Electric membrane fusion

Tsong, T. Y. Tomita, M. Lo, M. M. S. Pre-selection of B-lymphocytes by antigen for fusion to myeloma cells by pulsed electric field method. In Molecular Mechanisms of Membrane Fusion Oki, S. et al., Eds. Plenum New York pp 223-236. 

H. D. Hurwitz Professor Chismadjev did not limit himself to the problem of membrane breakdown and membrane fusion which is also tackled by Professor Berg s contribution. Other important aspects of bioelectrochemistry have been treated by Professor Chimadjev, like, for example, the determination of membrane potential, also considered in Professor Adams contribution, the electric field across membrane, the influence of antibiotics and of other vectors for the ion transport. I wish to ask the audience if there are any comments which concern such aspects of the selectivity of cell membranes to ions and the role of peptides. 

The development of cell manipulation methods is of considerable current interest. There have been intensive investigations of dielectric effects on living cells, which has led to the commercialization of an apparatus for electric cell fusion . Exposure of a cell suspension to an electric field or to an electric pulse on the order of 10 V-cm" results in the breakdown of the cellular membrane. ... 

In recent years work was started in which this sort of phenomena are smdied at the cell level, particularly the electrical breakdown of cell membranes and the fusion of neighboring cells under the influence of electric fields. These studies are of great value, in particular, for the further development of cell engineering and gene technology. 

There is considerable evidence that the release of 5-HT occurs by exocytosis, i.e. by the discharge from the cell of the entire content of individual storage vesicles. First, 5-HT is sufficiently ionized at physiological pH so that it does not cross plasma membranes by simple diffusion. Second, most intraneuronal 5-HT is contained in storage vesicles and other contents of the vesicle including SPB are released together with serotonin. By contrast, cytosolic proteins do not accompany electrical stimulation-elicited release of 5-HT. Third, the depolarization-induced release of 5-HT occurs by a calcium-dependent process indeed, it appears that the influx of extracellular calcium ions with or without membrane depolarization can increase the release of 5-HT. Calcium stimulates the fusion of vesicular membranes with the plasma membrane (see Chs 9,10). 

Above we noted that the fusion of synaptic vesicles or secretory granules with the plasma membrane of the synaptic terminal was caused by the arrival of an electrical signal flowing from the cell body down the axon. What is the nature of this electrical signal ... 

In the presynaptic cell, the neurotransmitters are stored in vesicles. On arrival of an electrical signal (action potential, see 16.2), an influx of Ca takes place into the presynaptic cell as voltage-gated Ca channels are opened. The increase in Ca concentration leads to fusion of the vesicles with the membrane of the postsynaptic cell. The neurotransmitters are released into the synaptic cleft and diffuse to a corresponding receptor on the surface of the postsynaptic cell. Binding of the nemotransmitter to the receptor induces opening of an ion channel that is a component of the receptor. The type of ions that can enter depends on the selectivity of the ion channel. There are Na K, Ca and Cf specific ion channels. The ion flux creates an electric signal in the post-... 

Physical methods of gene transfer. Genes can often be transferred without the use of a cloning vehicle. This is especially important for certain plant cells, such as those of cereal grains, for which transfer of genes via the Ti plasmid has been difficult.167 If DNA, which may be in a plasmid, is coprecipitated with calcium phosphate, it can often be taken up directly either by animal cells or by plant protoplasts.168 169 Polycations also facilitate DNA uptake cationic liposomes seem to be especially effective.170 In the widely used electroporation technique a short electrical pulse of a few hundred volts / cm is applied to create transient pores in the plasma membrane through which the DNA can enter a cell.111 8,171 175 Chromosomes can be transferred by cell fusion and either... 

In the following, a physical fusion method is described based on the electrical breakdown of membranes. No attempt is made to explain this procedure in full detail (for an extensive review see 61)), but rather a qualitative picture is given in order to provide a general understanding of the process. 

During the fusion process the relative surface area decreases with increasing volume indicating a loss of membrane material (about 22% in Fig. 51). In analogy to the fusion process of protoplasts it can be assumed that the excess lipid is removed in form of small, submicroscopic vesicles (Fig. 52). The electric breakdown in the membrane contact zone leads to the formation of several pores in which lipid molecules are randomly oriented (Fig. 52 b). The molecules reorient forming submicroscopic vesicles and the new membrane of the fused vesicle (Fig. 52c). Thus, fused giant liposomes should contain small, submicroscopic vesicles. This could possibly be proven by using fluorescence-labelled lipids for liposome fusion. 

Fig. 52a-c. Scheme of the fusion process of giant liposomes and the formation of small unilamellar vesicles (SUV) at the interface, a) lipid bilayers in contact b) pores generated by electric breakdown and lipid reorientation forming SUVs c) reconstitution of lipid membranes formation of a fused giant liposome and SUVs . 

The process of information flow between neurons is termed synaptic transmission, and in its most basic form it is characterized by unidirectional communication from the presynaptic to postsynaptic neuron. The process begins with the initiation of an electrical impulse in the axon of the presynaptic neuron. This electrical signal—the action potential—propagates to the axon terminal, which thereby stimulates the fusion of a transmitter-fllled synaptic vesicle with the presynaptic terminal membrane. The process of synaptic vesicle fusion is highly regulated and involves numerous biochemical reactions it culminates in the release of chemical neurotransmitter into the synaptic cleft. The released neurotransmitter diffuses across the cleft and binds to and activates receptors on the postsynaptic site, which thereby completes the process of synaptic transmission. 

The mechanism of fusion is not understood. It is known that when application of an external electric field causes the potential difference across the separating membrane to reach a certain threshold value, the membrane becomes reversibly transformed from the rest state to a fusion-susceptible state, particularly in the contact zone between adjacent cells. The membrane excitation in a broad sense is observed... 

The electrofusion technique is a significant new tool for research and production of controlled systems in the life sciences. The study of electric-field-induced membrane and cell phenomena on a molecular level will contribute to fundamental understanding both of cell-to-cell fusion and of membrane structure and function. 

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