Kinetics of influenza hemagglutinin-mediated membrane fusion as a function of technique

Reliable techniques are required to evaluate the plausibility of proposed membrane fusion mechanisms. Here we have studied the kinetics of establishing the lipidic connection between hemagglutinin-expressing cells (HA-cells) and red blood cells (RBC) labeled with octadecylrhodamine, R18, using three different experimental approaches: (1) the most common approach of monitoring the rate of the R18 dequenching in a cuvette with a suspension of RBC/HA-cell complexes; (2) video fluorescence microscopy (VFM) to detect the waiting times before the onset of R18 redistribution, not dequenching, for each RBC attached to an adherent HA-cell; and (3) a new approach based on blockage of RBC fusion to an adherent HA-cell at different time points by lysophosphatidylcholine (LPC), so that only the cell pairs which, at the time of LPC application, had fused or were irreversibly committed to fusion contributed to the final extent of lipid mixing. The LPC blockage and VFM gave very similar estimates for the fusion kinetics, with LPC monitoring also those sites committed to the lipid mixing process. In contrast, R18 dequenching in the cuvette was much slower, i.e., it monitors a much later stage of dye redistribution.