Surface shape change during fusion of erythrocyte membranes is sensitive to membrane skeleton agents.

We previously reported that the induction of membrane fusion between pairs of erythrocyte ghosts is accompanied by the formation of a multipore fusion zone that undergoes an area expansion with condition-dependent characteristics. These characteristics allowed us to hypothesize substantial, if not major, involvement of the spectrin-based membrane skeleton in controlling this expansion. It was also found that the fusion zone, which first appears in phase optics as a flat diaphragm, has a lifetime that is also highly condition-dependent. We report here that 2,3-diphosphoglycerate, wheat germ agglutinin, diamide, and N-ethylmaleimide, all known to have binding sites primarily on skeleton components (including spectrin), have condition-dependent effects on specific components of the fusion zone diameter versus time expansion curve and the flat diaphragm lifetime. We also report a pH/ionic strength condition that causes a dramatic stabilization of flat diaphragms in a manner consistent with the known pH/ionic strength dependence of the spectrin calorimetric transition, thus further supporting the hypothesis of spectrin involvement. Our data suggest that the influence of the membrane skeleton on cell fusion is to restrain the rounding up that takes place after membrane fusion and that it may have variable, rather than fixed, mechanical properties. Data show that WGA, a known ligand for sialic acid, and DPG, a known metabolite, influences the flat diaphragm stability and late period expansion rates, raising the possibility that some of these mechanical properties are biologically regulated.     

Membrane skeleton involvement in cell fusion kinetics: a parameter that correlates with erythrocyte osmotic fragility.

Spectrin levels in erythrocytes have been related to several biomechanical and biophysical membrane properties essential to the survival and function of the cell. Populations of erythrocytes display a natural and finite range of sensitivities to osmotic shock that has been directly correlated, in studies from other laboratories, to the presence of spectrin. We used a procedure to isolate subpopulations of 1) the osmotically most sensitive and 2) the osmotically most resistant erythrocyte membranes in an attempt to select for membranes enriched and depleted in spectrin (and/or a related component). The mechanical function of the spectrin-based membrane skeleton was further explored in these two subpopulations by searching for any effect on the time-dependent increase in fusion zone diameter in pairs of electrofused erythrocyte ghosts as a model for cell fusion. The results clearly show that the diameter expansions in fusions of membranes from osmotically resistant erythrocytes are faster in the early stage (up to 9 to 10 s after fusion) but do not thereafter expand as far as in fusions of membranes from osmotically sensitive membranes.     

Evidence that the spectrin network and a nonosmotic force control the fusion product morphology in electrofused erythrocyte ghosts.

The conversion of the membrane area in the "contact zones" shared by erythrocyte ghosts held in contact by dielectrophoresis into a fusion product by electrofusion was studied by both light and electron microscopy. Fusion products fell into two categories: (a) those with a freely expanding open lumen which ended in the "giant cell morphology" and with considerable internal vesicle membrane fragments, and (b) linear chains of polyghosts with long term stability but having planar diaphragms at the ghost-ghost junctions. Thin section electron microscopy showed each of these planar diaphragms to be a double membrane septum multiply-perforated with fusion pores. Heat and low ionic strength treatments known to denature or detach spectrin caused the stable planar diaphragms to dissolve, thereby quickly converting the polyghost chains to the giant cell morphology, thereby suggesting that spectrin restricts fusion zone diameter expansion if it is intact. Other indications suggest that the expansion of the open lumens appears to take place as a result of one or more membrane-specific forces with a nonosmotic origin but this tendency to expansion can be overcome if the spectrin network on only one side of a contact zone is intact.     

A long-lived fusogenic state is induced in erythrocyte ghosts by electric pulses

Treatment of erythrocyte ghosts in random positions in a suspension with membrane fusion-inducing direct current electric field pulses causes the membranes to become fusogenic. Significant fusion yields are observed if the membranes are dielectrophoretically aligned into membrane-membrane contact with a weak alternating electric field as much as 5 min after the application of the pulses. This demonstrates that a long-lived membrane structural alteration is involved in this fusion mechanism. Other experiments indicate that the areas on the membrane which become fusogenic after treatment with the pulses may be very highly localized. The locations of these fusogenic areas coincide with where the trans-membrane electric field strength was greatest during the pulse. The fusogenic membrane alteration, or components thereof, in these areas laterally diffuses very slowly or not at all, or, to be fusogenic, must be present at concentrations in the membrane above a certain threshold. The loss of soluble 0.9-3-nm-diameter fluorescent probes from resealed cytoplasmic compartments of randomly positioned erythrocyte ghosts occurs through electric field pulse-induced pores only during a pulse but not between pulses or after a train of pulses if the probe diameter is 1.2 nm or greater. For a given pulse treatment of membranes in random positions in suspensions, an increase in ionic strength of the medium results in (a) a decrease in loss during the pulse, (b) no difference in loss between pulses, and (c) an increase in fusion yield when membrane-membrane contact is established. The latter two results (b and c) are incompatible with a fusion mechanism that proposes a simple relationship between electric field-induced pores and fusion.     

Membrane transformation during malaria parasite release from human red blood cells

Three opposing pathways are proposed for the release of malaria parasites from infected erythrocytes: coordinated rupture of the two membranes surrounding mature parasites; fusion of erythrocyte and parasitophorus vacuolar membranes (PVM); and liberation of parasites enclosed within the vacuole from the erythrocyte followed by PVM disintegration. Rupture by cell swelling should yield erythrocyte ghosts; membrane fusion is inhibited by inner-leaflet amphiphiles of positive intrinsic curvature, which contrariwise promote membrane rupture; and without protease inhibitors, parasites would leave erythrocytes packed within the vacuole. Therefore, we visualized erythrocytes releasing P. falciparum using fluorescent microscopy of differentially labeled membranes. Release did not yield erythrocyte ghosts, positive-curvature amphiphiles did not inhibit release but promoted it, and release of packed merozoites was shown to be an artifact. Instead, two sequential morphological stages preceded a convulsive rupture of membranes and rapid radial discharge of separated merozoites, leaving segregated internal membrane fragments and plasma membrane vesicles or blebs at the sites of parasite egress. These results, together with the modulation of release by osmotic stress, suggest a pathway of parasite release that features a biochemically altered erythrocyte membrane that folds after pressure-driven rupture of membranes.     

Sendai Virus Fusion Activity as Modulated by Target Membrane Components

We have studied the differences between erythrocytes and erythrocyte ghosts as target membranes for the study of Sendai virus fusion activity. Fusion was monitored continuously by fluorescence dequenching of R18-labeled virus. Experiments were carried out either with or without virus/target membrane prebinding. When Sendai virus was added directly to a erythrocyte/erythrocyte ghost suspension, fusion was always lower than that obtained when experiments were carried out with virus already bound to the erythrocyte/erythrocyte ghost in the cold, since with virus prebinding fusion can be triggered more rapidly. Although virus binding to both erythrocytes and erythrocyte ghosts was similar, fusion activity was much more pronounced when erythrocyte ghosts were used as target membranes. These observations indicate that intact erythrocytes and erythrocyte ghosts are not equivalent as target membranes for the study of Sendai virus fusion activity. Fusion of Sendai virus with both target membranes was inhibited when erythrocytes or erythrocyte ghosts were pretreated with proteinase K, suggesting a role of target membrane proteins in this process. Treatment of both target membranes with neuraminidase, which removes sialic acid residues (the biological receptors for Sendai virus) greatly reduced viral binding. Interestingly, this treatment had no significant effect on the fusion reaction itself.     

Viral and non-viral induced fusion of pronase-digested human erythrocyte ghosts.

Human erythrocyte ghosts but was able to fuse only iso-human erythrocyte ghosts. Iso- and hypo-human erythrocyte ghosts were incubated with the proteolytic enzyme pronase under isotonic (iso-human erythrocyte ghosts) or hypotonic (hypo-human erythrocyte ghosts) conditions. Gel electrophoresis and electron microscope (freeze-etching) studies revealed that most of the erythrocyte membrane polypeptides were hydrolyzed by pronase under hypotonic conditions. Sendai virus readily agglutinated both pronase-digested iso-human erythrocyte ghosts and hypo-human erythrocyte ghosts were fused by the non-viral fusogenic agent glyceromonooleate. Freeze-etching studies revealed that during fusion the membranes of pronase-digested human erythrocyte ghosts are intermixed.     

Effect of erythrocyte transbilayer phospholipid distribution on fusion with vesicular stomatitis virus

To identify the specific component(s) in the target membrane involved in fusion of vesicular stomatitis virus (VSV), we examined the interaction of the virus with human erythrocyte membranes with asymmetric and symmetric bilayer distributions of phospholipids. Fusion was monitored spectrofluorometrically by the octadecylrhodamine dequenching assay. Fusion of VSV with lipid-symmetric erythrocyte ghosts was rapid at 37 degrees C and low pH, whereas little or no fusion was observed with lipid-asymmetric ghosts. Conversion of phosphatidylserine in the lipid-symmetric ghost membrane to phosphatidylethanolamine by means of the enzyme phosphatidylserine decarboxylase did not alter the target membrane's susceptibility to VSV fusion. Spin-labeled phospholipid analogues with phosphatidylserine, phosphatidylethanolamine, and phosphatidylcholine headgroups incorporated into the outer leaflet of lipid-asymmetric erythrocytes did not render those membranes fusogenic. Electron spin resonance spectra showed an increased mobility of a phosphatidylcholine spin-label incorporated into the outer leaflet of lipid-symmetric erythrocyte ghosts as compared to that of lipid-asymmetric ghosts. These results indicate that the susceptibility to VSV fusion is not dependent on any particular phospholipid but rather is related to packing characteristics of the target membrane.     

Action of polyethylene glycol on the fusion of human erythrocyte membranes

Summary Factors affecting the polyethylene glycol (PEG)-induced membrane fusion were examined. Human erythrocyte membrane ghosts, cytoskeleton-free vesicles budded from erythrocytes, mechanically disrupted erythrocyte vesicles, and recombinant vesicles from glycophorin and egg phosphatidylcholine were used as models. Fusion was monitored by darkfield light microscopy and by freeze-fracture electron microscopy. Osmotic swelling was found necessary for fusion between membrane ghosts following PEG treatment. The sample with the highest fusion percentage was sealed ghosts incubated in hypotonic media after at least 5 min of treatment in <25% PEG. At similar osmolarity, glycerol, dextran and PEG produced progressively more pronounced intramembranous particle (IMP) patching, correlating with their increasing fusion percentages. The patching of IMP preceded cell-cell contact, and occurred without direct PEG-protein interaction. The presence of cytoskeletal elements in small vesicles had no significant effect on fusion, nor on the aggregation of intramembranous particle (IMP) upon PEG treatment. Disrupting the membrane by lysolecithin, dimethylsulfoxide, retinol or mild sonication resulted in the fragmentation of ghosts without an increase in fusion percentage. The purity of the commercial PEG used had no apparent effect on fusion. We concluded that the key steps in PEG-induced fusion of cell membrane are the creation of IMP-free zones, and the osmotic swelling of cells after the formation of bilayer contacts during the PEG treatment. Cell cytoskeleton affects PEG-induced fusion only to the extent of affecting IMP patching.     

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.     

The Effect of PS Content on the Ability of Natural Membranes to Fuse with Positively Charged Liposomes and Lipoplexes

Supramolecular aggregates containing cationic lipids have been widely used as transfection mediators due to their ability to interact with negatively charged DNA molecules and biological membranes. First steps of the process leading to transfection are partly electrostatic, partly hydrophobic interactions of liposomes/lipoplexes with cell and/or endosomal membrane. Negatively charged compounds of biological membranes, namely glycolipids, glycoproteins and phosphatidylserine (PS), are responsible for such events as adsorption, hemifusion, fusion, poration and destabilization of natural membranes upon contact with cationic liposomes/lipoplexes. The present communication describes the dependence of interaction of cationic liposomes with natural and artificial membranes on the negative charge of the target membrane, charges which in most cases were generated by charging the PS content or its exposure. The model for the target membranes were liposomes of variable content of PS or PG (phosphatidylglycerol) and erythrocyte membranes in which the PS and other anionic compound content/exposure was modified in several ways. Membranes of increased anionic phospholipid content displayed increased fusion with DOTAP (1,2-dioleoyl-3-trimethylammoniumpropane) liposomes, while erythrocyte membranes partly depleted of glycocalix, its sialic acid, in particular, showed a decreased fusion ability. The role of the anionic component is also supported by the fact that erythrocyte membrane inside-out vesicles fused easily with cationic liposomes. The data obtained on erythrocyte ghosts of normal and disrupted asymmetry, in particular, those obtained in the presence of Ca²⁺, indicate the role of lipid flip-flop movement catalyzed by scramblase. The ATP-depletion of erythrocytes also induced an increased sensitivity to hemoglobin leakage upon interactions with DOTAP liposomes. Calcein leakage from anionic liposomes incubated with DOTAP liposomes was also dependent on surface charge of the target membranes. In all experiments with the asymmetric membranes the fusion level markedly increased with an increase of temperature, which supports the role of membrane lipid mobility. The decrease in positive charge by binding of plasmid DNA and the increase in ionic strength decreased the ability of DOTAP liposomes/lipoplexes to fuse with erythrocyte ghosts. Lower pH promotes fusion between erythrocyte ghosts and DOTAP liposomes and lipoplexes. The obtained results indicate that electrostatic interactions together with increased mobility of membrane lipids and susceptibility to form structures of negative curvature play a major role in the fusion of DOTAP liposomes with natural and artificial membranes.     

The effect of the membrane skeleton of non-nucleated erythrocytes on the properties of transport ATPases]

Removal of spectrin and other proteins of membrane skeleton from rat erythrocyte membranes resulted in a significant loss of Na,K-ATPase and Ca-ATPase activities, and even more of respective phosphatase activities. At the same time the modulating influence of ATP and Ca2+ on the enzymes disappeared. These ATPase activities were reconstituted by addition of concentrated spectrin to spectrin-depleted membranes. The activating influence of Ca2+ on ouabain-resistant and ouabain-sensitive phosphatases in ghosts could be discovered only in the presence of ATP. The highest activities of both the phosphatases were revealed when both ATP (0.5 mM) and Ca2+ (10-30 mM) were present simultaneously in the incubation medium. These data show that the functioning of transport ATPases in non-nuclear erythrocyte membranes is related to the membrane skeleton: regulating influence of intracellular ATP and Ca2+ on enzymes seems to be realized through the proteins of the skeleton.     

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.     

The lens membrane skeleton contains structures preferentially enriched in spectrin-actin or tropomodulin-actin complexes

The spectrin-based membrane skeleton plays an important role in determining the distributions and densities of receptors, ion channels, and pumps, thus influencing cell shape and deformability, cell polarity, and adhesion. In the paradigmatic human erythrocyte, short tropomodulin-capped actin filaments are cross-linked by spectrin into a hexagonal network, yet the extent to which this type of actin filament organization is utilized in the membrane skeletons of nonerythroid cells is not known. Here, we show that associations of tropomodulin and spectrin with actin in bovine lens fiber cells are distinct from that of the erythrocyte and imply a very different molecular organization. Mechanical disruption of the lens fiber cell membrane skeleton releases tropomodulin and actin-containing oligomeric complexes that can be isolated by gel filtration column chromatography, sucrose gradient centrifugation and immunoadsorption. These tropomodulin-actin complexes do not contain spectrin. Instead, spectrin is associated with actin in different complexes that do not contain tropomodulin. Immunofluorescence staining of isolated fiber cells further demonstrates that tropomodulin does not precisely colocalize with spectrin along the lateral membranes of lens fiber cells. Taken together, our data suggest that tropomodulin-capped actin filaments and spectrin-cross-linked actin filaments are assembled in distinct structures in the lens fiber cell membrane skeleton, indicating that it is organized quite differently from that of the erythrocyte membrane skeleton.     

Fusion of erythrocyte ghosts induced by calcium phosphate. Kinetic characteristics and the role of Ca2+, phosphate and calcium-phosphate complexes

Using an assay which allows continuous monitoring of the mixing of aqueous contents during membrane fusion, we have investigated the kinetics of calcium-phosphate-induced fusion of erythrocyte ghosts. In the presence of 10 mM phosphate, the threshold concentration for Ca2+-induced fusion was 1.25 mM, while the optimal concentration was approx. 1.75 mM Ca2+. Further enhancement of the cation concentration (greater than or equal to 2 mM) inhibited fusion of the ghosts. Initiation of fusion required the addition of phosphate prior to the addition of Ca2+, indicating that the combined interaction of Ca2+ and phosphate in or at the plane of the bilayer was a prerequisite for the induction of fusion. Furthermore, fusion was greatly facilitated upon transformation of calcium phosphate in the bulk medium from an amorphous to a solid, crystalline phase. It is suggested that membrane aggregation, and hence fusion, is facilitated by the formation of crystalline calcium phosphate nucleating on the ghost membrane. La3+, Mg2+ and Mn2+ did not trigger the fusion process, although aggregation of the ghosts did occur. Under conditions where calcium phosphate precipitation was inhibited, lanthanum phosphate precipitates facilitated fusion after prior treatment of ghosts with phosphate and Ca2+. These results indicated that fusion-prone conditions were induced prior to calcium phosphate precipitation. It is proposed that prior to calcium phosphate precipitation membrane changes are induced by separate interaction of Ca2+ and phosphate with the ghost membrane. Such an interaction could then render the ghosts susceptible to fusion and as soon as conditions are provided allowing close contact between adjacent membranes, fusion will be observed.     

The in vitro influence of the external electrostatic field on the physical parameters of erythrocyte membranes

The in vitro influence of external electrostatic fields with 200 kV/m tension on the biophysical parameters of the erythrocyte membranes and their ghosts of white outbred rats was studied. The investigation on the parameters of erythrocyte membranes and their ghosts, particularly, their microviscosity, the amount and degree of membrane proteins submersion in lipids, polarity in depth of the membrane bilayer and its viscosity was carried out by the spectrofluorimeteric method using pyrene as a hydrophobic fluorescent probe. The analyses of literature data, findings of the current study and their comparison with the results of our previous works allow of concluding that the in vitro influence of external electrostatic fields with 200 kV/m tension on the erythrocyte membranes and their ghosts occurs at different sites of membrane. It is shown that the preliminary exposure of erythrocytes in external electrostatic fields leads to the changes of the parameters both of a membrane surface layer and the intra-membrane domains. So, the decrease in the strength of peripheral proteins binding to the erythrocyte membranes and the increase in the microviscosity of the lipid bilayer are observed. The influence of the field on the ghosts of intact erythrocytes results in alterations of the studied parameters only of the membrane surface.     

The effect of the lipid-binding site of the ankyrin-binding domain of erythroid β-spectrin on the properties of natural membranes and skeletal structures

It was previously shown that the beta-spectrin ankyrin-binding domain binds lipid domains rich in PE in an ankyrin-dependent manner, and that its N-terminal sequence is crucial in interactions with phospholipids. In this study, the effect of the full-length ankyrin-binding domain of β-spectrin on natural erythrocyte and HeLa cell membranes was tested. It was found that, when encapsulated in resealed erythrocyte ghosts, the protein representing the full-length ankyrin-binding domain strongly affected the shape and barrier properties of the erythrocyte membrane, and induced partial spectrin release from the membrane, while truncated mutants had no effect. As found previously (Bok et al. Cell Biol. Int. 31 (2007) 1482–94), overexpression of the full-length GFP-tagged ankyrin-binding domain aggregated and induced aggregation of endogenous spectrin, but this was not the case with overexpression of proteins truncated at their N-terminus. Here, we show that the aggregation of spectrin was accompanied by the aggregation of integral membrane proteins that are known to be connected to spectrin via ankyrin, i.e. Na⁺K⁺ATP-ase, IP3 receptor protein and L1 CAM. By contrast, the morphology of the actin cytoskeleton remained unchanged and aggregation of cadherin E or N did not occur upon the overexpression of either full-length or truncated ankyrin-binding domain proteins. The obtained results indicate a substantial role of the lipid-binding part of the β-spectrin ankyrin-binding domain in the determination of the membrane and spectrin-based skeleton functional properties.     

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)     

Stomatocytosis of latex particles (0.26 micron) by rat erythrocytes by the electrical breakdown technique

The uptake of macromolecules by erythrocytes can be achieved with the electrical breakdown technique [2, 4]. In this technique the erythrocyte membranes are subjected to a high external electrical field pulse for a short period. Local, reversible breakdowns of the cell membrane occur above a critical field strength which lead to a time-dependent increase in the permeability of the membrane. By this means, human erythrocyte membranes can be made permeable to DNA, pharmaceutical compounds, and latex particles following an electrical field pulse [1, 3, 5]. Larger particles should also be taken up by erythrocytes using this method. Vienken et al. [5] demonstrated the entrapment of latex particles with a diameter of 0.091 micron in human erythrocyte ghosts, although this was shown with only a single electron micrograph which does not prove that the ghost membrane was intact. In our experiments in order to entrap latex particles with a diameter of 0.26 micron rat erythrocytes were subjected to an electrical field pulse of 12 kV/cm with a decay time of 60 microseconds. Experiments using the electron microscope show that after such an electrical field pulse the uptake of latex particles by rat erythrocytes follows the stomatocytotic pathway. We show further that using electron microscopic techniques, a single section cannot demonstrate the completed uptake of a latex particle by the erythrocyte.     

Fusion events and nonfusion contents mixing events induced in erythrocyte ghosts by an electric pulse.

The mechanism of membrane fusion was studied by using human erythrocyte ghosts held in close contact by alternating current-induced dielectrophoresis and inducing fusion with a single electric field pulse. Individual fusion events were followed visually using either 1,1'-dihexadecyl-3,3,3',3'-tetramethylindo carbocyanine perchlorate as a membrane-mixing label or 10-kD fluorescein isothiocyanate-dextran as a contents-mixing label. However, over a range of variables, the number of contents-mixing events usually considerably exceeded the number of membrane-mixing events, although the discrepancy was less at higher ionic strength. However, when the dielectrophoretic force holding the membranes in contact was turned off after the pulse, Brownian motion caused some of the groups of ghosts in which contents mixing occurred to eventually separate from one another, showing that they could not represent fusion events. Separate experiments showed, conversely, that fusion did occur in the groups that did not separate after the dielectrophoresis was turned off.