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'-tetramethylindocarbocyanine 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 degrees 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].(ABSTRACT TRUNCATED AT 250 WORDS)