irreversible sickle erythrocytes

The major feature of sickle cell anemia is the tendency of erythrocytes to sickle when exposed to decreased oxygen tension and to unsickle when reoxygenated. Irreversible sickle cells (ISCs) are sickle erythrocytes which retain bipolar elongated shapes despite reoxygenation. ISCs are believed to owe their biophysical abnormalities to acquired membrane alterations which decrease membrane deformability. While increased membrane surface viscosity has been measured in ISCs, the lateral dynamics of membrane lipids in these cells have not heretofore been examined. We have measured the lateral diffusion of the lipid analog 3,3'-dioctadecylindocyanine iodide (DiI) in the plasma membrane of intact normal erythrocytes, reversible sickle cells (RSCs), and irreversible sickle cells by fluorescence photobleaching recovery (FPR). The diffusion coefficients +/- standard errors of the mean of DiI in intact normal red blood cells (RBCs), RSCs, and ISCs at 37 degrees C are (8.06 +/- 0.29) X 10(-9) cm2 X s-1, (7.74 +/- 0.22) X 10(-9) cm2 X s-1, and (7.29 +/- 0.24) X 10(-9) cm2 X s-1, respectively. A similar decrease in the diffusion coefficient of DiI in the plasma membranes of the three cell types was observed at 4, 10, 17, 23, and 30 degrees C. ANOVA analysis of the changes in DiI diffusion showed significant differences between the RBC and ISC membranes at all temperatures examined. The characteristic breaks in Arrhenius plots of the diffusion coefficients for the RBCs, RSCs, and ISCs occurred at 20, 19, and 18.6 degrees C, respectively. Photobleaching recovery data were used to estimate (Boullier, J.A., Melnykovich, G. and Barisas, B.G. (1982) Biochim. Biophys. Acta 692, 278-286) the microviscosities of the plasma membranes of the three cell types at 25 degrees C. We find significant differences between our microviscosity values and those obtained in previous fluorescence depolarization studies. However, both methods indicate qualitatively similar differences in membrane microviscosity among the various cell types.     

Kinetics and mechanism of cell membrane electrofusion.

A new quantitative approach to study cell membrane electrofusion has been developed. Erythrocyte ghosts were brought into close contact using dielectrophoresis and then treated with one square or even exponentially decaying fusogenic pulse. Individual fusion events were followed by lateral diffusion of the fluorescent lipid analogue 1,1'-dihexadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (Dil) from originally labeled to unlabeled adjacent ghosts. It was found that ghost fusion can be described as a first-order rate process with corresponding rate constants; a true fusion rate constant, k(f), for the square waveform pulse and an effective fusion rate constant, k(ef), for the exponential pulse. Compared with the fusion yield, the fusion rate constants are more fundamental characteristics of the fusion process and have implications for its mechanisms. Values of k(f) for rabbit and human erythrocyte ghosts were obtained at different electric field strength and temperatures. Arrhenius k(f) plots revealed that the activation energy of ghost electrofusion is in the range of 6-10 kT. Measurements were also made with the rabbit erythrocyte ghosts exposed to 42 degrees C for 10 min (to disrupt the spectrin network) or 0.1-1.0 mM uranyl acetate (to stabilize the bilayer lipid matrix of membranes). A correlation between the dependence of the fusion and previously published pore-formation rate constants for all experimental conditions suggests that the cell membrane electrofusion process involve pores formed during reversible electrical breakdown. A statistical analysis of fusion products (a) further supports the idea that electrofusion is a stochastic process and (b) shows that the probability of ghost electrofusion is independent of the presence of Dil as a label as well as the number of fused ghosts.     

Reduced lateral mobility of a fluorescent lipid probe in cholesterol-depleted erythrocyte membrane

The effect of cholesterol depletion of the human erythrocyte membrane on the lateral diffusion rate of a fluorescent lipid probe is reported. At low temperatures (?5 to 5°C), the diffusion of the probe is 50% slower in the cholesterol-depleted membrane than in non-depleted membrane. At high temperatures (30 to 40° C), probe mobility is not affected by cholesterol depletion. These results suggest that cholesterol suppresses aspects of phospholipid phase changes in animal cells in a manner consistent with its behavior in artificial bilayers and multilayers.Whole erythrocytes were depleted of 30–50% of their cholesterol by incubation with a sonicated dispersion of dipalmitoyl phosphatidylcholine. Cells were then labeled with 3,3′-dioctadecylindocarbocyanine (diI), a phospholipid-like fluorescent dye, and hemolyzed into spherical ghosts. The rate of lateral motion of diI was measured by observing the fluorescence recovery after local photobleaching with a focused laser spot.The diffusion rate of the lipid probe in both control and cholesterol-depleted erythrocyte membrane is substantially smaller than in any cell or model membrane previously measured.     

Plasma membrane lipid diffusion and composition of sea urchin egg membranes vary with ocean temperature

A diverse and complex array of lipids plays a vital role in structuring and organizing cell membranes. However, the details of lipid requirements for global membrane organization are poorly understood. One obstacle to this understanding is the difficulty of accurately manipulating the lipid composition of commonly studied mammalian cells. In contrast, the lipid composition of cells of ectotherms changes with changes in environmental temperatures. Thus, comparison of lipid probe diffusion in cells from animals living at different temperatures, together with biochemical analysis, can be used toward understanding membrane organization. We used two dialkyindocarbocyanine iodide (DiI) probes, of differing chain length, to probe lipid organization in terms of their lateral diffusion in eggs of the sea urchin Strongylocentrotus purpuratus. The lateral diffusion of our probes changed in urchins developing in the year of an "El Ni?o" weather event, which raised the ocean temperature by several degrees, suggesting alterations in membrane domain composition and structure. Indeed the changes in lateral diffusion were correlated with lower levels of unsaturated fatty acids and cholesterol in animals of the "El Ni?o" year than in animals of the preceding or following years. We found similar trends comparing DiI diffusion in membranes of eggs from 15 degrees C waters with those from 10 degrees C. Our findings establish a new approach for manipulating and studying membrane organization.     

Transbilayer movement of cholesterol in the human erythrocyte membrane

The rate of transbilayer movement of cholesterol was measured in intact human erythrocytes. Suspended erythrocytes were incubated briefly with [3H]cholesterol in ethanol at 4 degrees C, or with liposomes containing [3H]cholesterol over 6 hr at 4 degrees C to incorporate the tracer into the outer leaflet of erythrocyte plasma membranes. The erythrocytes were then incubated at 37 degrees C to allow diffusion of cholesterol across the membrane bilayer. Cells were treated briefly with cholesterol oxidase to convert a portion of the outer leaflet cholesterol to cholestenone, and the specific radioactivity of cholestenone was determined over the time of tracer equilibration. The decrease in specific radioactivity of cholestenone reflected transbilayer movement of [3H]cholesterol. The transbilayer movement of cholesterol had a mean half-time of 50 min at 37 degrees C in cells labeled with [3H]cholesterol in ethanol, and 130 min at 37 degrees C in cells labeled with [3H]cholesterol exchanged from liposomes. The cells were shown, by the absence of hemolysis, to remain intact throughout the assay. The presence of 1 mM Mg2+ in the assay buffer was essential to prevent hemolysis of cells treated with cholesterol oxidase perturbed the cells, resulting in an accelerated rate of apparent transbilayer movement. Our data are also consistent with an asymmetric distribution of cholesterol in erythrocyte membranes, with the majority of cholesterol in the inner leaflet.     

The Rh polypeptide is a major fatty acid-acylated erythrocyte membrane protein

The erythrocyte Rh antigens contain an Mr = 32,000 integral protein which is thought to contribute in some way to the organization of surrounding phospholipid. To search for possible fatty acid acylation of the Rh polypeptide, intact human erythrocytes were incubated with [3H]palmitic acid prior to preparation of membranes and sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography. Several membrane proteins were labeled, but none corresponded to the glycophorins or membrane proteins 1-8. An Mr = 32,000 band was prominently labeled on Rh (D)-negative and -positive erythrocytes and could be precipitated from the latter with anti-D. No similar protein was labeled on membranes from Rhmod erythrocytes, a rare phenotype lacking Rh antigens. Labeling of the Rh polypeptide most likely represents palmitic acid acylation through thioester linkages. The 3H label was not extracted with chloroform/methanol, but was quantitatively eluted with hydroxylamine and co-chromatographed with palmitohydroxamate and free palmitate by thin layer chromatography. The fatty acid acylations occurred independent of protein synthesis and were completely reversed by chase with unlabeled palmitate. It is concluded that the Rh polypeptide is fatty acid-acylated, being a major substrate of an acylation-deacylation mechanism associated with the erythrocyte membrane.     

Application of fluorescence photobleaching recovery to assess complex formation between the two envelope proteins of Sendai virus in membranes of fused human erythrocytes

Fusion of human erythrocytes by Sendai virions is accompanied by lateral mobilization of the viral envelope proteins (F, the fusion protein, and HN, the hemagglutinin/neuraminidase protein) in the target cell membrane; the dynamic parameters characterizing the lateral diffusion of F and HN in the fused cell membrane are identical [Henis, Y. I., & Gutman, O. (1987) Biochemistry 26, 812-819; Aroeti, B., & Henis, Y. I. (1988) Biochemistry 27, 5654-5661]. This identity raised the possibility that F and HN diffuse together in the cell membrane in mutual heterocomplexes. In order to investigate the possible formation of F-HN complexes in the target cell membrane, which could be important for the fusion process mediated by the viral envelope proteins, we combined fluorescence photobleaching recovery (FPR) measurements of the lateral mobility of the viral glycoproteins with antibody-mediated cross-linking of F or HN. After fusion, one viral glycoprotein type was immobilized by cross-linking with highly specific bivalent polyclonal IgG. The other glycoprotein type was labeled with fluorescence monovalent Fab' fragments that do not induce cross-linking, and its mobility was measured by FPR. Neither the mobile fraction nor the lateral diffusion coefficient of the Fab'-labeled viral glycoproteins was affected by immobilization of the second viral envelope protein, demonstrating that F and HN diffuse independently in the target cell membrane and are not associated in mutual complexes.     

Transient alterations in the lateral mobility of erythrocyte membrane components during Sendai virus-mediated fusion.

Destabilization of the target membrane structure by fusion-promoting viral glycoproteins is assumed to be an essential part of the fusion mechanism. To explore this possibility, we employed fluorescence photobleaching recovery to investigate changes in the lateral mobility of native membrane constituents in human red blood cells (RBCs) during the course of Sendai virus-mediated fusion. The mobile fraction of RBC membrane proteins labeled with 5-(4,6-dichloro-5-triazin-2-yl)aminofluorescein increased significantly in the course of fusion, relaxing back to the original values upon completion of the fusion process. A different effect was observed on the lateral mobility of a fluorescent lipid probe, N-(7-nitro-2,1,3-benzoxadiazol-4-yl)phosphatidylethanolamine, incorporated initially into the external monolayer. In this case, the lateral diffusion coefficient (rather than the mobile fraction) increased during fusion; this increase was permanent in the absence of Mg-ATP and transient in its presence. An active viral fusion protein was required to mediate the effects on both protein and lipid mobility. These effects, which take place on the same time scale as that of the fusion process, suggest that the organization of the RBC membrane is perturbed during fusion and that the observed changes may be related to the fusion mechanism.     

Heat-induced alterations in monkey erythrocyte membrane phospholipid organization and skeletal protein structure and interactions

Rhesus monkey erythrocytes were subjected to heating at 50 degrees C for 5-15 min, and the heat-induced effects on the membrane structure were ascertained by analysing the membrane phospholipid organization and membrane skeleton dynamics and interactions in the heated cells. Membrane skeleton dynamics and interactions were determined by measuring the Tris-induced dissociation of the Triton-insoluble membrane skeleton (Triton shells), the spectrin-actin extractability at low ionic strength, spectrin self-association and spectrin binding to normal monkey erythrocyte membrane inside-out vesicles (IOVs). The Tris-induced Triton shell dissociation and spectrin-actin extractability were markedly decreased by the erythrocyte heating. Also, the binding of the heated erythrocyte membrane spectrin-actin with the IOVs was much smaller than that observed with the normal erythrocyte spectrin-actin. Further, the spectrin structure was extensively modified in the heated cells, as compared to the normal erythrocytes. Transbilayer phospholipid organization was ascertained by employing bee venom and pancreatic phospholipases A2, fluorescamine, and Merocyanine 540 as the external membrane probes. The amounts of aminophospholipids hydrolysed by phospholipases A2 or labeled by fluorescamine in intact erythrocytes considerably increased after subjecting them to heating at 50 degrees C for 15 min. Also, the fluorescent dye Merocyanine 540 readily stained the 15-min-heated cells but not the fresh erythrocytes. Unlike these findings, the extent of aminophospholipid hydrolysis in 5-min-heated cells by phospholipases A2 depended on the incubation time. While no change in the membrane phospholipid organization could be detected in 10 min, prolonged incubations led to the increased aminophospholipid hydrolysis. Similarly, fluorescamine failed to detect any change in the transbilayer phospholipid distribution soon after the 5 min heating, but it labeled greater amounts of aminophospholipids in the 5-min-heated cells, as compared to normal cells, after incubating them for 4 h at 37 degrees C. These results have been discussed to analyse the role of membrane skeleton in maintaining the erythrocyte membrane phospholipid asymmetry. It has been concluded that both the ATP-dependent aminophospholipid pump and membrane bilayer-skeleton interactions are required to maintain the transbilayer phospholipid asymmetry in native erythrocyte membrane.     

A delay in membrane fusion: lag times observed by fluorescence microscopy of individual fusion events induced by an electric field pulse

Low light level video microscopy of the fusion of DiI- (1,1'-dihexadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate) labeled rabbit erythrocyte ghosts with unlabeled rabbit erythrocyte ghosts, held in stable apposition by dielectrophoresis in sodium phosphate buffers, showed reproducible time intervals (delays) between the application of a single fusogenic electric pulse and the earliest detection of fluorescence in the unlabeled adjacent membranes. The delay increased over the range 0.3-4 s with a decrease in (i) the electric field strength of the fusion-inducing pulse from 1000 to 250 V/mm, (ii) the decay half-time of the fusogenic pulse in the range 1.8-0.073 ms, and (iii) the dielectrophoretic force which brings the membranes into close apposition. A change in the buffer viscosity from 1.8 to 10 mP.s caused the delay to increase from 0.36 to 3.7 s (in glycerol solutions) or to 5.2 s (in sucrose solutions). The delay decreased 2-3 times with an increase in temperature from 21 to 37 degrees C. It did not differ significantly for "white" ghosts [0.013 mM hemoglobin (Hb)] or "red" ghosts (0.15 mM Hb) or buffer strength over the range 5-60 mM (sodium phosphate, pH 8.5). The calculated activation energy, 17 kcal/mol, does not depend on the field strength. The yield of fused cells was high when the delay was short. The delay in electrofusion resembles the delays in pH-dependent fusion of vesicular stomatitis viruses with erythrocyte ghosts [Clague, M. J., Schoch, C., Zech, L., & Blumenthal, R. (1990) Biochemistry 29, 1303-1308] and of fibroblasts expressing influenza hemagglutinin and red blood cells [Morris, S. J., Sarkar, D.P., White, J. M., & Blumenthal, R. (1989) J. Biol. Chem. 264, 3972-3978].(ABSTRACT TRUNCATED AT 250 WORDS)     

Thallium and rubidium permeability of human and rat erythrocyte membrane

Transport of Tl+ and Rb+ in human and rat erythrocytes was investigated in the presence of ouabain. The chloride-dependent cotransport of Tl+, Rb+ and Na+ was precluded by replacement of Cl- by NO3-. The inward and outward rate constants for the residual fluxes of the cations were determined by measuring the transport of 204Tl and 86Rb in double label experiments. The rate of passive transport of Tl+ exceeded that of Rb+ by one-two orders of magnitude in human as well as rat erythrocytes. The membrane barrier which contributes to the maintenance of ion gradients was shown not to be a barrier for Tl+ which easily penetrates the membrane by an unknown mechanism. In rat erythrocytes the barrier for Rb+ was 10-15 times weaker than that in human red blood cells, while the corresponding ratio of rat/human Tl+ permeabilities was about 1.8-2.0. It follows that Tl+ permeability is only slightly affected by factors modifying the permeability to alkali cations. The increase of temperature from 20 degrees to 37 degrees C resulted in a three-fourfold stimulation of the passive transport of Tl+ both in human and rat erythrocytes. The movement of Tl+ and Rb+ through the erythrocyte membrane differed substantially from their diffusion along the excitable membrane channels characterized both by poor Tl+/K+ selectivity and weak temperature dependence.     

Outside-inside translocation of aminophospholipids in the human erythrocyte membrane is mediated by a specific enzyme

When human erythrocytes are incubated with spin-labeled analogues of sphingomyelin, phosphatidylcholine, phosphatidylserine, or phosphatidylethanolamine, with a short beta chain (C5) bearing a doxyl group at the fourth carbon position, the labeled lipids incorporate readily in the outer monolayer. The incorporation is followed in fresh erythrocytes by a selective inward diffusion of the amino derivatives. This observation led us to postulate the existence of a selective ATP-dependent system that would flip aminophospholipids from the outer to the inner monolayer [Seigneuret, M., & Devaux, P. F. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 3751-3755]. This study further examines the nature of this selective transport and demonstrates that it is mediated by a specific membrane protein. By measurement of the initial rate of transverse diffusion of spin-labeled lipids incorporated at various concentrations in the membrane outer leaflet of packed erythrocytes, apparent Km values were determined for the phosphatidylserine and phosphatidylethanolamine analogues. A ratio of approximately equal to 1/9.4 [corrected] was obtained (KmPS/KmPE). Using spin-labels bearing either a 14N or a 15N isotope, we have carried out competition experiments allowing us to measure simultaneously the transport of two different phospholipids. By this procedure, we show that phosphatidylserine and phosphatidylethanolamine compete for the same transport site but that phosphatidylserine has a higher affinity, in agreement with a lower apparent Km. On the other hand, the slow diffusion of the phosphatidylcholine or sphingomyelin analogues has no influence on the transport of phosphatidylserine or phosphatidylethanolamine. Experiments carried out in ghosts loaded with ATP enabled us to determine the activation energies for phosphatidylserine and phosphatidylcholine transverse diffusion.(ABSTRACT TRUNCATED AT 250 WORDS)     

Distinct mechanical relaxation components in pairs of erythrocyte ghosts undergoing fusion.

It was previously reported (Chernomordik and Sowers, 1991) that erythrocyte ghosts which were exposed to a 42 degrees C, 10-min heat treatment would, upon electrofusion, produce over 15-20 s a fusion product with an "open lumen" (i.e., the fusion product became converted to one large sphere), while electrofusion of ghost membranes not so exposed would lead to chains of polyghosts. In phase optics the chains of polyghosts showed a "flat diaphragm" at virtually every ghost-ghost junction (i.e., the ghosts do not appear to be fused even though fluorescent-labeled lipid analogs can laterally diffuse from a labeled ghost to an adjacent unlabeled ghost). In the present study we found that the diameter increase in open lumen- and flat diaphragm-producing fusion processes both had a rapid but short early phase (0-5 s after fusion) which was exponential or nearly so and a slow but long late phase (5-120 s after fusion) which was essentially linear. Heat treatments at 39 or 42 degrees C caused a minor acceleration in only the late phase, while temperatures of 45 or 50 degrees C caused an immediate and dramatic acceleration in the rate of diameter increase (spheres in 1-2 s). Ghost membranes in the presence of glycerol at 20% (v/v) did not form open lumens when exposed to the 42 degrees C (but not the > or = 45 degrees C) heat treatment. This suggested that the heat treatment was denaturing a critical protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dynamics of membrane structure of frog erythrocyte ghosts measured with a nanosecond fluorometer

The dynamics of membrane microstructure was studied as molecular motions of phospholipids for bullfrog erythrocyte ghosts by the DPH fluorescence depolarization technique with a nanosecond fluorometer. The bullfrog erythrocyte ghosts were obtained by hypotonic lysis and collagenase treatment. The constituents of membrane proteins were confirmed by the disk gel electrophoresis. The viscosity of erythrocyte membrane ghosts was estimated to be 3.3 +/- 1.0 at 10 degrees C, and 2.1 +/- 0.1 at 20 degrees C and 1.3 +/- 0.2 at 30 degrees C in the unit of poise and the wobbling angle of lipid molecule was 35 +/- 1, 41 +/- 1 and 43 +/- 1 degree at the respective temperatures on an average and +/- S.D. The viscosity is lower than that of human erythrocytes. The relatively low viscous phospholipid bilayer may be one of the factors for the deformability of bullfrog erythrocytes.     

Rotational diffusion of the erythrocyte integral membrane protein band 3: effect of hemichrome binding.

Human erythrocyte band 3 was covalently labeled within the integral membrane domain by incubating intact erythrocytes with the phosphorescent probe eosinyl-5-maleimide. The rotational diffusion of band 3 in membranes prepared from these labeled cells was measured using the technique of time-resolved phosphorescence anisotropy. Three rotational correlation times ranging from 16 to 3800 microseconds were observed, suggesting that band 3 exists in different aggregate states within the plane of the membrane. The oxidizing agent phenylhydrazine was used to induce hemichrome formation within intact erythrocytes. The immobilization of band 3 in membranes prepared from these erythrocytes suggests that the binding of hemichromes induces clustering of band 3. The addition of purified hemichromes to erythrocyte ghosts leads to a similar effect. We have also examined the mobility of the cytoplasmic domain of band 3. This region was labeled indirectly using a phosphorescently labeled antibody which binds to an epitope within the cytoplasmic domain. We observed very rapid motion of the cytoplasmic region of band 3, which was only partially restricted upon hemichrome binding. This suggests that the integral and cytoplasmic domains of band 3 may be independently mobile.     

Effect of complement on the lateral mobility of erythrocyte membrane proteins. Evidence for terminal complex interaction with cytoskeletal components

The lateral mobilities of erythrocyte membrane proteins and terminal complement complexes (TCC) were measured on C-treated erythrocyte ghosts by the technique of fluorescence redistribution after photobleaching. Results showed that the lateral diffusion coefficient of the bulk membrane proteins decreased with the assembly of TCC on the membrane at low C dose and was significantly reduced with assembly of the full membrane attack complex (C5b-9), even in the absence of cell lysis. At high serum doses, the mobility of the membrane proteins increased slightly above that of the control cells. The diffusion coefficients of the TCC on the erythrocyte membrane range from 1.18 to 4.37 x 10(-11) cm2/s, values characteristic of anchored membrane proteins. Spectrin-depletion of the C-lysed erythrocytes results in 25- and 45-fold increases in the diffusion coefficients of the membrane proteins and the C5b-9 complex, respectively. Conversely, oxidative cross-linking of spectrin by diamide reduced the diffusion coefficients of both membrane and C proteins. These studies indicate that the deposition of TCC on an erythrocyte can result in a substantial change in the physical and structural properties of the target membrane, aside from the creation of functional lesions. The low mobilities of the terminal complexes on the target membrane suggest possible interactions with cytoskeletal elements or with anchored membrane proteins.     

Evidence that electrofusion yield is controlled by biologically relevant membrane factors

Rabbit + rabbit and human + human combinations of erythrocyte ghost membranes were fused under the same conditions with an electric pulse. Storage at 4 degrees C of ghost membranes from both rabbit and human erythrocytes showed no change with time but storage of the erythrocytes for various periods before ghost preparation showed consistent storage-dependent changes in fusion yield.     

The influenza virus-induced fusion of erythrocyte ghosts does not depend on osmotic forces

The role of osmotic forces and cell swelling in the influenza virus-induced fusion of unsealed or resealed ghosts of human erythrocytes was investigated under isotonic and hypotonic conditions using a recently developed fluorescence assay (Hoekstra, D., De Boer, T., Klappe, K., Wilschut, J. (1984) Biochemistry 23, 5675-5681). The method is based on the relief of fluorescence selfquenching of the fluorescent amphiphile octadecyl rhodamine B chloride (R18) incorporated into the ghost membrane as occurs when labeled membranes fuse with unlabeled membranes. No effect neither of the external osmotic pressure nor of cell swelling on virally mediated ghost fusion was established. Influenza virus fused unsealed ghosts as effectively as resealed ghosts. It is concluded that neither osmotic forces nor osmotic swelling of cells is necessary for virus-induced cell fusion. This is supported by microscopic observations of virus-induced fusion of intact erythrocytes in hypotonic and hypertonic media. A disruption of the spectrin-actin network did not cause an enhanced cell fusion at acidic pH of about 5 or any fusion at pH 7.4.