Cells expressing the hemagglutinin protein of influenza virus were fused to planar bilayer membranes containing the fluorescent lipid probes octadecylrhodamine (R18) or indocarbocyanine (DiI) to investigate whether spontaneous curvature of each monolayer of a target membrane affects the growth of fusion pores. R18 and DiI lowered the transition temperatures for formation of an inverted hexagonal phase, indicating that these probes facilitate the formation of negative curvature structures. The probes are known to translocate from one monolayer of a bilayer membrane to the other in a voltage-dependent manner. The spontaneous curvature of the
cismonolayer (facing the cells) or the transmonolayer could therefore be made more negative through control of the polarity of voltage across the planar membrane. Electrical admittance measurements showed that the open times of flickering fusion pores were shorter when probes were in transmonolayers and longer when in cismonolayers compared with times when probe was symmetrically distributed. Open times were the same for probe symmetrically distributed as when probes were not present. Thus, open times were a function of the asymmetry of the spontaneous curvature between the transand cismonolayers. Enriching the cismonolayer with a negative curvature probe reduced the probability that a small pore would fully enlarge, whereas enriching the transmonolayer promoted enlargement. Lysophosphatidylcholine has positive spontaneous curvature and does not translocate. When lysophosphatidylcholine was placed in transleaflets of planar membranes, closing of fusion pores was rare. The effects of the negative and positive spontaneous curvature probes do not support the hypothesis that a flickering pore closes from an open state within a hemifusion diaphragm (essentially a “flat” structure). Rather, such effects support the hypothesis that the membrane surrounding the open pore forms a three-dimensional hourglass shape from which the pore flickers shut.