Effects of spontaneous bilayer curvature on influenza virus-mediated fusion pores.
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 Molecular Biophysics and Physiology, Rush Medical College, Chicago, Illinois 60612, USA.
V I
VI
Razinkov
G B
GB
Melikyan
R M
RM
Epand
R F
RF
Epand
F S
FS
Cohen
D016475:3T3 Cells / Q000737:chemistry / Q000502:physiology; D000818:Animals; D002232:Carbocyanines / Q000494:pharmacology; D005456:Fluorescent Dyes / Q000494:pharmacology; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000502:physiology*; D015640:Ion Channel Gating / Q000187:drug effects / Q000502:physiology; D007700:Kinetics; D008051:Lipid Bilayers / Q000378:metabolism*; D008561:Membrane Fusion / Q000187:drug effects / Q000502:physiology*; D008565:Membrane Proteins / Q000502:physiology; D051379:Mice; D009975:Orthomyxoviridae / Q000502:physiology*; D018272:Porins / Q000737:chemistry / Q000187:drug effects / Q000502:physiology*; D011487:Protein Conformation; D012235:Rhodamines / Q000494:pharmacology
C101089:CM-DiI; D002232:Carbocyanines; D005456:Fluorescent Dyes; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D008051:Lipid Bilayers; D008565:Membrane Proteins; D018272:Porins; D012235:Rhodamines; C044542:octadecyl Rhodamine B chloride