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J. Gen. Physiol.,
Volume 112, Number 4, October 1, 1998 409-422
From the * Department of Molecular Biophysics and Physiology, Rush Medical College, Chicago, Illinois 60612; and 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 cis monolayer (facing the cells) or the trans monolayer 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 trans monolayers and longer when in cis
monolayers 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 trans and cis monolayers. Enriching the cis monolayer with a negative curvature probe reduced the probability that a small pore would fully enlarge, whereas enriching the trans monolayer
promoted enlargement. Lysophosphatidylcholine has positive spontaneous curvature and does not translocate.
When lysophosphatidylcholine was placed in trans leaflets 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.
Department of
Biochemistry, McMaster University, Health Sciences Centre, Hamilton, Ontario, Canada L8N 3Z5
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