Tension-voltage relationship in membrane fusion and its implication in exocytosis

AbstractIn this study, new methods are used to control cellular membrane tension to evaluate the role it plays in electrofusion. The data show that membrane tension present during the application of an electric field facilitates electro-induced membrane fusion. No enhancement was detected if the strain was applied after the pulse. Analysis of the electromechanical process of fusion revealed a synergy between the two kinds of constraints in the membrane fusion. Both mechanical and electrical constraints apparently play a key role in membrane fusion between the granule membrane and the plasma membrane, i.e. the exocytosis process

Recommendations guidelines on the key information to be reported in studies of application of PEF technology in food and biotechnological processes

The application of pulsed electric field (PEF) technology as a non-thermal cell membrane permeabilization treatment, was widely demonstrated widely to be effective in microbial inactivation studies, as well as to increase the rates of heat and mass transfer phenomena in food and biotechnological processes (drying, osmotic treatment, freezing, extraction, and diffusion). Nevertheless, most published papers on the topic do not provide enough information for other researchers to assess results properly. A general rule/guidance in reporting experimental data and most of all exposure conditions, would be to report details to the extent that other researchers will be able to repeat, judge and evaluate experiments and data obtained. This is what is described in the present recommendation paper. Industrial relevance: Pulsed electric field (PEF) treatment is a promising technology that has received considerable attention in food and biotechnology related applications food and biotechnology related applications of PEF include: i) “cold” pasteurization of liquid foods and disinfection of wastewater by microbial inactivation ii) PEF-assisted processing (drying, extraction or expression

Spontaneous lipid vesicle fusion with electropermeabilized cells

AbstractFusion is obtained between electropermeabilized mammalian cells and intact large unilamellar lipid vesicles. This is monitored by a fluorescence assay. Prepulse contact is obtained by Ca2+ when negatively charged lipids are present in the liposomes. The mixing of the liposome content in the cell cytoplasm is observed under conditions preserving cell viability. Electric conditions are such that free liposomes are not affected by the external field. Therefore destabilization of only one of the two membranes of the partners is sufficient for fusion. The comparison between the efficiency of dye delivery for different liposome preparations (multilamellar vesicles, large unilamellar vesicles, small unilamellar vesicles) is indicative that more metastable liposomes are more fusable with electropulsated cells. This observation is discussed within the framework of the recent hypothesis that occurrence of a contact induced electrostatic destabilization of the plasma membrane is a key step in the exocytosis process

Control by Osmotic Pressure of Voltage-Induced Permeabilization and Gene Transfer in Mammalian Cells

AbstractCells can be transiently permeabilized by a membrane potential difference increase induced by the application of high electric pulses. This was shown to be under the control of the pulsing buffer osmotic pressure, when short pulses were applied. In this paper, the effects of buffer osmotic pressure during electric treatment and during the following 10min were investigated in Chinese hamster ovary cells subjected to long (ms) square wave pulses, a condition needed to mediate gene transfer. No effect on cell permeabilization for a small molecule such as propidium iodide was observed. The use of a hypoosmolar buffer during pulsation allows more efficient loading of cells with β-galactosidase, a tetrameric protein, but no effect of the postpulse buffer osmolarity was observed. The resulting expression of plasmid coding for β-galactosidase was strongly controlled by buffer osmolarity during as well as after the pulse. The results, tentatively explained in terms of the effect of osmotic pressure on cell swelling, membrane organization, and interaction between molecules and membrane, support the existence of key steps in plasmid-membrane interaction in the mechanism of cell electrically mediated gene transfer

Electroporator with automatic change of electric field direction improves gene electrotransfer in-vitro

<p>Abstract</p> <p>Background</p> <p>Gene electrotransfer is a non-viral method used to transfer genes into living cells by means of high-voltage electric pulses. An exposure of a cell to an adequate amplitude and duration of electric pulses leads to a temporary increase of cell membrane permeability. This phenomenon, termed electroporation or electropermeabilization, allows various otherwise non-permeant molecules, including DNA, to cross the membrane and enter the cell. The aim of our research was to develop and test a new system and protocol that would improve gene electrotransfer by automatic change of electric field direction between electrical pulses.</p> <p>Methods</p> <p>For this aim we used electroporator (EP-GMS 7.1) and developed new electrodes. We used finite-elements method to calculate and evaluate the electric field homogeneity between these new electrodes. Quick practical test was performed on confluent cell culture, to confirm and demonstrate electric field distribution. Then we experimentally evaluated the effectiveness of the new system and protocols on CHO cells. Gene transfection and cell survival were evaluated for different electric field protocols.</p> <p>Results</p> <p>The results of <it>in-vitro </it>gene electrotransfer experiments show that the fraction of transfected cells increases by changing the electric field direction between electrical pulses. The fluorescence intensity of transfected cells and cell survival does not depend on electric field protocol. Moreover, a new effect a shading effect was observed during our research. Namely, shading effect is observed during gene electrotransfer when cells are in clusters, where only cells facing negative electro-potential in clusters become transfected and other ones which are hidden behind these cells do not become transfected.</p> <p>Conclusion</p> <p>On the basis of our results we can conclude that the new system can be used in <it>in-vitro </it>gene electrotransfer to improve cell transfection by changing electric field direction between electrical pulses, without affecting cell survival.</p

Effect of electric field vectoriality on electrically mediated gene delivery in mammalian cells

AbstractElectropermeabilization is a nonviral method used to transfer genes into living cells. Up to now, the mechanism is still to be elucidated. Since cell permeabilization, a prerequired for gene transfection, is triggerred by electric field, its characteristics should depend on its vectorial properties. The present investigation addresses the effect of pulse polarity and orientation on membrane permeabilization and gene delivery by electric pulses applied to cultured mammalian cells. This has been directly observed at the single-cell level by using digitized fluorescence microscopy. While cell permeabilization is only slightly affected by reversing the polarity of the electric pulses or by changing the orientation of pulses, transfection level increases are observed. These last effects are due to an increase in the cell membrane area where DNA interacts. Fluorescently labelled plasmids only interact with the electropermeabilized side of the cell facing the cathode. The plasmid interaction with the electropermeabilized cell surface is stable and is not affected by pulses of reversed polarities. Under such conditions, DNA interacts with the two sites of the cell facing the two electrodes. When changing both the pulse polarity and their direction, DNA interacts with the whole membrane cell surface. This is associated with a huge increase in gene expression. This present study demonstrates the relationship between the DNA/membrane surface interaction and the gene transfer efficiency, and it allows to define the experimental conditions to optimize the yield of transfection of mammalian cells

Caspase-10-Dependent Cell Death in Fas/CD95 Signalling Is Not Abrogated by Caspase Inhibitor zVAD-fmk

Upon CD95/Fas ligation, the initiator caspase-8 is known to activate effector caspases leading to apoptosis. In the presence of zVAD-fmk, a broad-spectrum caspase inhibitor, Fas engagement can also trigger an alternative, non-apoptotic caspase-independent form of cell death, which is initiated by RIP1. Controversy exists as to the ability of caspase-10 to mediate cell death in response to FasL (CD95L or CD178). Herein, the role of caspase-10 in FasL-induced cell death has been re-evaluated