Membranes electrofusion

The phenomenon of membrane electrofusion is of particular interest, because of its widespread use in cell biology and biotechnology (e.g., [162-164] and the references cited therein). The application of electrofusion to cells can lead to the creation of multinucleated viable cells with new properties (this phenomenon is also known as hybridization) (e.g., [164]). In addition, electroporation and electrofusion are often used to introduce molecules like proteins, foreign genes (plasmids), antibodies, and drugs into cells. 

This article is concerned with the control of membrane electrofusion. This technique has several attractive features compared to other artificial methods. Because it is based entirely on the electrical properties of the cells, the detailed biochemical and biological properties of the cells have little influence on the fusion process. Therefore, the method is applicable to a majority of cells. The only... 

Electrical breakdown of the membrane sets an upper limit on the fields that can be applied. Controlled electrical breakdown of the membrane is a fairly well understood process which is used in the techniques of electrofusion and electroporation but it is clearly undesirable in the devices under discussion. Surprisingly, fibroblasts can be cultivated for days with no apparent adverse effects in fields inducing trans-membrane potentials of 80% of the breakdown voltage (unpublished data). 

Although the mechanisms of electroporation, electrofusion, and electroinsertion are not known, biophysical data suggest that the primary field pulse effect is interfacial polarization by ion accumulation at the membrane surfaces. The resulting transmembrane electric field causes rearrangements of the lipids such that pores are formed1718. Electropores anneal slowly (over a period of minutes) when the pulse is switched off. 

The physical properhes of hpid bilayers are those that define their response to external perturbations. Knowing the mechanical and rheological characteristics of hpid membranes will prepare us to tackle problems related to stress induced in bilayers by electric fields and the phenomena that it triggers, for example, dynamics of vesicle and cell deformahon, bilayer instability, electroporation, and electrofusion. 

In this sechon some applicahon aspects of giant vesicle electroporation are considered. In parhcular, it will be demonstrated that creahng macropores in GUVs and observing their closing dynamics can be successfully apphed to the evaluation of material properties of membranes. While in Section 7.4.2 we saw that such experiments can be used to characterize membrane stability in terms of the crihcal porahon potenhal f c, here we will find out how one can also evaluate the edge tension of porated membranes. In addition, another apphcation based on electro-porahon, namely vesicle electrofusion, is introduced whereby the use of GUVs as microreactors suitable for the synthesis of nanoparhcles is demonstrated. 

When a DC pulse is applied to a couple of fluid-phase vesicles, which are in contact and oriented in the direction of the field, electrofusion can be observed. Vesicle orientation (and even alignment into pearl chains) can be achieved by application of an AC field to a vesicle suspension. This phenomenon is also observed with cells [164, 165] and is due to dielectric screening of the field. When the suspension is dilute, two vesicles can be brought together via the AC field and aligned. A subsequent application of a DC pulse to such a vesicle couple can lead to fusion. The necessary condition is that poration is induced in the contact area between the two vesicles. The possible steps of the electrofusion of two membranes are schematically illustrated in Figure 7.8a. In Sections 7.5.2.1 and 7.5.2.2, consideration will be given to the fusion of vesicles with different membrane composition or different composition of the enclosed solutions. 

Figure 7.9 Creating a multidomain vesicle by electrofusion of two vesicles with different membrane composition as observed with fluorescence microscopy, (a, b) Images acquired with confocal microscopy scans nearly at the equatorial plane of the fusing vesicles, (a) Vesicle 1 is made of sphingomyelin and cholesterol (7 3) and labeled with one fluorescent dye (green). Vesicle 2 is composed of dioleoylphosphatidylcholine and cholesterol (8 2) and labeled with another...

Riske, K.A., Bezlyepkina, N., Lipowsky, R., and Dimova, R. (2005) Electrofusion of model lipid membranes viewed with high temporal resolution. Biophysical Review Letters, 1 (4), 387 400. 

Chang, D.C. (1992) Structure and dynamics of electric field-induced membrane pores as revealed by rapid-freezing electron microscopy, in Guide to Electroporation and Electrofusion (eds D.C. Chang, B.M. Chassy, J.A. Saunders, and A.E. Sowers), Academic Press, San Diego, pp. 9-27. 

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. 

Use of a high field to activate a membrane enzyme was first reported by Witt et al. (25) in 1976. They used dc pulses of approximately 1 kV/cm and of 1-ms duration to induce ATP synthesis by the chloroplast ATPase. Following this initial work, there have been many reports on 1-kV/ cm dc field-induced ATP synthesis in different ATP synthetic systems (see the literature cited in references 13 and 14). The main conclusion from these studies is that an applied field-induced transmembrane potential can facilitate ATP release from the enzyme whether a PEF can affect enzyme turnover is not clear. Because 1-kV/ cm dc fields also cause severe Joule heating of a sample suspension, thermal effects cannot be easily avoided except when very short electric pulses (microseconds) are used. Thus, the method has limited utility for electroactivation experiments. The PEF method is, however, quite popular for the study of electroporation and electrofusion of cell membranes (see the chapter by J. Weaver in this volume), electroinsertion of membrane proteins (26), and electrotransfection of cells (27). 

Tsong, T. Y. Electroporation of cell membranes Its mechanisms and applications. In Electroporation and Electrofusion in Cell Biology Neumann, E. et al., Eds. Plenum New York pp 149-163. 

At the cellular level, the cell membranes of polarized cells are of the order of 10 kV/mm, and additional field strengths may easily bring the membrane into a nonlinear region even without cell excitation. Cell excitation, the opening of membrane channels, and the creation of an action potential are the result of nonlinear processes. Electroporation and electrofusion of cells in vitro (Section 10.11) are also processes in the nonlinear region. 

Electrofusion is the connection of two separate cell membranes into one by a similar pulse. It is believed that the process is based on the same field-induced restructuring of the bilayer lipid membranes, a process that may be reversible or irreversible. 

Weaver, J.C., Barnett, A., 1992. Guide to Electroporation and Electrofusion in Progress toward a Theoretical Model of Electroporation Mechanism Membrane Electrical Behavior and Molecular Transport, 26. 

Techaumnat B, Washizu M (2007) Analysis of the effects of an orifice plate on the membrane potential in electroporation and electrofusion of cells. J Phys D ApplPhys 40 1831-1837... 

Since its first appearance, the technique has successfully been used in many different applications (Potter, 1988, 1993 Neumann et al., 1989 Chang et al., 1992 Shikegawa and Dower, 1988 Chassy, 1988) transient and stable transfection of bacteria, plant, and mammalian cells with exogenous DNA (electrotransfection) insertion of enzymes, antibodies, biochemical reagents, viruses, and particles into cells deposition of macromolecules in cell membranes and electrofusion of whole cells. 

For AU = 1 V the breakdown of membrane in field direction occurs more easily the larger r at a given E, or in other words, for electrofusion, a higher E is needed for small ceSls[6,7]. 

Breakdown of membrane by sudden protein orientation and movement in the field direction before electrofusion of cells can occur. 

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