The long-lived fusogenic state induced in erythrocyte ghosts by electric pulses is not laterally mobile.

The long-lived fusogenic state induced in spherical-shaped erythrocyte ghosts by electric field pulses (Sowers, A.E. 1984. J. Cell Biol. 99:1989-1996; Sowers, A.E. 1986. J. Cell Biol. 102:1358-1362) was studied in terms of how the fusion yield depended on both (a) the location where membrane-membrane contact took place with respect to the orientation of the electric pulse and (b) the time interval between the pulse treatment and membrane-membrane contact. Fusion yields were greater for membrane-membrane contact locations closer to where the pulse-induced transmembrane voltage was expected to be greatest and showed a time interval-dependent accelerating decay. The portion of the membrane that became fusogenic included the area up to a latitude of approximately 38 degrees of arc towards the equators of the membranes. A time interval-dependent increase or decrease in rate of decay in the fusion yield for membrane-membrane contacts induced closer to the equator of the membranes did not occur showing that the pulse-induced fusogenic state is immobile in the early 5-45-s interval after induction and has a rate of decay, which does not permit long time interval changes in lateral position to be measured.     

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.     

Determination of electric field threshold for electrofusion of erythrocyte ghosts. Comparison of pulse-first and contact-first protocols.

Rabbit erythrocyte ghosts were fused by means of electric pulses to determine the electrofusion thresholds for these membranes. Two protocols were used to investigate fusion events: contact-first, and pulse-first. Electrical capacitance discharge (CD) pulses were used to induce fusion. Plots of fusion yield vs peak field strength yielded curves that intersected the field strength axis at positive values (pseudothresholds) which depended on the protocol and decay half time of the pulses. It was found that plots of pseudothreshold vs reciprocal half time were linear for each protocol; when extrapolated to reciprocal half time = 0 (i.e., t----infinity), these lines intersected the ordinate at values of the field strength considered to be the true electrofusion thresholds. In this fashion, the contact-first protocol gave an electrofusion threshold of 46.5 +/- 11.5 V/mm for hemoglobin-free ghosts (white ghosts) and 40.9 +/- 8.8 V/mm for ghosts with fractional hemoglobin (pink ghosts), while the threshold for the pulse-first protocol applied to pink ghosts was determined to be 93.4 +/- 11.0 V/mm. Although the thresholds depended on the electrofusion protocol, plots of critical field strength vs reciprocal time had the same slopes, i.e., approximately 24 Vs/mm. The results suggest that the fusogenic state induced by an electric pulse in either the contact-first protocol or the pulse-first protocol (long-lived fusogenic state) may in fact share a common mechanism, if the two states are not actually identical.     

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.     

Correlation between electric field pulse induced long-lived permeabilization and fusogenicity in cell membranes.

Electric field pulses have been reported to induce long-lived permeabilization and fusogenicity on cell membranes. The two membrane property alterations are under the control of the field strength, the pulse duration, and the number of pulses. Experiments on mammalian cells pulsed by square wave form pulses and then brought into contact randomly through centrifugation revealed an even stronger analogy between the two processes. Permeabilization was known to affect well-defined regions of the cell surface. Fusion can be obtained only when permeabilized surfaces on the two partners were brought into contact. Permeabilization was under the control of the pulse duration and of the number of pulses. A similar relationship was observed as far as fusion is concerned. But a critical level of local permeabilization must be present for fusion to take place when contacts are created. The same conclusions are obtained from previous experiments on ghosts subjected to exponentially decaying field pulses and then brought into contact by dielectrophoresis. These observations are in agreement with a model of membrane fusion in which the merging of local random defects occurs when the two membranes are brought into contact. The local defects are considered part of the structural membrane reorganization induced by the external field. Their density is dependent on the pulse duration and number of pulses. They support the long-lived permeabilization. Their number must be very large to support the occurrence of membrane fusion.     

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)     

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.     

Human erythrocyte electrofusion kinetics monitored by aqueous contents mixing.

The kinetics of electrically induced fusion of human erythrocyte ghosts were monitored by the Tb/DPA and ANTS/DPX fluorescence fusion assays. Ghosts were aligned by dielectrophoresis using a 3-MHz 350-V/cm alternating field and were fused by single 15- or 50-microseconds electric field pulses of amplitude 2.5-5.0 kV/cm. Fusion was detected immediately after the pulse. The peak fluorescence change due to fusion was always obtained within 7 s of pulse application, and was highest for a 5.0 kV/cm 15-microseconds pulse. Probe leakage was measured separately and became apparent only 2-3 s after the initiation of fusion. Increasing pulse amplitudes produced higher fusion yields but produced more leakage from the fusion products. 50-microseconds pulses produced less fusion, resulting from a disruption of the dielectrophoretic alignment by fluid turbulence immediately after pulse application. Probe leakage was observed only when pulse application was preceded by dielectrophoresis, suggesting that close membrane positioning allows for additional membrane destabilization caused by the high field pulse. The fluorescence kinetics are interpreted using a simplified model depicting three major types of events: (a) fusion without observable leakage, (b) fusion followed by probe leakage, and (c) contact-related leakage from ghosts which do not undergo contents mixing.     

Electrofusion of dissimilar membrane fusion partners depends on additive contributions from each of the two different membranes

Rabbit erythrocyte ghost (REG) membranes and human erythrocyte ghosts (HEG) were aligned into contact by dielectrophoresis and fused with an electric pulse in REG + REG, HEG + HEG, and REG + HEG combinations. REG + HEG fusion yields were approximately midway between fusion yields for REG + REG and HEG + HEG over a wide range of pulse characteristics.     

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.     

Low concentrations of macromolecular solutes significantly affect electrofusion yield in erythrocyte ghosts

Electrofusion yields in rabbit erythrocyte ghosts containing various amounts of hemoglobin, bovine serum albumin, or dextran at low concentrations were measured as a function of pulse field strength and pulse decay half-time. The presence of any of the macromolecules in low concentrations caused fusion yields to be significantly higher than when the ghosts were white (i.e., containing only buffer). The fusion yield enhancement was also critically dependent on the parameters of the electric field pulse. The fusion yield was also significantly affected by small changes in the concentration of hemoglobin when it was present outside the ghost membranes in the suspension buffer.     

The Systematic Study of the Electroporation and Electrofusion of B16-F1 and CHO Cells in Isotonic and Hypotonic Buffer

The fusogenic state of the cell membrane can be induced by external electric field. When two fusogenic membranes are in close contact, cell fusion takes place. An appropriate hypotonic treatment of cells before the application of electric pulses significantly improves electrofusion efficiency. How hypotonic treatment improves electrofusion is still not known in detail. Our results indicate that at given induced transmembrane potential electroporation was not affected by buffer osmolarity. In contrast to electroporation, cells’ response to hypotonic treatment significantly affects their electrofusion. High fusion yield was observed when B16-F1 cells were used; this cell line in hypotonic buffer resulted in 41?±?9?% yield, while in isotonic buffer 32?±?11?% yield was observed. Based on our knowledge, these fusion yields determined in situ by dual-color fluorescence microscopy are among the highest in electrofusion research field. The use of hypotonic buffer was more crucial for electrofusion of CHO cells; the fusion yield increased from below 1?% in isotonic buffer to 10?±?4?% in hypotonic buffer. Since the same degree of cell permeabilization was achieved in both buffers, these results indicate that hypotonic treatment significantly improves fusion yield. The effect could be attributed to improved physical contact of cell membranes or to enhanced fusogenic state of the cell membrane itself.     

Electrofusion between heterogeneous-sized mammalian cells in a pellet: potential applications in drug delivery and hybridoma formation.

High-efficiency electrofusion between cells of different sizes was achieved by application of fusing electric pulses to cells in centrifuged pellets. Larger target cells (Chinese hamster ovary or L1210 cells) were stacked among smaller human erythrocytes or erythrocyte ghosts by sequential centrifugation at 700 g to form five-tier pellets in a specially designed centrifugation-electrofusion chamber. The membranes of erythrocytes and ghost were labeled with fluorescent membrane dye (1,1' dioctadecyl-3,3,3'3'-tetramethylindocarbocyanine (Dil)), and the contents of ghosts were loaded with water-soluble fluorescent dye (42-kDa fluorescein isothiocyanate dextran (FITC-dextran)), to monitor heterogeneous cell fusion. Fusion efficiency was assayed by the extent of either membrane dye mixing or contents (FITC-dextran) mixing with target cells. Four rectangular electric pulses at 300 V and 80 microseconds each were found to give the optimal fusion results of approximately 80% heterogeneous fusion by the content-mixing assay and approximately 95% by the membrane-dye-mixing assay. Cell viability remained greater than 80% after electrofusion. Because of the electric breakdown of cell membranes at the beginning of the pulse, the pellet resistance and hence the partial voltage across the pellet reduced rapidly during the remaining pulse time. This voltage redistribution favored the survival of fused cells. The limited colloidal-osmotic swelling of cells in pellets enhanced cell-cell contact and increased the pellet resistance after each pulse. As a result, the partial voltage across the pellet was restored when the next pulse was applied. This redistribution of pulse voltage in the pellet system permitted the breakdown of cell membranes at a lower applied voltage threshold than that required for electrofusion of cells in suspension or in dielectrophoretic cell chains. The cell viability and soluble dye retention within cells (FITC-dextran) remained at the same high levels for 3 h when the cells were incubated in respective culture media with serum at 37 degrees C. Viability and dye retention decreased significantly within 30 min when cells were incubated in phosphate-buffered saline without serum. The pellet technique was applied to form hybridomas by fusion of larger SP2/0 murine myelomas with smaller naive mouse lymphocytes. An optimum of 173 +/- 70 hypoxanthine aminopterin thymidine (HAT)-selected clones of the hybridomas was obtained from 40,000 SP2/0 cells and 1.5 x 10(6) lymphocytes used in each trial. This high-efficiency fusion technique may be adapted to mediate drug and gene transfer to target cells ex vivo as well as to form hybrid cells with limited cell sources.     

Temperature and external electric field influence in membrane permeability of Babesia infected erythrocytes

1. Modification of erythrocyte membrane properties infected by Babesia canis was studied using the effect of electric pulses of short duration. 2. This process induces the formation of pores in the membrane and the releasing of hemoglobin and other cytoplasmic proteins into the external medium. 3. The rate of molecular permeation across the electrically perforated membranes depends on several factors: electric-field strength, pulse number, pulse duration, temperature and cellular concentration. 4. Even for low parasitemia, differences in the effect of these parameters were observed between infected and non-infected erythrocytes. 5. Here we describe an influence of electric field intensity and temperatures on the opening pores.     

Membrane electroporation--fast molecular exchange by electroosmosis.

Human and rabbit erythrocyte ghosts loaded with FITC-dextran (mol. mass = 10 kDa) and NBD-glucosamine (mol. mass = 342 Da) in buffers of different ionic strength and composition were subjected to electric pulses (intensity 0.7 kV/mm and decay half-time 1 ms) at 7-10 degrees C and 20-24 degrees C. The transfer of the fluorescent dyes from the interior of the ghosts through the electropores was observed by low light level video microscopy. The pulses caused the fluorescence to appear outside the membranes as a transient cylindrical cloud directed toward the negative electrode during the first video frame (17 ms). It was similar in both rabbit and human erythrocyte ghosts and at both temperatures but differs for the two dyes, the fluorescence cylinder is long and tall for the FITC-dextran and relatively short and thick for the NBD-glucosamine. The molecular exchange was 2-3 orders of magnitude faster within the first 17 ms after the pulse than the diffusional exchange. It decreased with increasing ionic strength. Formulae for the transfer of molecules by electroosmotic flow through the pores are in agreement with these observations. They allow estimation of the total area of pores with radii larger than that of the fluorescent dye during the pulse. The major conclusion is that electroosmosis is the dominating mechanism of molecular exchange in electroporation of erythrocyte ghosts.     

Phospholipid diversity: correlation with membrane-membrane fusion events

The transport of various metabolically important substances along the endocytic and secretory pathways involves budding as well as fusion of vesicles with various intracellular compartments and plasma membrane. The membrane-membrane fusion events between various sub-compartments of the cell are believed to be mainly mediated by so-called "fusion proteins". This study shows that beside the proteins, lipid components of membrane may play an equally important role in fusion and budding processes. Inside out (ISO) as well as right side out (RSO) erythrocyte vesicles were evaluated for their fusogenic potential using conventional membrane fusion assay methods. Both fluorescence dequenching as well as content mixing assays revealed fusogenic potential of the erythrocyte vesicles. Among two types of vesicles, ISO were found to be more fusogenic as compared to the RSO vesicles. Interestingly, ISO retained nearly half of their fusogenic properties after removal of the proteins, suggesting the remarkable role of lipids in the fusion process. In another set of experiments, fusogenic properties of the liposomes (subtilosome), prepared from phospholipids isolated from Bacillus subtilis (a lower microbe) were compared with those of erythrocyte vesicles. We have also demonstrated that various types of vesicles upon interaction with macrophages deliver encapsulated materials to the cytosol of the cells. Membrane-membrane fusion was also followed by the study, in which a protein synthesis inhibitor ricin A (that does not cross plasma membrane), when encapsulated in the erythrocyte vesicles or subtilosomes was demonstrated to gain access to the cytosol.     

Membrane skeleton restraint of surface shape change during fusion of erythrocyte membranes: evidence from use of osmotic and dielectrophoretic microforces as probes.

The role of the spectrin-based membrane skeleton in cell fusion was studied by following the condition-dependent diameter versus time expansion signature of the fusion zone in electrofused pairs of erythrocyte ghost membranes. Previous work showed that the presence of the dielectrophoresis-inducing alternating electric field, which is used to bring membranes into contact through pearl chain formation, had a detectable promoting effect on fusion zone expansion. Two new dielectrophoresis protocols were used in the present work to utilize this externally generated and controllable microforce field to probe the forces intrinsic to the system that drives the expansion of the fusion zone. First, fusion zones expanded to a greater diameter in a strong AC field compared to a weak AC field, and they expanded to a greater diameter if erythrocyte ghosts received a prior heat treatment (42 degrees C, 20 min). Furthermore, flat diaphragm fusion zones broke down into open lumen fusion zones sooner (i.e., had shorter lifetimes) when they were expanding more quickly. Second, changing the AC field strength at specific times during the fusion zone expansion led to an immediate visco-elastic response. However, shifting the AC field strength to zero after 5 s of fusion zone expansion resulted in a subsequent decrease in the average fusion zone diameter. This suggests not only that the spectrin-based membrane skeleton actually tends to prevent the rounding up process but that it may be capable of generating an antirounding force, which has broad implications for the role of the membrane skeleton in cell fusion. These results are consistent with the hypothesis that flat diaphragm fusion zones induced in heat-treated membranes were very easily stretched and that membrane-based forces that control or drive the expansion process must originate from membrane area that is outside rather than inside the fusion zone. Lastly, when an outward-directed osmotic pressure-based microforce was present at the time that erythrocyte ghosts were fused, the fusion zone diameter underwent a greater expansion in the 0-1 s interval after fusion. This suggests that an osmotic pressure-based microforce can be used to experimentally calibrate the dielectrophoretic force.     

Attraction, deformation and contact of membranes induced by low frequency electric fields

The force of attraction between erythrocyte ghosts induced by low frequency electric fields (60 Hz) was measured as a function of the intermembrane separation. It varied from 10(-14) N for separation of the order of the cell diameter to 10(-12) N for close approach and contact in 20 mM sodium phosphate buffers (conductivity 260 mS/m, pH 8.5). For large separations the interaction force followed a dependence on separation as predicted for dipole-dipole interactions. For small separation an empirical formula was obtained. The membranes deformed at close approach (less than 1 microns) before making contact. The contact area increased with time until reaching the final equilibrium state. The ghosts separated reversibly after switching off the electric field. The membrane tension induced by the ghost interaction at contact was estimated to be of the order of 0.1 mN/m. These first quantitative measurements of the force/separation dependence for intermembrane interactions induced by low frequency electric fields indicate that attractive forces, membrane deformation and contact area of cells depend strongly on intermembrane separation and field strength. The quantitative relationship between them are important for measuring membrane surface and mechanical properties, intermembrane forces and understanding mechanisms of membrane adhesion, instability and fusion in electric fields and in general.     

Phospholipid diversity: Correlation with membrane-membrane fusion events

The transport of various metabolically important substances along the endocytic and secretory pathways involves budding as well as fusion of vesicles with various intracellular compartments and plasma membrane. The membrane-membrane fusion events between various sub-compartments of the cell are believed to be mainly mediated by so-called “fusion proteins”. This study shows that beside the proteins, lipid components of membrane may play an equally important role in fusion and budding processes. Inside out (ISO) as well as right side out (RSO) erythrocyte vesicles were evaluated for their fusogenic potential using conventional membrane fusion assay methods. Both fluorescence dequenching as well as content mixing assays revealed fusogenic potential of the erythrocyte vesicles. Among two types of vesicles, ISO were found to be more fusogenic as compared to the RSO vesicles. Interestingly, ISO retained nearly half of their fusogenic properties after removal of the proteins, suggesting the remarkable role of lipids in the fusion process. In another set of experiments, fusogenic properties of the liposomes (subtilosome), prepared from phospholipids isolated from Bacillus subtilis (a lower microbe) were compared with those of erythrocyte vesicles. We have also demonstrated that various types of vesicles upon interaction with macrophages deliver encapsulated materials to the cytosol of the cells. Membrane-membrane fusion was also followed by the study, in which a protein synthesis inhibitor ricin A (that does not cross plasma membrane), when encapsulated in the erythrocyte vesicles or subtilosomes was demonstrated to gain access to the cytosol.