Chapter 18. Electroporation and electrofusion of membranes
D.S. Dimitrov
Section on Membrane Structure and Function, National Cancer Institute,
National Institutes of Health, Bethesda, Maryland 20892, USA
and
Central Laboratory of Biophysics,
Bulgarian Academy of Sciences, Sofia 1113, Bulgaria
1. Introductory remarks
1.1. Membrane fusion ensures specific, controlled transfer of molecules in life processes and is important for biotechnology and biomedical research
Fusion of membranes is a physical process which was of critical importance for the rapid evolution of life on earth and is involved in a variety of life processes today. One could imagine that billions of years ago the mechanical and/or electrical interactions in the ancient ocean mediated close apposition and destabilization of membranes which resulted in their fusion. Fusion of parts of the same membrane led to cell division and fusion of different membranes resulted in cell fusion. Both phenomena led to transfer and exchange of molecules which contributed to the rapid acceleration of the evolution of life on earth. One could further speculate that the 'first' fusion reactions were non-specific and any membranes could fuse provided appropriate conditions, including molecular contact, membrane destabilization and proper composition. Subsequently, specialized proteins appeared which led to the specific fusion of membranes and to controlled transfer of molecules. Today, the specific fusion of membranes seems to be the predominant mode of fusion in biological systems; however, the ability of membranes to fuse non-specifically, e.g., by external electric fields, provides information about the fundamental mechanisms of fusion and is important for biotechnology, medicine and research in biology.
1.2. Manifestations of cell fusion were observed nearly two centuries ago, while electrofusion was discovered in the late 1970's
The first manifestations of a fusion phenomenon were observed almost two centuries ago by the German biologist Johannes Muller (see [1]). He discovered multinucleated giant cells in histological specimen while studying pathological conditions. By the turn of the nineteenth century, the medical literature contained several reports of 'polykaryocytosis' as symptomatic of a variety of diseases, including tuberculosis, variola, varicella, and rubeola. This raised the question whether these giant cells originated from successive mitoses or from fusion of mononucleated cells [2]. Later fusion was clearly established [3] as a mode of their formation, but it was not until the 1960's, when the studies of fusion 'exploded'. During a relatively short period of time a number of interesting fusion phenomena were discovered and characterized by using light microscopy (for history see [1]): (i) viruses can induce formation of giant multinucleated cells (syncytia) [4-6], (ii) during fertilization the acrosomal membrane interdigitates and then coalesces with the egg membranes [7], (iii) mononucleated myoblasts fuse to form myotubes, at least in vitro [8, 9], and (iv) cell hybrids can be formed in vitro by spontaneous cell fusion [10, 11]. The interest in studying fusion grew in the next decades mainly because of its importance for production of hybrid cells. In the early 1970's polyethylene glycol was introduced as a fusion agent for plant protoplasts [12, 13] and animal cells [14] (see also the chapter of K. Arnold in this book). In the late 1970's groups from Japan and Germany reported that cell fusion can be also induced by external electric fields (electrofusion) [15-18].
1.3. Electric fields can induce fusion of a wide variety of membranes
During the last decade numerous studies have shown that external electric fields can induce fusion of a wide variety of cell and artificial membranes (for review see [19-23]. This experimental observation is a demonstration of an inherent ability of membranes to fuse if appropriate conditions are provided and indicates the existence of properties of membrane systems, related to fusion, which are largely independent of the type of membranes. These properties include membrane stability and adhesion. Membranes are designed by nature to be stable and resist external constraints. They must be destabilized, i.e. they must be forced to change their structure to molecular conformations appropriate for fusion. The destabilized membranes must be at close apposition to allow merging of their lipid matrix. Therefore, understanding of fusion mechanisms requires understanding mechanisms of membrane destabilization and establishment of contact. External direct current (DC) fields can destabilize membranes and induce formation of pores (electroporation) (for recent review see [23]). External alternating current (AC) fields can induce membrane approach and contact predominantly by a process termed dielectrophoresis [24]. In the following chapter, I will focus on membrane fusion induced by electric fields (electrofusion) but I will also discuss electroporation and dielectrophoresis which are closely related to electrofusion.
I first briefly discuss polarization as one of the basic mechanisms of interactions of membranes with electric fields, leading to electroporation, dielectrophoresis and electrofusion, and then summarize observations on electroporation and dielectrophoresis related to electrofusion. In the rest of the chapter, I focus on observations of electrofusion and on the current concepts of its mechanisms. Why electrofusion may be important for understanding biological fusion is also briefly discussed.
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