Characterization of electric field-induced fusion in erythrocyte ghost membranes.

Abstract

Fusion has been reported to occur in a variety of membrane systems in response to the application of certain electric currents to the medium (Zimmermann, U., 1982, Biochim. Biophys. Acta., 694:227-277). The application of a weak but continuous alternating current causes the membranes in suspension to become rearranged into the "pearl-chain" formation. Fusion can then be induced by one or more strong direct current pulses that cause pore formation. This results in the conversion of individual membranes in the "pearl-chain" formation to a single membrane with one or more hourglass constrictions that form lumens which connect the cytoplasmic compartments. As the diameter of the lumens increases, the overall membrane shape grows to one large sphere. To further characterize electric field-induced fusion, experiments were conducted using the erythrocyte ghost as a model membrane, and a new combination of electrical circuit and fusion chamber that is simpler and improved over previous systems. All odd-shaped ghosts (collapsed or partly collapsed spherical shapes, echinocytes, discocytes, and stomatocytes) in 30 mM phosphate buffer was first converted to spherocytes and then fused with increasing yields by increasing the number of pulses. After fusion, the lateral diffusion of a fluorescent lipid soluble label (Dil) from labeled to unlabeled membranes was observed to occur both with and without the appearance in phase-contrast optics of distinct communication (lumens) between cytoplasmic compartments of the fused membranes. Connections between cytoplasmic compartments, however, were unmistakable with the instant transfer of a fluorescent water-soluble label (fluorescein isothiocyanate-dextran) from labeled to unlabeled cytoplasmic compartments upon fusion. Although pulses still resulted in the lateral diffusion of Dil to unlabeled membranes, the presence of glycerol in the medium strongly reduced the yield of lumens observable by phase-contrast optics in fusion events. The presence of glycerol also inhibited the conversion of membranes to spherocytes, but did not inhibit the lateral diffusion of Dil from labeled to unlabeled membranes.