Electrical fusion for optimal formation of protoplast heterokaryons in Nicotiana

The electrical fusion of protoplasts has been studied in order to maximize the formation of heterokaryons for culture. Heterokaryons of Nicotiana tabacum L. mesophyll protoplasts and N. plumbaginifolia Viviani supension-cell protoplasts were identified in fixed and stained as well as living material; a quantitative fusion index was thereby developed. With this index the efficiencies of various electric fields and fusion-chamber designs have been determined. Optimal fusion was obtained with an alternating-current (AC) field of 150 V/cm and direct-current (DC) square-wave pulses of 1000 V/cm. A new, simple-to-use, largescale fusion chamber is described in which batches of up to 5·10⁵ protoplasts (0.5 ml of cells at 10⁶/ml) can be fused in 5–7 min with efficiencies approaching 40%. Half of the fusion products are heterokaryons, thus fusion is random. Of the fusion products, 60% are bi- or trinucleate. Using fusion procedures similar to those described here Bates and C. Hasenkampf (1985, Theor. Appl. Genet., in press) have recovered viable somatic hybrids which have been regenerated.Abbreviations AC alternating current - DC direct current - PEG polyethylene glycol     

Hyperosmolality inhibits exocytosis in sea urchin eggs by formation of a granule-free zone and arrest of pore widening

Summary Hyperosmolality is known to inhibit membrane fusion during exocytosis. In this study cortical granule exocytosis in sea urchin eggs is used as a model system to determine at what step this inhibition occurs.Strongylocentrotus purpuratus eggs were incubated in hyperosmotic seawater (Na₂SO₄, sucrose or sodium HEPES used as osmoticants), the eggs activated with 20 m A23187 to trigger exocytosis, and then quick frozen or chemically fixed for electron microscopy. Thin sections and freeze-fracture replicas show that at high osmolality (2.31 osmol/kg), there is a decrease in cortical granule size, a 90% reduction in granule-plasma membrane fusion, and formation of a granulefree zone between the plasma membrane and cortical granules. This zone averages 0.64 m in thickness and prevents the majority of granules from docking at the plasma membrane. The remaining granules (10%) exhibit early stages of fusion which appear to have been stabilized; the matrix of these granules remains intact. We conclude that exocytosis is blocked by two separate mechanisms. First, the granule-free zone prevents granule-plasma membrane contact required for fusion. Second, in cases where fusion does occur, opening of the pocket and dispersal of the granule contents are slowed in hyperosmotic media.     

Fusion of enveloped viruses with cells and liposomes

The fusion of viruses with cells and liposomes is reviewed with focus on the analysis of the final extents and kinetics of fusion.Influenza virus andSendai virus exhibit 100% of fusion capacity with cells at pH 5 and pH 7.5, respectively. On the other hand, there may be in certain cases, a limit on the number of virions that can fuse with a single cell, that is significantly below the limit on binding. It still remains to be resolved whether this limit reflects a limited number of possible fusion sites, or a saturation limit on the amount of viral glycoproteins that can be incorporated in the cellular membrane, like the case of virus fusion with pure phospholipid vesicles, in which the fusion products were shown to consist of a single virus and several liposomes. Both viruses demonstrate incomplete fusion activity towards liposomes of a variety of compositions. In the case ofSendai virus, fusion inactive virions bind essentially irreversibly to liposomes. Yet, preliminary results revealed that such bound, unfused virions can be released by sucrose gradient centrifugation. The separated unfused virions subsequently fuse when incubated with a “fresh” batch of liposomes. We conclude, therefore, that the fraction of initially bound unfused virions does not consist of dective particles, but rather of particles bound to liposomes via “inactive” sites. Details of the low pH inactivation of fusion capacity ofinfluenza virus towards cells and liposomes are presented. This inactivation is caused by protonation and exposure of the hydrophobic segment of HA₂, and affects primarily the fusion rate constants. Some degree of inactivation also occurs when virions are bound to cellular membranes.     

Time-sequence of nuclear and chloroplast fusions in the zygote of Chlamydomonas reinhardii

Contradictory data have been published concerning the time-sequence of nuclear and chloroplast fusions in the zygote of Chlamydomonas. In the present study, adjacent ultrathin sections of Chlamydomonas reinhardii zygotes of various ages were examined with the electron microscope. These sections clearly reveal that nuclear fusion precedes chloroplast fusion.     

Fine structure of fusion products from soybean cell culture and pea leaf protoplasts

Protoplasts from pea (Pisum sativum L.) leaves and cultured soybean (Glycine max L.) cells were fused by means of polyethylene glycol and subsequently cultured for one week. Both agglutinated protoplasts and cultured fusion products were examined by electron microscopy. Agglutination occurred over large areas of the plasma membranes. The membrane contanct was discontinuous and irregularly spaced. Many cultured fusion products regenerated cell walls and divided to form cell clusters. Fusion of pea and soybean interphase nuclei occurred in some cells. The detection of heterochromatin typical of pea in the synkaryon, even after division, suggests the cells were hybrids. The cytoplasm of the cells from the fusion products contained both soybean leucoplasts and pea chloroplasts. The chloroplasts had apparently ceased dividing and some showed signs of degenerating. Large multinucleate fusion products developed cell walls but failed to divide.Abbreviations PEG polyethylene glycol - SEM scanning electron microscopy - TEM transmission electron microscopy Supported by National Research Council of Canada, Grant A6304     

Heterokaryon formation in the basidiomycete Schizophyllum commune by electrofusion of protoplasts

Summary Conditions for high frequency electrofusion of protoplasts from the basidiomycete Schizophyllum commune are described. Visual inspection revealed up to 30% of the protoplasts engaged in fusion. Using complementing nutritional mutations, nearly 7% of the regenerated protoplasts could be recovered as heterokaryotic mycelia. The method is probably equally applicable to other basidiomycetes such as Agaricus bisporus, permitting the recovery of fusion products in the absence of selection markers.     

Cytoskeleton,cell surface and the development of invasive plasmodial tapetum inTradescantia virginiana L.

Summary The anther tapetum inTradescantia virginiana L. is of the invasive plasmodial type: the cells lose their walls during early spore meiosis and develop long invasion processes which invade the loculus to penetrate spaces between the sporogenous cells. Fusion to form a syncytium is delayed and conventional ultra-thin sections and the Thiéry reaction reveal the presence of a loose fibrillar extracellular cell coat on the free surfaces of tapetal cells and their invasion processes. Cell fusion involves formation of apposition areas characterized by an absence of cell coat and the local appearance of microtubular arrays. Conspicuous membrane sacs, associated closely with microtubules, were found to migrate to and accumulate at the plasma membranes near the fusion sites and sporogenous cells. Microtubules are always present in the cortical regions of the tapetal cells and their invasion processes. It is surmised that microtubules are not responsible either for initiating or guiding tapetal invasion of the loculus; instead they may help to sustain the form of the invasion processes, help in the migration of membrane sacs, and participate in cell fusion. The cell coat disappears with syncytium formation towards the end of meiosis, and the developing spore cells become surrounded by a perispore membrane, which, derived from the original plasma membranes and augmented by membrane sacs, forms labyrinthine membrane reservoirs that are described further in the accompanying paper.     

Parasexual fusion products in green algae: Enteromorpha and Ulvaria (Ulvales,Chlorophyta)

Enteromorpha linza and Ulvaria oxysperma in North Carolina reproduce exclusively by asexual zoospores. Calcofluor white staining indicated that newly released zoospores lack significant cellulose cell wall material, making them suitable for treatment as protoplasts in a parasexual fusion process using high pH-Ca²⁺, PEG and centrifugation. Presumptive fusion products were identified by their larger size, twin chloroplasts and eyespots, and presence of fluorescence labelled and unlabelled portions. Parasexual fusion and karyogamy were confirmed by elevated levels of nuclear DNA in fusion cell germlings. In addition, aceto-orcein staining of fusion cell products revealed a diploid chromosome complement of 2N = 20 in Enteromorpha linza. Fusion cells were isolated by killing the more numerous adjacent unfused zoospores with 2-3 min exposure to blue light (410–490 nm). Unexposed fusion cells could be readily distinguished and recovered by micropipette at the 10-day stage.Center for Marine Science Research UNC-W Contribution No. 008.     

Improvement of the cell volume ofCandida blankii through protoplast fusion

Summary The yeastCandida blankii ESP-94, capable of utilizing xylose as substrate, was isolated for the production of single-cell protein (SCP) on bagasse hydrolysates. However, the small cell volume of strain ESP-94 would complicate harvesting of the cells during a continuous fermentation process. Auxotrophic mutants of strain ESP-94 were generated and intraspecific protoplast fusion experiments performed in an attempt to increase the cell volume of strain ESP-94. The fusion products were characterised with respect to cell volume, DNA content and genetic stability. Six genetically stable fusants with bigger cell volumes and higher DNA contents were obtained. One such fusant, fusant F17, had a cell volume 3-times that of strain ESP-94, while exhibiting similar growth rates to strain ESP-94 ond-xylose as carbon source.     

Membrane events involved in fusion of uterine epithelial cells in pseudopregnant rabbits

Summary During pseudopregnancy in the rabbit some uterine epithelial cells undergo conversion into symplasmata. This event serves as a model for studies of membrane apposition, fusion and fission of the lateral membranes with the use of different ultrastructural techniques. Apposition of lateral membranes occurs by means of proliferation of the tight-junctional belt and macular tight junctions. Membrane fusion is characterized in freeze-fracture replicas by continuously running fracture planes between neighboring membrane leaflets of epithelial cells, in general without reorganization of the particles. It is suggested that the reorganization of particles as well as the blebs or vesicles of smooth membranes, which are occasionally observed, may be artefacts. Membrane fission occurs simultaneously with fusion resulting in irregularly shaped membrane holes on freeze-fracture replicas. These events are rarely seen in thin sections. Staining with tannic acid reveals that only the layers of the plasma membrane are accessible to this agent. The fusion-fission process starts in the lower region of the lateral membranes, whereas the luminal portion with the broad tight-junctional belt remains intact.Dedicated to Professor Dr. med. Dr. phil. Karl-Heinrich Knese, Stuttgart-Hohenheim, in honour of his 70th birthday     

Intra-specific fusions in Solanum tuberosum

Summary Plants were regenerated from callus arising from protoplast fusion of two S. tuberosum diploids. Tetraploid progeny from the fusion of the two diploid partners had increased vigor. Isozyme analysis confirmed the presence of proteins from both partners in the fusion progeny. Pigmentation of tubers and anthers was heightened substantially in the fusion products. This fusion, the first intra-specific fusion within S. tuberosum, indicates that somatic fusion may be useful for transferring traits within this group.     

Induction of fast-growing and morphologically different strains through intergeneric protoplast fusions of Ulva and Enteromorpha (Ulvales,Chlorophyta)

Isolated protoplasts of Ulva pertusa and Enteromorpha prolifera were electrically fused. Treatment of protoplasts in 1% protease for 15–20 min prior to fusion enhanced fusion ability. Protoplasts from each fusion partner were mixed together in 1:1 ratio in low conductivity electrofusion solution at a density of 1 × 10⁵ cells ml⁻¹ before subjecting them to electrofusion. The protoplasts were aligned in AC field (1MHz, 25 V for 10–15 s) and subsequently fused by a high intensity single DC pulse of 250 V for 25 μs duration. Fusion buffer supplemented with 1 mM calcium and 1 mM magnesium yielded optimum fusion frequencies (about 18–24%). Entrapment of fusion treated cells inside agarose/agar plate facilitated marking and regeneration of fusion products. The regeneration patterns of fused protoplasts were similar to normal (unfused) protoplast development. Most of the regenerated plants from fusion products had a thallus similar to either U. pertusa type or E. prolifera type. Although some of the plants of the former were morphologically similar to U. pertusa, but most had a higher growth rate (1.9 to 1.5 times) than U. pertusa. Furthermore the thallus of some plants had a characteristic irregular and dentate margin, which was never observed in the parental type.     

Cellular dynamics of conjugation in the ciliate euplotes aediculatus. II. Cellular membranes

The formation and subsequent dissolution of a common bridge of cytoplasm between conjugating ciliated protozoan cells provides an excellent opportunity to follow the dynamics of the cellular membrane systems involved in this process. In particular, separation of conjugant partners offers the chance to observe, at a fixed site on the cell surface, how the ciliate surface complex of plasma and alveolar membranes (collectively termed the “pellicle”) is constructed. Consequently, cortical and cellular membranes of Euplotes aediculatus were studied by light and electron microscopy through the conjugation sequence. A conjugant fusion zone of shared cytoplasm elaborates between the partner cells within their respective oral fields (peristomes) to include microtubules, cytosol, and a concentrated endoplasmic reticulum (heavily stained by osmium impregnation techniques) that may also be continuous with cortical ER of each cell. Cortical membranes displacd by fusion are autolyzed in acid phosphatase-positive lysosomes in the fusion zone. As conjugants separate, expansion of the plasma membrane may occur through the fusion of vesicles with the plasma membrane, presumably at bare membrane, presumably at bare membrane patches near the fusion zone. The underlying cortical alveolar membranes and their plate-like contents are reconstructed beneath the plasma membrane, apparently by multiple fusions of dense-cored alveolar precursor vesicles (APVs). These precursor vesicles themselves appear to condense directly from the smooth ER present in the fusion zone. No Golgi apparatus was visible in the fusion zone cytoplasm, and no step of APV maturation that might involve the Golgi complex was noted.     

Intergeneric fusion of Zygnemataceae protoplasts

In intergeneric fusion fromMougeotia andZygnema protoplasts, the fate of fusion products, as well as nuclei and chloroplasts, could be classified according to the number of protoplasts involved from the two algae. Stable elongation growth occurred only in products of groups involving one protoplast from one alga and several protoplasts from the other alga. The features of the elongating products were those of the alga more numerously represented. The different nuclei combined by fusion failed to co-exist. In the groups involving one protoplast from one alga and several from the other, the nucleus from the former degenerated in an early period and only the nuclei from the latter were maintained. Also, the different chloroplasts combined did not co-exist. The genus of the chloroplasts maintained coincided with that of the nuclei maintained. The chloroplasts from the other genus degenerated gradually. An early morphological change in the degenerating chloroplasts was seen in the quantity of starch grains. Later, the chloroplasts generally became rounded, In degeneratingZygnema chloroplasts, thylakoid stacking was prominent. Without collapse of the thylakoid or accumulation of plastoglobules, the degenerating chlorplasts showed rupture of the chloroplast envelope.     

Membrane fusion: Lipid vesicles as a model system

In many cellular functions the process of membrane fusion is of vital importance. It occurs in a highly specific and strictly controlled fashion. Proteins are likely to play a key role in the induction and modulation of membrane fusion reactions. Aimed at providing insight into the molecular mechanisms of membrane fusion, numerous studies have been carried out on model membrane systems. For example, the divalent-cation induced aggregation and fusion of vesicles consisting of negatively charged phospholipids, such as phosphatidylserine (PS) or cardiolipin (CL), have been characterized in detail. It is important to note that these systems largely lack specificity and control. Therefore conclusions derived from their investigation can not be extrapolated directly to a seemingly comparable counterpart in biology. Yet, the study of model membrane systems does reveal the general requirements of lipid bilayer fusion. The most prominent barrier to molecular contact between two apposing bilayers appears to be due to the hydration of the polar groups of the lipid molecules. Thus, dehydration of the bilayer surface and fluctuations in lipid packing, allowing direct hydrophobic interactions, are critical to the induction of membrane fusion. These membrane alterations are likely to occur only locally, at the site of intermembrane contact. Current views on the way membrane proteins may induce fusion under physiological conditions also emphasize the notion of local surface dehydration and perturbation of lipid packing, possibly through penetration of apolar amino acid segments into the hydrophobic membrane interior.     

Incorporation of hygromycin resistance in Brassica nigra and its transfer to B. napus through asymmetric protoplast fusion

Summary With the idea to develop a selection system for asymmetric somatic hybrids between oilseed rape (Brassica napus) and black mustard (B. nigra), the marker gene hygromycin resistance was introduced in this last species by protoplast transformation with the disarmed Agrobacterium tumefaciens strain C58 pGV 3850 HPT. The B. nigra lines used for transformation had been previously selected for resistance to two important rape pathogens (Phoma lingam, Plasmodiophora brassicae). Asymmetric somatic hybrids were obtained through fusion of X-ray irradiated (mitotically inactivated) B. nigra protoplasts from transformed lines as donor with intact protoplasts of B. napus, using the hygromycin resistance as selection marker for fusion products. The somatic hybrids hitherto obtained expressed both hygromycin phosphotransferase and nopaline synthase genes. Previous experience with other plant species had demonstrated that besides the T-DNA, other genes of the donor genome can be co-transferred. In this way, the produced hybrids constitute a valuable material for studying the possibility to transfer agronomically relevant characters — in our case, diseases resistances — through asymmetric protoplast fusion.     

Veratridine triggers exocytosis in Paramecium cells by activating somatic Ca channels

Paramecium tetraurelia wild-type (7S) cells respond to 2.5 mm veratridine by immediate trichocyst exocytosis, provided [Ca²⁺] _o (extracellular Ca²⁺ concentration) is between about 10^–4 to 10^–3 m as in the culture medium. Exocytosis was analyzed by light scattering, light and electron microscopy following quenched-flow/ freeze-fracture analysis. Defined time-dependent stages occurred, i.e., from focal (10 nm) membrane fusion to resealing, all within 1 sec.Veratridine triggers exocytosis also with deciliated 7S cells and with pawn mutants (without functional ciliary Ca channels). Both chelation of Ca²⁺ _o or increasing [Ca²⁺] _o to 10^–2 m inhibit exocytotic membrane fusion. Veratridine does not release Ca²⁺ from isolated storage compartments and it is inefficient when microinjected. Substitution of Na⁺ _o for N-methylglucamine does not inhibit the trigger effect of veratridine which also cannot be mimicked by aconitine or batrachotoxin. We conclude that, in Paramecium cells, veratridine activates Ca channels (sensitive to high [Ca²⁺] _o ) in the somatic, i.e., nonciliary cell membrane and that a Ca²⁺ influx triggers exocytotic membrane fusion. The type of Ca channels involved remains to be established.We thank Dr. C. Kung (Madison, WI) for providing the pawn mutant, Drs. G. Lehle and R. Waldschütz-Schüppel (Konstanz, Germany) for their help with light scattering experiments, and Ms. E. Dassler and D. Bliestle for continuous help during the extensive photographic documentation. This work has been supported by Deutsche Forschungsgemeinschaft, Schwerpunkt Neue mikroskopische Techniken für Biologie und Medizin (grant P178/11) and SFB156/B4.     

The lid forming apparatus in cysts of the green algaAcetabularia mediterranea

Summary Cross sections and cross tangential sections of 1 to 3-day-old cysts (gametangia) ofAcetabularia mediterranea were examined by electron microscopy. In a defined zone of the peripheral cytoplasm of the cysts, where the lid is to be formed, a characteristic circular band-like structure, the putative lid forming apparatus, can be identified. In 1 -day-old cysts this structure is characterized by two electron dense amorphous layers close and parallel to the plasma membrane. In 3-day-old cysts the lower layer consists of rod-like structures. The position of the circular band-like lid forming apparatus is correlated to the position of the cyst organizing secondary nucleus which occupies a non central position. Usually the center of the lid forming apparatus lies on the shortest line between the secondary nucleus and the cyst wall. This suggests that the cyst organizing secondary nucleus plays an important role in the formation of the cyst lid.     

Fertility and chromosome stability in Brassica napus resynthesised by protoplast fusion

Summary Fertile somatic hybrids between Brassica campestris and B. oleracea have been produced by protoplast fusion. Fusion products were identified by their intermediate protoplast morphology. Heterokaryons were isolated either with micropipettes using a micromanipulator or by flow sorting. About 2% of the obtained calli differentiated to shoots. Of the shoots obtained from manually selected heterokaryons, 100% were true hybrids as confirmed by isozyme analysis while 87% of the flow sorted ones showed a hybrid pattern. Ploidy level of the hybrid plants was determined by chromosome counting and relative DNA-content analysis. The sum of the chromosome number (38) from the two fusion partners were found in 30% of the hybrids; 9% had fewer and 61% had more chromosomes. Pollen viability and seed set varied with ploidy level. Compared to natural B. napus, a pollen viability of 52%–93% and a fertility of 1%–40% was found for the somatic hybrids with normal chromosome number. Restriction enzyme analysis of chloroplast-DNA showed that either B. campestris or B. oleracea chloroplasts were present in the somatic hybrid plants. Of 11 hybrid plants 5 had the campestris and 6 had the oleracea type (11 ratio).     

Dynamic changes in plasma membrane properties of semliki forest virus infected cells related to cell fusion

The mechanism of the processes leading to membrane fusion is as yet unknown. In this report we demonstrate that changes in membrane potential and potassium fluxes correlate with Semliki Forest virus induced cell-cell fusion at mildly acidic pH. The changes observed occur only at pH's below 6.2 corresponding to values required to trigger the fusion process. A possible role of these alterations of the plasma membrane related to membrane fusion phenomena is discussed.