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Department of Molecular Biophysics and Physiology, Rush Medical College, Chicago, Illinois 60612
Cells that express wild-type influenza hemagglutinin (HA) fully fuse to RBCs, while cells that
express the HA-ectodomain anchored to membranes
by glycosylphosphatidylinositol, rather than by a transmembrane domain, only hemifuse to RBCs. Amphipaths were inserted into inner and outer membrane
leaflets to determine the contribution of each leaflet in
the transition from hemifusion to fusion. When inserted
into outer leaflets, amphipaths did not promote the
transition, independent of whether the agent induces
monolayers to bend outward (conferring positive spontaneous monolayer curvature) or inward (negative curvature). In contrast, when incorporated into inner leaflets, positive curvature agents led to full fusion. This
suggests that fusion is completed when a lipidic fusion
pore with net positive curvature is formed by the inner
leaflets that compose a hemifusion diaphragm. Suboptimal fusion conditions were established for RBCs bound
to cells expressing wild-type HA so that lipid but not
aqueous dye spread was observed. While this is the
same pattern of dye spread as in stable hemifusion, for
this "stunted" fusion, lower concentrations of amphipaths in inner leaflets were required to promote transfer of aqueous dyes. Also, these amphipaths induced larger pores for stunted fusion than they generated
within a stable hemifusion diaphragm. Therefore, spontaneous curvature of inner leaflets can affect formation
and enlargement of fusion pores induced by HA. We
propose that after the HA-ectodomain induces hemifusion, the transmembrane domain causes pore formation
by conferring positive spontaneous curvature to leaflets
of the hemifusion diaphragm.
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