Kinetics of Influenza Hemagglutinin-Mediated Membrane Fusion as a Function of Technique Aditya Mittal,* Eugenia Leikina,† Joe Bentz,* and Leonid V. Chernomordik† ,1 *Department of Bioscience & Biotechnology, Drexel University, Philadelphia, Pennsylvania 19104; and †Section on Membrane Biology, The Laboratory of Cellular and Molecular Biophysics, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892 Received August 22, 2001; published online March 6, 2002 Reliable techniques are required to evaluate the plausibility of proposed membrane fusion mecha- nisms. Here we have studied the kinetics of establish- ing the lipidic connection between hemagglutinin-ex- pressing cells (HA-cells) and red blood cells (RBC) labeled with octadecylrhodamine, R18, using three different experimental approaches: (1) the most com- mon approach of monitoring the rate of the R18 de- quenching 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 ap- proach based on blockage of RBC fusion to an adher- ent HA-cell at different time points by lysophosphati- dylcholine (LPC), so that only the cell pairs which, at the time of LPC application, had fused or were irre- versibly 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. Membrane fusion is a ubiquitous biological event and understanding its molecular mechanism is central to biology. Based on the current knowledge of crystal structures of fragments of some fusogenic proteins and similarities between the dependencies of diverse fusion processes on the composition of the membrane lipid bilayers, it is hypothesized that fusion processes cata- lyzed by different proteins share a basic molecular mechanism (1–3). Elucidation of these mechanisms re- quires coupling of membrane fusion kinetics, and its analysis, with different aspects of the proteins in- volved, like conformational and mutational studies. HA 2 -mediated fusion involves three distinct kineti- cally significant events, which are detected using dif- ferent experimental approaches. Electrophysiological measurements detect the establishment of the earliest fusion pore, a local aqueous connection between fusing cells that allows the first transfer of ions. Lipid mixing between the merged membranes is monitored as fluo- rescent lipid dye redistribution between labeled and unlabeled membranes. Similarly, aqueous contents mixing between membrane compartments can be mea- sured as a spread of aqueous fluorescent probes within the fusing cells. Much of our knowledge on membrane fusion mecha- nisms is based on different lipid mixing assays because of their relative simplicity in the technique. There have been a number of studies on the reliability of different probes that can be utilized for these lipid mixing assays (4 – 8). However, the question of how to best assay redistribution of any given probe has not been solved (9 –11). Here, we studied low pH-triggered fusion of HA- expressing cells and red blood cells (RBCs) labeled by the same fluorescent lipid probe, octadecylrhodamine (R18) using three different lipid mixing assays. We note that R18, which is a self-quenching probe, is not always a reliable reporter of transmembrane lipid flow. While in some cases the analysis of the total dequench- ing signal as a measure of fusion is complicated by the slow transfer of the probe without fusion and by R18 1 To whom correspondence should be addressed at Building 10, Room 10D04, 10 Center Drive, MSC 1855 Bethesda, MD 20892-1855. Fax: (301) 480-2916. E-mail: lchern@helix.nih.gov. 2 Abbreviations used: HA, hemagglutinin; RBC, red blood cell; VFM, video fluorescence microscopy; LPC, lysophosphatidylcholine; PBS, phosphate-buffered saline; FDQ, fluorescence dequenching. 0003-2697/02 $35.00 145 Analytical Biochemistry 303, 145–152 (2002) doi:10.1006/abio.2002.5590, available online at http://www.idealibrary.com on