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Abstract

When cells of Dictyostelium discoideum are exposed to electric pulses they are induced to fuse, yielding motile polykaryotic cells. By combining electron microscopy and direct recording of fluorescent cells, we have studied the emergence of fusion pores in the membranes and the localization of actin to the cell cortex. In response to electric pulsing, the plasma membranes of two contiguous cells are turned into tangles of highly bent and interdigitated membranes. Live-imaging of cells double-labeled for membranes and filamentous actin revealed that actin is induced to polymerize in the fusion zone to temporarily bridge the gaps in the vesiculating membrane. The diffusion of green fluorescent protein (GFP) from one fusion partner to the other was scored using spinning disc confocal microscopy. Fusion pores that allowed intercellular exchange of GFP were formed after a delay, which lasted up to 24 seconds after exposure of the cells to the electric field. These data indicate that the membranes persist in a fusogenic state before pores of about 3 nm diameter are formed.