Movement of a fluorescent lipid label from a labeled erythrocyte membrane to an unlabeled erythrocyte membrane following electric-field-induced fusion

A short burst of electric field pulses was used to induce nearly simultaneous fusion among 50% or more of a population composed of unlabeled erythrocytes and erythrocytes labeled with the fluorescent lipid analogue DiI (1,1'-dihexadecyl-3,3,3'',3'-tetra-methylindo carbocyanine perchlorate). Fusion products that ended in an hourglass shape were selected for analysis. The net movement of the label from the labeled membrane to the adjacent unlabeled membrane in each of the hourglass-shaped fusion products was recorded by micrography at various known times after the fusion took place, but before equilibrium was achieved. The lateral concentration gradients were measured by densitometry and compared with predictions based on Huang's model (Huang, H.-W., 1973, J. Theor. Biol., 40:11–17) for lateral diffusion on a spherical membrane. The average lateral diffusion coefficients, 3.8 and 8.1 X 10(-9) cm2/s in pH 7.4 isotonic phosphate buffer at 23–25 degrees C and 35–37 degrees C, respectively, compare very favorably with the results of three published photobleaching studies of the lateral diffusion of DiI in erythrocyte membranes. While the fusion approach to measuring lateral diffusion is not new, it has not enjoyed widespread use because of the uncertainty in the degree of fusion synchrony and low fusion yield. This study shows that the use of pulsed electric fields to induce synchronous fusion is a promising approach to overcome both of these drawbacks and yield results comparable to those obtainable by the photobleaching approach.