Electric Field-induced Fusion Research Articles

Agents facilitating the electric field-induced fusion of intact rabbit erythrocytes

In the recent years, the role of specific membrane active agents in the electrofusion process has started to draw attention, and it has been found that the presence of various substances in the cell medium can affect the fusion process either in a positive or negative way. In this work, the effect of several proteins, bivalent cations and antibiotics was tested with respect to their ability to protect intact erythrocytes from hemolysis and facilitate the fusion process. The effect of different sugars was also studied. Among the different proteins, pronase and proteinase were found to be the most effective. With respect to bivalent cations, Ca 2+ and Mn 2+ were more effective while Mg 2+ was less important. From the antibiotics, penicillin caused a negative effect while streptomycin acted positively. Finally, glucose medium was found to be the most effective compared to all sugars tested. The results indicated that there are strong differentiations of the induced effects caused by each substance, and some possible mechanisms of action of these agents on the erythrocyte membrane were discussed.

Ultrastructure of Fusion Product between Protoplasts from C3 and C4 Species of Amaranthaceae

SummaryThis paper describes the ultrastructure of the electric field-induced fusion products of C3 and C4 species of Amaranthaceae at the early developmental stage. Protoplasts of C3 species were isolated from a Ceiosia cristata L. cell suspension and, those of C4 species were isolated from an Amaranthus tricolor L. cotyledon. Incompatibility occurred in the C3/C4 hybrid. The incompatibility reactions were detected in the newly formed hybrid cells accompanied with significant changes in the nucleolus (segregation of nucleolar components) and plastids (cup-like shape or amoeboid plastid enclosing cytoplasmic materials) of C3 species parent. The structural changes in the organelles of the C4 partner were less marked. After 5 days of culture, most organelles showed high cellular activity, and a normal dedifferentiation process of mesophyll chloroplasts was observed. At this stage nucleolar segregation was not detected and the C3 species plastids were difficult to distinguish from the proplastids formed from mesophyll chloroplasts. In addition, some mitochondria showed bursting-like structure. However, under the culture condition used these somatic incompatibility did not seem to impair further growth of fusion products since they were still proliferating well resulting in callus formation.

Efficient immortalization of rheumatoid synovial tissue B-Iymphocytes. A comparison between the techniques of electric field-induced and PEG fusion

In this study, B-cells isolated from rheumatoid synovial tissue were immortalized, without prior in vitro stimulation, by means of electric-field induced fusion and conventional PEG fusion in order to compare the efficiency of these methods. Two myeloma cell lines were used as fusion partners, the murine myeloma Ag8 and the murine-human heteromyeloma HAB-1. The results of seven fusion experiments performed simultaneously with identical cell populations showed that fusion frequencies obtained by electrofusion were 4 to 35 times higher than by the PEG fusion technique. The morphological and immunohistochemical evaluation of synovial tissues used for fusion showed that only tissues exhibiting a follicular distribution of B-cells with a high percentage of CD 22-positive lymphocytes gave rise to high fusion yields and produced B-cell clones, whereas synovial tissues with the same percentage of plasma cells but lower percentages of CD 22 lymphocytes yielded very low fusion rates. In conclusion, electrofusion is more efficient for immortalizing small amounts of synovial tissue B-lymphocytes than PEG fusion, since high fusion frequencies could be obtained by this technique without the need for prior in vitro stimulation. Synovial tissue exhibiting a follicular distribution of B-lymphocytes with high percentages of CD 22-positive lymphocytes gave rise to high hybridoma yields and therefore an ideal source of human rheumatoid B-cell clones.

Intra- and Interspecific Electrofusion of Dunaliella Cells and Yeast Protoplasts

Summary For the first time intra- and interspecific electric field-induced fusion of the extremely salt tolerant unicellular green algae Dunaliella parva and Dunaliella bardawil , the extremely acid resistant Dunaliella acidopbila (optimal growth and photosynthesis of pH 1.0) and of yeast protoplasts to Dunaliella species is described. The scientific potential of such fusion products is discussed.

PEG-enhanced, electric field-induced fusion of tonoplast and plasmalemma of grape protoplasts

Cultured grape cells accumulate anthocyanins in vacuoles rather than secreting them into the nutrient medium. Therefore, grape cells that contain tonoplast segments in their plasmalemma should be capable of excreting anthocyanins rather than sequestering them in their vacuoles. In initial attempts to construct such ‘novel’ cells, small vacuoles were fused with the plasmalemma of cultured plant cells. Protoplasts were isolated from grape calluses that produce and accumulate anthocyanins. Small vacuoles were formed by gently rupturing vacuoles isolated from grape protoplasts. Although small vacuoles and protoplasts became aligned in an AC field, the tonoplast and plasmalemma did not readily fuse when subjected to 3 DC pulses of 1200 V cm−1 for 50 μs each. Changes in the intensity, number and/or duration of the DC pulses had no effect on the fusion process. When 1.0% polyethylene glycol was added to the electrofusion buffer, however, small vacuoles and protoplasts fused within a few minutes after the DC pulses were applied. These ‘novel’ grape cells remained viable for several hours.

Electric field-induced fusion of mitochondria

Fusion of mitochondria in H-medium from rat liver was induced by the application of square-wave voltage with electric field strengths of 1–2.5 kV/cm and duration 100 μs. Electron micrographs showed that the newly fused mitochondria could contain up to five mitoplasts. The fusion yield was close to 12% and respiratory activity was enhanced. The electric field lines did not go through the inner membrane, however, when the electric field strength was greater than 3 kV/cm they did so, resulting in total destruction of the mitochondria.

Human Monoclonal Antibodies to Human Cytomegalovirus

Human monoclonal antibodies (HMAbs) to human cytomegalovirus (HCMV) have been developed by using electric field-induced cell fusion of human B lymphocytes to the human-mouse cell line SBC-H20. By this procedure, multiple hybridomas have been produced that secrete IgG 1 HMAbs with distinct patterns of indirect immunofluorescence on HCMV-infected cells. HMAbs Z01 and X20 immunoprecipitated a major protein at 64 kDa. HMAb Z02 immunoprecipitated a major protein of 48-50 kDa. HMAb Z10 identified a single protein at 65 kDa and HMAb X16 identified proteins at 100, 65, and 36-38 kDa. The HMAbs demonstrated varying degrees of virus-neutralizing activity. The production of HMAbs to HCMV provides an important approach to studying the human host response to HCMV by elucidating biologically relevant antigens and epitopes. In addition, HMAbs are a potentially unlimited source of relevant human antibodies for treating life-threatening HCMV infection.

The Cell Cycle Governs the Onset of Spherulation of Xenopus Eggs Fused by an Electric Field: (amphibian egg/cell cycle/cortex/electic field-induced fusion).

The mechanism of electric field-induced fusion has been studied in detail (Zimmermann, Biochim. Biophys. Acta. 694 (1982) 227), but little is known about the process by which the two fused cells become a single entity, a process we term spherulation. We observe a clear difference between activated and unactivated Xenopus eggs in the time after electic field (EF) application when spherulation starts, and in the time required for spherulation to be completed. In unactivated eggs, spherulation started 7 min after EF application and was completed within 5 1/2 min. In activated eggs, the lag between EF application and the start of spherulation increased with the cell cycle. At the end of the first cell cycle spherulation started 78 min after EF application and was competed 30 min later. The lag period is not due to delayed fusion, for electric coupling between activated eggs can be recorded before the start of spherulation. The morphology of the contact zone between paired eggs, as observed by light and electron microscopy, is also described. We suggest that the difference in the timing of spherulation reflects a difference in the lability of the cytoskeleton through the cell cycle.

Loss of functional sperm entry into Xenopus eggs after activation correlates with a reduction in surface adhesivity

In Xenopus, the plasma membrane of the unactivated egg is receptive to sperm only in the animal hemisphere ( R. Grey, M. Bastiani, D. Webb, and E. Schertel, 1982, Dev. Biol. 89, 475–487 ). The reinsemination experiments of investment-free eggs reported in this paper demonstrate that functional sperm entry is lost after activation. Supernumerary sperm were excluded even though the fertilization envelope was absent and the membrane potential had returned to the level found in the unfertilized egg. Even when the electrical block to polyspermy was suppressed by 40 m M NaI (which reduces the membrane potential), polyspermy could be induced only if denuded eggs were initially inseminated in this medium. We estimate that the loss of functional sperm entry, independent of the electrical block, occurs during the first 10 min following fertilization. Sperm readily adhere to the surface of the animal hemisphere of unactivated eggs divested of their extracellular coats, but they do not adhere to the surface of activated eggs. Denuded eggs also adhere to each other, with the surface of the animal hemisphere of unactivated eggs exhibiting the greatest degree of adhesivity. We used electric field-induced fusion (EFIF), without prior dielectrophoresis, to quantify the regional and temporal adhesiveness of eggs. At electric field strengths greater than 8 V/cm, the probability of fusion during EFIF is highest with the animal hemisphere of unactivated eggs, moderate with both the vegetal hemisphere of unactivated eggs and the animal hemisphere of activated eggs, and lowest with the vegetal hemisphere of activated eggs. When pairs of eggs are constructed with different hemispheres in contact, the fusion characteristics of the pair are similar to the more adhesive member of the pair. The regional and temporal differences in the adhesiveness of the Xenopus egg surface correlate with its receptivity to sperm and could possibly account for the plasma membrane's activation-induced loss of functional sperm entry.

Protoplast fusion of Lactobacillus acidophilus and Streptococcus lactis via electric field or chemical induction.

Protoplast formation and regeneration procedures were developed for lactic acid bacteria. Initial attempts to fuse the protoplasts using an electric field were unsuccessful. No fusion was observed and the protoplasts lysed when the electric field voltage was increased to 24kV/cm. However, fusion products were obtained when the protoplasts were suspended in subfusion concentrations (<20%) of polyethylene glycol (PEG) and exposed to D. C. current. Also, the PEG protected protoplasts against lysis in current fields up to 72kV/cm. The frequency of hybrid cell formation (i.e. plasmid transfer) via the electric field-induced fusion was equivalent to the plasmid transfer frequency obtained via conjugation. Chemically induced fusion resulted in a transfer frequency of an order of magnitude greater than that of conjugation.

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.

Plant-animal cell fusions.

The successful fusion of plant protoplasts and animal cells depends on a variety of factors. A method using high pH, high Ca2+ concentration and raised temperature has advantages over the polyethylene glycol method first used to fuse plant and animal cells. Electric field-induced fusion will perhaps be superior to both methods. The readiness with which the two different cytoplasms fuse was found to influence the success of interkingdom fusions; the high pH/high Ca2+ method leads to more rapid and complete cytoplasmic fusion than the polyethylene glycol method. Immunofluorescence studies of the extent of plasma membrane interaction in plant-animal heterokaryons have been made, using antibodies to animal cell membrane components. The influence of the culture medium on the survival of plant-animal heterokaryons and on the dominance of one or other cell partner has also been investigated. Further studies should indicate whether both genotypes are functioning in such interkingdom heterokaryons.

Fusion of mitochondrial inner membranes by electric fields produces inside-out vesicles: Visualization by freeze-fracture electron microscopy

The fusion of vesicular-shaped mitochondrial inner membranes was observed by a new approach which combines freeze-fracture electron microscopy and electric field-induced fusion. Results show that membrane events caused by the exposure to the electric field can be time-coordinated with sample freezing for subsequent analysis by freeze-fracture electron microscopy.

Electric field-induced fusion of large liposomes from natural and polymerizable lipids

Electric field-induced fusion of large liposomes from natural and polymerizable lipids

Transfer of mitochondrial function into a cytoplasmic respiratory-deficient mutant of Saccharomyces yeast by electro-fusion

Electric field-induced fusion was induced between Saccharomyces cerevisiae protoplasts from the ρ (-) heterozygous diploid strain 2114 and the respiratory-competent diploid strain 3441, carrying chromosomal markers. Close membrane contact between the cells of the two different strains (ratio 1:1) was achieved by dielectrophoresis in a weak inhomogeneous alternating field (about 1 kV/cm, 2 MHz). Due to dielectrophoresis pearl chains of two or more cells of the two strains are formed between the electrodes. Cell fusion was induced by application of two single square field pulses sufficiently high to induce reversible electrical breakdown in the membrane contact zone between cells within a pearl chain (about 7 to 8 kV/cm field strength and 40 Ms duration). The two subsequent pulses were applied at an interval of about 10 s.Hybrids could be isolated on selection medium in a high yield (compared with conventional fusion techniques). The hybrids were diploid, respiratory-competent and produced prototrophic spores. Thus, the fused hybrids contained only the chromosomal markers of strain 2114 and the cytoplasmic marker for respiratory competence from strain 3441; electro-fusion thus resulted mainly in plasmogamy.

Electric field-induced fusion: electro-hydraulic procedure for production of heterokaryon cells in high yield

Electric field-induced fusion: electro-hydraulic procedure for production of heterokaryon cells in high yield

Giant culture cells by electric field-induced fusion

Giant culture cells by electric field-induced fusion

Electric Field-induced Fusion of Sea Urchin Eggs.

An electrical method is described which permits the fusion of denuded eggs of the sea urchin species Paracentrotus lividus. In a nearly non-conductive medium, containing 1.2 M glucose at pH 6.0-8.4, eggs or fertilized stages are brought into close membrane contact by dielectrophoresis arising from the application of a highly inhomogeneous alternating electric field. During this process the eggs aligne parallel to the field forming "egg-chains". In well pigmented eggs pigmentcapping is observed in the areas of cell contact. After completion of the alignment, the application of an additional single high field pulse of μs duration induces fusion of two or more eggs. The mechanism underlying the fusion process is the reversible electric breakdown of membranes in the zones of cell-to-cell contact. Fusion proceeds within 1-10 min at 10-20°C. Fused eggs have intact nuclei, can be fertilized, but undergo abortive cleavage.