Electrically Stimulated Fusion of Different Plant Cell Protoplasts : MESOPHYLL CELL AND GUARD CELL PROTOPLASTS OF VICIA FABA

Cell fusion is induced between guard cell and mesophyll cell protoplasts of Vicia faba by electrical field application. The process of fusion is initiated by electrical breakdown of the cell membrane. Prior to the application of an external electrical field pulse which brings about reversible breakdown of the membrane, the cells (suspended in a low-conducting medium) are brought into close contact with one another by exposing them to an external alternating, nonuniform field (5 volts, electrode distance, 200 micrometers; 500 kiloHertz). During this process, they form “pearl chains” which may become sufficiently long to form bridges between the electrodes. The process is reversible as long as this voltage is not exceeded. Cell fusion is initiated as a result of an electrical field pulse of 50 microseconds duration and of sufficiently high intensity to induce reversible electrical breakdown of the membranes. The process of fusion is completed within 40 minutes or less in the case of guard cell protoplasts, as well as in the case of fusion between guard cell and mesophyll cell protoplasts. The fused cells are spherical in shape, if the fusion product consists only of two or three cells.     

The hydraulic conductivity as a criterion for the membrane integrity of protoplasts fused by an electric field pulse

The hydraulic conductivity of the membrane, Lp, of fused plant protoplasts was measured and compared to that for unfused cells, in order to identify possible changes in membrane properties resulting from the fusion process. Fusion was achieved by an electric field pulse which induced breakdown in the membranes of protoplasts in close contact. Close membrane contact was established by dielectrophoresis. In some experiments pronase was added during field application; pronase stabilizes protoplasts against high field pulses and long exposure times to the field. The Lp-values were obtained from the shrinking and swelling kinetics in response to osmotic stress. The Lp-values of fused mesophyll cell protoplasts of Avena sativa L. and of mesophyll and guard cell protoplasts of Vicia faba L. were found to be 1.9±0.9·10^-6, 3.2±2.2·10^-6, and 0.8±0.7·10^-6 cm·bar^-1·s^-1, respectively. Within the limits of error, no changes in the Lp-values of fused protoplasts could be detected in comparison to unfused protoplasts. The Lp-values are in the range of those reported for walled cells of higher plants, as revealed by the pressure probe.Abbreviations GCP guard cell protoplast - Lp hydraulic conductivity - MCP mesophyll cell protoplast     

High frequency fusion of plant protoplasts by electric fields

Mesophyll cell protoplasts of Vicia faba were collected by dielectrophoresis in a highly inhomogeneous alternating electric field (sine wave, 5 to 10 V peak-to-peak value, 500 kHz, electrode distance 200 m). Under these conditions, the cells formed aggregates of two or three on the electrodes or bridges consisting of 4 to 6 protoplasts between the electrodes. This pearl chain arrangement of the cells was only stable for the duration of the applied field. By the additional application of a high single field pulse (square wave, 15 V, 50 s), it was possible to induce cell fusion within the aggregates or bridges. This electrically stimulated fusion of cells proceeded at room temperature and under physiological pH-conditions, without the use of chemical reagents, and gave a high yield. Smaller fused aggregates formed spheres within a few minutes. During the dielectrophoretically induced adhesion of the protoplasts to one another, the field strength must be chosen such that dielectric breakdown of the membrane is avoided, but at the same time, the strength of the subsequently applied single field pulse must be high enough to induce dielectric breakdown at the sites of contact between the protoplast membranes. From these results, one can conclude that in addition to close contact between membranes, the prerequisite for electrically stimulated cell fusion is dielectric breakdown which leads to changes in the membrane conductance, permeability, and probably fluidity.Presented at II Congress FESPP, Santiago de Compostela, Spain, 27.7.–1.8.1980, and Gordon Research Conference of Bioelectrochemistry, Tilton, New Hampshire, USA, 4.8.–8.8.1980     

Electric field-induced fusion of isolated vacuoles and protoplasts of different developmental and metabolic provenience

Using an electric field pulse technique, we induced fusion between vacuoles and protoplasts of Kalanchoë daigremontiana , between protoplasts from etiolated and green leaf mesophyll, and between mesophyll protoplasts from plants of different physiological properties ( Avena sativa : C₃ mechanism of photosynthesis, Kalanchoë daigremontiana : crassulacean acid metabolism). Close membrane contact amongst protoplasts or between protoplasts and vacuoles (as required for fusion) was achieved by the application of an alternating, non-uniform electric field to the suspension. Due to the dielectrophoresis effect the cells attach to each other along the field lines. The fusion process is initiated by the injection of an electric field pulse of high intensity and short duration (μs range). The field intensity has to be sufficiently high to induce reversible breakdown in the area of close membrane contact. After the application of the field pulse, the fusion process is initiated and completed within seconds to a few minutes, depending on the material investigated.
Fusion occurs between protoplasts and vacuoles as well as between protoplasts of different species. Both tonoplast and plasma membranes completely intermingled, indicating that in contrast to suggestions in the literature these membranes are compatible. Furthermore the cytoplasms of etiolated and green protoplasts obviously do not mix after fusion is completed, as etioplasts and chloroplasts kept separated from each other. In all experiments the volume of the fusion product equalled the sum of the compartments that underwent fusion. The wide spectrum of possible applications resulting from these fusion experiments in relation to metabolic problems is discussed.     

Electrorotation of Oat Protoplasts before and after Fusion

This paper describes an experimental chamber suitable for inducingcell fusion by an electric field and for measuring the rotationalbehaviour of single protoplasts and fusion products in high-frequencyrotating fields. Intact protoplasts from Avena sativa L. leavesrotate before, during and after fusion, as demonstrated by therotation spectra of cells. The electrorotation technique allowselectrical properties of fused cells to be examined within asecond after applying the fusion pulse. (Received June 23, 1986; Accepted June 19, 1987)     

Electro-fusion of haploid Saccharomyces yeast cells of identical mating type

Yeast protoplasts from the haploid strains 21 a and 111a were exposed to an inhomogeneous alternating field (about 1 kV/cm, 2 MHz). Due to dielectrophoretic aggregation two or more cells with close membrane contact are formed between the electrodes. Cell fusion was observed by application of two field pulses (11 kV/cm, 7 s duration) applied at an interval of 1 s. The intensity of the field pulses is sufficiently high to induce reversible electrical breakdown at membrane sites oriented in the field direction. After a 8 to 14 days incubation period on selection medium two types of fusion products could be isolated: 1) Hybrids with a haploid constitution, respiratory-competent and auxotrophic for histidine. 2) Cells with a diploid cell size and prototrophic for histidine. The genetic analysis for mating types and auxotrophic markers show that in the second case plasmogamy followed by karyogamy had occurred.     

Ultrastructural and electrophysiological correlates of cell coupling and cytoplasmic fusion during myogenesis in vitro

An electrophysiological monitoring strategy involving iontophoretic application of acetylcholine and intracellular recording has been employed in an investigation of the time course of cell fusion during myogenesis in vitro. Pairs of closely apposed, acetylcholine-sensitive rat or chick myogenic cells were continuously monitored. The onset of high-efficiency electrical coupling between members of such pairs corresponded to the moment at which cytoplasmic continuity was established. Ultrastructural analysis of the serial section record of two rat myotubes, fixed 2–3 min after fusion began, demonstrated the rapid disappearance of surface membranes in the fusion area at an average rate > 1 μm² of membrane per second. Ten pairs of acetylcholine-sensitive cells were also observed to form lower-efficiency electrical connections. Such cells were not fused but were associated via specialized close membrane appositions resembling gap junctions. These membrane appositions apparently persist through the early events of cell fusion, for their remnants were found in recently fused cells. Possible roles of electrical coupling and of these close junctions in the fusion process are considered.     

In vitro double fertilization in Nicotiana tabacum (L.): polygamy compared with selected single pair somatic protoplast and chloroplast fusions

In vitro polygamy was studied mainly by using isolated sperm and central cells of tobacco in order to elucidate the mechanism that might be involved in preventing in vivo polygamy. In 17.5% 4000 M.W. polyethylene glycol, only when two sperm cells were made close enough to each other and adhered to a female cell simultaneously was polygamy possible. If one sperm cell fused with the egg or central cell, within 30 min another sperm cell could not fuse with the same egg or central cell. Similar phenomena were found in selected single somatic cell fusion. When more than two protoplasts adhered to each other simultaneously, fusion was always successful; after two protoplasts fused, within 30 min the fusion products could not fuse with another protoplast under the same conditions. This comparative study revealed this characteristic to be shared by both sexual and somatic cell fusion. However, after cytoplasm reorganization was complete in the fusion product, it was possible for the fusion product to fuse with the third protoplast. This indicates that the obstruction to additional fusion was present only during a certain period after the preceding fusion under certain condition. The possible reason for the effect is discussed. Received: 7 March 2000 / Revision accepted: 15 June 2000     

Vesicle formation during electro-fusion of mesophyll protoplasts of Kalanchoë daigremontiana

Following electro-fusion of plant protoplasts the volume of the fused cell is the sum of the volumes of the parent cells. As shown for mesophyll protoplasts from leaves of Kalanchoë daigremontiana, the excess in membrane material arising from the reduction in membrane area is removed-at least to a larger extent — by the formation of vesicles which are visible in the light microscope. These vesicles, which may have been formed by the fusion of sub-microscopic vesicles, are observed in the contact zone of the fusing cells. The mechanism of the formation of vesicles during electro-fusion is discussed.     

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.     

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.     

Electric pulse-induced fusion of mouse lymphoma cells: Roles of divalent cations and membrane lipid domains

Summary Mouse leukemic lymphoblasts (L5178Y) brought into close contact by dielectrophoresis underwent cell fusion following the application of electrical pulses in the presence of electrolytes. The electrically fused cells became spherical after switching off the dielectrophoretic field. Fusion between a cell vitally stained with Janus Green and that with Neutral Red resulted in the homokaryon with a mixed color. Intracellular potentials simultaneously recorded from the two cells located on both sides of the homokaryon were identical. The fusion efficiency was remarkably dependent upon temperature, displaying a discontinuity at about 11°C in the Arrhenius plot. The extracellular application of phospholipase-A₂ or-C suppressed the fusion yield. Thus, it appears that the phospholipid domains play a crucial role in the electric pulse-induced cell fusion. Treatment of the cells with proteolytic enzymes markedly enhanced the fusion yield, presumably due to removing the glycocalix and/or giving rise to fusion-potent, protein-free lipid domains. The presence of millimolar concentrations of divalent cations (irrespective of Mg²⁺ or Ca²⁺) as well as of micromolar concentrations of Ca²⁺ (but not Mg²⁺) was prerequisite to the resealing of membranes suffered from electrical breakdown upon exposure to electric pulses. In addition, extracellular Ca²⁺ (but not Mg²⁺) ions at more than micromolar concentrations were indispensable for the cell fusion.     

Dielectrophoretic forces and potentials induced on pairs of cells in an electric field.

A combined numerical/experimental study is reported of the membrane potentials and dielectrophoretically induced forces between cells, membrane pressures, and velocity of attraction of cells under the influence of an electric field. This study was designed to explore electrical and mechanical effects produced by a field on cells in close proximity or undergoing electrically induced fusion. Laplace's equation for pairs of membrane-covered spheres in close proximity was solved numerically by the boundary element method, and the electrically induced forces on the cells and between cells were obtained by evaluating the Maxwell stress tensor. The velocity of approach of erythrocyte ghosts or fused ghosts in a 60-Hz field of 6 V/mm was measured experimentally, and the data were interpreted by using Batchelor's theory for hydrodynamic interaction of hard spheres. The numerical results show clearly the origin of the dielectrophoretic pressures and forces in fused and unfused cells and the effects of a nearby cell on the induced membrane potentials. The experimental results agree well with predictions based on the simple electrical model of the cell. The analysis shows the strong effect of hydrodynamic interactions between the cells in determining their velocity of approach.     

Modification of cell membranes with viral envelopes during fusion of cells with HVJ (Sendai virus) : I. Interaction between cell membranes and virus in the early stage

A large number of viral materials are associated with the surface of cells after cell fusion with HVJ at 37 °C for 30 min. This is due to fusion of viral envelopes with the cell membrane. Studies were made on the process from viral adsorption to cell-cell, or cell-viral envelope fusion. On incubation at low temperatures, such as 0–15 °C, no envelope fusion or cell fusion was observed, although there was some interaction between the virus and cells. This interaction resulted in loss of hemadsorption (HA) activity of the cells and partial damage of the ion barrier of the cell membrane. The viral particles seem to come close to the lipid layer of the cell membrane at the low temperatures and to distort the non-flexible membrane structure. On incubation of the cell-virus complex at 37 °C, the cells rapidly became HA-positive and the HA activity was maximal within 5 min. At this stage there was much leakage of ions through the cell membrane. On further incubation the damage to the ion barrier of the cell membrane was repaired completely with completion of cell fusion. This process may be correlated with fusion of viral envelopes with cell membranes and restoration of the cell membrane fused with them.     

Application of electric field fusion in plant tissue culture

Manipulation of protoplasts via fusion and organelle transfer is expected to be facilitated by the technique known as electric field fusion. Construction and use are described of three flow-through fusion chambers that incorporate flat-sided electrodes in a manner that makes fusion of protoplasts possible througout the chambers' total volume (4, 49 or 110 μl) under constant electrical, chemical and physical conditions. Brassica napus L. protoplasts subjected to fusogenic conditions, that is, application of voltages that induce reversible membrane breakdown, were capable not only of survival but also of cell wall resynthesis, cell division and subsequent growth and development. Intraspecific ( B. napus × B. napus ), interspecific. ( B. napus × B. campestris L.) and intergeneric ( B. napus × Primula acaulis L.) fusion and engulfment events were followed by using on the one hand autofluorescence and fluorescein isothiocynate as respective markers or on the other hand autofluorescence and vacuolar anthocyanin ( Primula ). Properties and merits of flat-sided versus cylindrical electrodes are discussed.     

Modification of cell membranes with viral envelopes during fusion of cells with HVJ (Sendai virus).

A large number of viral materials are associated with the surface of cells after cell fusion with HVJ at 37 °C for 30 min. This is due to fusion of viral envelopes with the cell membrane. Studies were made on the process from viral adsorption to cell-cell, or cell-viral envelope fusion. On incubation at low temperatures, such as 0–15 °C, no envelope fusion or cell fusion was observed, although there was some interaction between the virus and cells. This interaction resulted in loss of hemadsorption (HA) activity of the cells and partial damage of the ion barrier of the cell membrane. The viral particles seem to come close to the lipid layer of the cell membrane at the low temperatures and to distort the non-flexible membrane structure. On incubation of the cell-virus complex at 37 °C, the cells rapidly became HA-positive and the HA activity was maximal within 5 min. At this stage there was much leakage of ions through the cell membrane. On further incubation the damage to the ion barrier of the cell membrane was repaired completely with completion of cell fusion. This process may be correlated with fusion of viral envelopes with cell membranes and restoration of the cell membrane fused with them.     

Behavior and viability of tobacco protoplasts in response to electrofusion parameters

This investigation examines responses of protoplasts in a systematic and quantitative way to the various electrical treatments used to achieve electrofusion and their individual and cumulative effect on protoplast viability. Mesophyll and cell suspension protoplasts from two species of the same genera, Nicotiana tabacum and N. rustica var brasilia were used in these experiments. Optimal frequencies for alignment of tobacco protoplasts were between 500 kilohertz and 2 megahertz at 100 volts per centimeter. Variations in frequency and voltage of the alternating current (AC) field caused predictable movements of protoplasts within an electrofusion chamber. AC frequencies below 10 hertz or above 5 megahertz significantly decreased the viability of protoplasts in the fusion chamber as estimated by fluorescein diacetate staining 1 hour after treatment. Although the direct current (DC) pulse appeared to have a slight detrimental effect on protoplast viability, this effect was not significantly different from untreated control preparations.

Protoplasts from both leaf mesophyll cells and suspension cells were induced to fuse with one or more 10 to 30 microseconds DC square wave pulses of approximately 1 kilovolt per centimeter after the protoplasts had been closely appressed with an AC field.

     

Electron microscopic study of plant-animal cell fusion

Summary Avian erythrocytes and protoplasts isolated from mesophyll cells of tobacco plants were suspended in 1% protease, agglutinated with polyethylene glycol (PEG) and subsequently fused upon elution of the PEG. The fusion reaction was monitored by scanning (SEM) and transmission (TEM) electron microscopy. SEM studies showed a marked difference in the topography of agglutinated cells. During, and subsequent to fusion, the markedly different surfaces of the two cell types became homogeneous and lines of demarcation between the cells were no longer visible. TEM revealed that adhesion occurred over the entire membrane area between agglutinated cells. Incipient fusion was evidenced by the appearance of vacuoles at the intermembrane surfaces. During initial elution of the PEG, cytoplasmic channels between erythrocytes and protoplasts were evident. With continued elution of the PEG, starch-containing plant chloroplasts and starch grains were seen within erythrocytes and homogenous erythrocyte cytoplasm was present inside plant protoplasts. Cytoplasmic mixing between the two cell types occurred within 3 hours of elution. The frequency of interkingdom fusion was estimated to be 0.5–1%.     

Electrical fusion of protoplasts from sycamore mutants and hybrid selection on hypoxanthine-aminopterin-thymidine (HAT) medium

Summary An electrical fusion method has been used to form somatic hybrids between protoplasts of two mutant cell lines of sycamore tissue culture cells. Both mutants will not grow in a hypoxanthine-aminopterin-thymidine (HAT) medium. It was possible to select the fused hybrids from homospecific fusion products and nonfused protoplasts by the use of HAT medium. In this way the viability and regeneration of the fused cells during the first few weeks of culture could be evaluated. An electron microscopic examination of the fusion process showed that it occurred at a series of points along the surface of the plasmalemma. Cytoplasmic bridges between the two cells were formed separated by vesicles which later dispersed to give complete cytoplasmic continuity between the cells.     

Electro-acoustic fusion of erythrocytes and of myeloma cells

Mammalian cells can be concentrated in a sound field. A method is introduced, which combines the reversible aggregation of cells in a sound field with the electrical breakdown of cell membranes to fuse cells, which are in contact. Human red blood cells and mouse myeloma cells are fused by means of that procedure.