A DELAY IN MEMBRANE-FUSION - LAG TIMES OBSERVED BY FLUORESCENCE MICROSCOPY OF INDIVIDUAL FUSION EVENTS INDUCED BY AN ELECTRIC-FIELD PULSE

Low light level video microscopy of the fusion of DiI- (1.1′-dihexadecyl-3,3,3′, 3′-tetra- methylindocarbocyanine perchlorate) labeled rabbit erythrocyte ghosts with unlabeled rabbit erythrocyte ghosts, held in stable apposition by dielectrophoresis in sodium phosphate buffers, showed reproducible time intervals (delays) between the application of a single fusogenic electric pulse and the earliest detection of fluorescence in the unlabeled adjacent membranes. The delay increased over the range 0.3–4 s with a decrease in (i) the electric field strength of the fusion-inducing pulse from 1000 to 250 V/mm, (ii) the decay half-time of the fusogenic pulse in the range 1.8–0.073 ms, and (iii) the dielectrophoretic force which brings the membranes into close apposition. A change in the buffer viscosity from 1.8 to 10 mP•s caused the delay to increase from 0.36 to 3.7 s (in glycerol solutions) or to 5.2 s (in sucrose solutions). The delay decreased 2–3 times with an increase in temperature from 21 to 37 °C. It did not differ significantly for “white” ghosts [0.013 mM hemoglobin (Hb)] or “red” ghosts (0.15 mM Hb) or buffer strength over the range 5–60 mM (sodium phosphate, pH 8.5). The calculated activation energy, 17 kcal/mol, does not depend on the field strength. The yield of fused cells was high when the delay was short. The delay in electrofusion resembles the delays in pH-dependent fusion of vesicular stomatitis viruses with erythrocyte ghosts [Clague, M. J., Schoch, C., Zech, L., & Blumenthal, R. (1990) Biochemistry 29, 1303–1308] and of fibroblasts expressing influenza hemagglutinin and red blood cells [Morris, S. J., Sarkar, D. P., White, J. M., & Blumenthal, R. (1989) J. Biol. Chem. 264, 3972-3978]. The delay reflects the lifetime of a long-lived intermediate state in fusion. It may be partly due to the time of mutual approach of membranes needed to reach molecular contact and/or the time of relatively slow molecular rearrangements leading to formation of intermembrane connections. © 1990, American Chemical Society. All rights reserved.