Early events in calcium-induced liposome fusion

Calcium-induced interaction of liposomes composed of pure phosphatidylserine (PS) has been studied using a rapid-mixing, rapid-freeze device. Freeze-fracture electron microscopy of this material revealed that liposomes react very rapidly after addition of calcium ions. After only 10 ms (the resolution of the technique) vesicle fusion was apparent. At the same time, however, vesicles also collapsed, and appeared as aggregates of flattened membranes. This may explain controversies which have arisen over vesicle fusion studied with more indirect methods.     

Importance of mammalian sperm metalloendoprotease activity during the acrosome reaction to subsequent sperm-egg fusion: inhibitor studies with human sperm and zona-free hamster eggs.

We have previously shown that each of the metalloendoprotease (MEP) inhibitors phosphoramidon, diethylenetriaminepentaacetic acid, and carbobenzoxy-L-phenylalanine, when present only during the human sperm acrosome reaction (AR), will not inhibit the AR or sperm motility but will decrease the number of sperm that penetrate zona-free hamster eggs. The present study was designed to investigate whether this inhibition of penetration is due to an effect on sperm binding to the egg plasma membrane and/or to an effect on the actual membrane fusion event. In these studies we used ionomycin to initiate the AR and assayed binding in a Ca(2+)-free medium and fusion in Ca(2+)-containing medium in the same experiment. Eggs were loaded with the fluorescent dye Hoechst 33342, and the appearance of fluorescence in a sperm head indicated that fusion had occurred. The three MEP inhibitors reduced binding only slightly but inhibited the actual fusion step by 50-60% (determined with an equation that corrected for any inhibition of fusion due to inhibition of binding). MEP inhibitors present only during gamete interactions had little or no effect on fusion. We also found that phosphoramidon-inhibitable MEP activity was released during the ionomycin-initiated AR. Incubation of AR supernatant containing MEP activity with previously acrosome-reacted, phosphoramidon-treated sperm resulted in a large reversal of the phosphoramidon-inhibitory effect on sperm-egg fusion. These results support the hypothesis that the acrosomal phosphoramidon-inhibitable MEP released during the AR acts directly or indirectly during that event to increase the fusibility of the sperm plasma membrane region required for subsequent sperm-egg fusion.     

Expression and quantification of firefly luciferase under control of Rhizobium meliloti symbiotic promoters

We have tested the use of firefly luciferase for monitoring regulated symbiotic nitrogen fixation gene expression. Broad-host-range plasmids carrying translational fusions of Rhizobium meliloti nifH, fixA and nifA promoters were constructed. Despite low levels of promoter activity the absence of Escherichia coli endogenous luminescence and the high sensitivity of the bioluminescent assay for firefly luciferase allowed rapid screening for functional luciferase expression. Plasmids containing symbiotic promoter-luc fusions were established in R. meliloti. Luciferase activity was detected and measured in both vegetative and symbiotic cells giving comparable results with those obtained by beta-galactosidase assays. In addition, the luciferase assay was quicker, more sensitive and could be carried out with unrestricted cells. Furthermore, bioluminescence was high enough in alfalfa nodules containing nifH—luc fusion to be observed by a dark-adapted eye and photographed.     

Macromolecules Involved in the Amoeba-Bacteria Symbiosis1

ABSTRACT. In the Amoeba-bacteria symbiosis, rod-shaped Gram-negative bacterial endosymbionts reside within symbiosomes in the host cytoplasm, and the host and symbionts are mutually dependent for survival. Three proteins and one group of lipopolysaccharides (LPS) synthesized by the bacterial endosymbionts and two proteins derived from the host cells have been found to be involved in the host-symbiont interactions, although their respective roles are not yet fully known. The symbiont-derived molecules included proteins with molecular weights of 29 kDa, 67 kDa and 96 kDa and LPS. The 29-kDa protein was most abundant in the host cytoplasm, while the 96-kDa protein and LPS were found mostly on the symbiosome membranes. The 67-kDa protein was a GroEL analog and stayed within the symbionts. The host-derived 43-kDa protein, actin, was selectively accumulated by the symbionts, while the 220/225-kDa protein, spectrin, was attached to the symbiosome membranes. The symbiont genes coding for the 29-kDa and 67-kDa proteins were cloned and sequenced. The 29-kDa protein gene was unique with no relation to any known DNA sequences but has a leucine zipper-like motif, suggesting a possible DNA-binding function. The DNA sequence of the 67-kDa protein gene showed a 70% identity with heat-shock-protein genes of Escherichia coli and Coxiella burnetii.     

Expression of aChlamydia anticarbohydrate single-chain antibody as a maltose binding fusion protein

A single-chain antibody fragment has been constructed for an antibody that binds to theChlamydia specific carbohydrate structure of the lipopolysaccharide. Single-chain protein was expressed and secreted into the periplasmic space ofE. coli as a fusion protein with the maltose binding protein. The fusion protein was purified in one step by virtue of its ability to bind to maltose. In a sandwich ELISA, the eluted protein boundChlamydia lipopolysaccharide, which demonstrates that the single-chain protein domain will function as part of a fusion protein. The expression of maltose binding fusion proteins into the periplasmic space could be used for production of other single-chain antibodies or protein fragments requiring appropriate folding and disulfide bond formation.     

Further characterization of the aggregation and fusion of chromaffin granules by synexin as a model for compound exocytosis

Synexin was isolated from bovine liver and found to aggregate adrenal chromaffin granules in the same Ca2+-dependent manner as previously described for adrenal synexin. The chromaffin granule aggregating activity of liver synexin was blocked in vitro by the addition of an antibody prepared to the 47,000 molecular weight band extracted from an SDS gel of an adrenal medullary synexin preparation. Chromaffin granules aggregated by synexin fused when exposed to cis-unsaturated fatty acids at concentrations comparable to those released from phospholipids by stimulated secretory cells. The synexin-induced aggregation reaction was blocked by Erythrosin B, a common food coloring, and by the phenothiazine antipsychotic trifluoperazine and promethazine. The aggregation and fusion of chromaffin granules thus appears to be a useful model system for studying synexin from diverse tissues and for testing pharmacologically or toxicologically active substances for effects on secretory systems.     

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.     

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