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. 1997 Mar 10;136(5):995-1005.
doi: 10.1083/jcb.136.5.995.

Inner but not outer membrane leaflets control the transition from glycosylphosphatidylinositol-anchored influenza hemagglutinin-induced hemifusion to full fusion

G B Melikyan  1 , S A Brener, D C Ok, F S Cohen
Affiliations

Inner but not outer membrane leaflets control the transition from glycosylphosphatidylinositol-anchored influenza hemagglutinin-induced hemifusion to full fusion

G B Melikyan et al. J Cell Biol. .

Abstract

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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Figures

Figure 1
Figure 1
Schematic representation of fusion progressing from a bound state to membranes connected by a stalk, followed by stalk expansion to form an HD, and then to pore formation that completes fusion. The lipids curve in opposite directions for the stalk and pore. A cone-shaped agent (as shown) facilitates formation of a stalk. Inverted cone-shaped, positive curvature agents (shown between hemifusion and fusion) inhibit formation of a stalk but promote formation of a pore within the HD. The HD is composed of cytoplasmic leaflets of both cells and separates the aqueous compartments of the two fusing cells.
Figure 2
Figure 2
Chlorpromazine induces complete fusion between hemifused GPI cells and RBCs. RBCs were colabeled with CF and R18. 5 min after fusion was triggered by a brief exposure of cell–RBC pairs to an acidic pH, the redistribution of R18 between RBCs and GPI cells was completed (A and B), whereas CF did not spread into the GPI cells (C and D). Transiently (for 1 min) exposing cells in the lefthand panels to 0.5 mM CPZ resulted in efficient CF redistribution without detectable lysis (E, compare with C). In contrast, similar treatment of cells shown in the righthand panels with 1 mM of membrane-impermeable M-CPZ did not lead to any changes in CF fluorescence pattern (F, same as D). The CPZ did not cause a redistribution of R18, but it did strongly quench the R18 fluorescence in a concentration-dependent fashion.
Figure 3
Figure 3
(A) Concentration dependence of hemifusion-to-fusion transition induced by phenothiazines (CPZ and TFP) and a local anesthetic (DB). The fusion efficiency at pH 7.0 was calculated as the ratio of cells stained with NDB-t to the total number of cells decorated with RBCs. The functional forms of extent of fusion vs concentration (note semilogarithmic scale) were similar for all three drugs, despite the differences in effective concentration ranges. Error bars show the standard errors for four to eight experiments. (B) Efficiency of hemifusion-to-fusion transition induced by CPZ as a function of pH. GPI cells hemifused to RBCs were briefly exposed to 0.4 mM CPZ buffered at the indicated pH values. Cells were returned to a CPZ-free solution at the same pH, and the fraction of cells stained with NBD-t was counted (circles, lefthand scale). The standard error was smaller than the size of the symbols. The ratios of the neutral (B, membranepermeable) and protonated (BH +) forms of CPZ were calculated assuming pK 9.3 and are shown by the solid line (righthand scale).
Figure 4
Figure 4
The effect of CPZ and DB on the propensity of lipids to form a pore in planar lipid bilayers. (Upper panel) Schematic illustration of the current (top trace) upon application of a voltage step (bottom trace) that results in irreversible breakdown of a planar membrane. The lifetime of the planar bilayer (τ) is defined as the time from stepping to a given voltage to the moment of the irreversible increase of current signifying membrane breakdown. (Lower panel) Semilogarithmic plot of τ as a function of applied voltage (V). Standard errors are shown for 10–12 experiments. The line tensions of lipidic pores were estimated by curve-fitting (solid lines) Eq. 1 to experimental data. The specific capacitance, Cm, and surface tension, σ, of DOPE/DOPC/PS bilayers were measured in the absence (control) and presence of amphipathic agents. Cm = 0.66 ± 0.05 μF/cm2 was not affected by the agents, but σ = 0.42 ± 0.04 mN/m was lowered significantly by 1 mM CPZ or 4 mM DB to 0.21 ± 0.02 and 0.15 ± 0.03 mN/m, respectively. σ = 0.36 ± 0.04 mN/m in the presence of 0.1 mM CPZ was not appreciably lower than control. (Filled circles) Control, γ = 16.0 pN (R2 = 0.96); (open diamonds) 0.1 mM CPZ, γ = 17.1 pN (R2 = 0.99); (filled squares) 4 mM dibucaine, γ = 4.1 pN (R2 = 0.85); (filled diamonds) 1 mM CPZ, γ = 3.7 pN (R2 = 0.98).
Figure 5
Figure 5
The transfer of membrane and aqueous dye between CF/R18-colabeled RBCs and WT cells. R18 redistributed efficiently between cells upon transient exposure to low pH (A and B), but only a small amount of CF (C and D) transferred into only a few cells (arrows) 5 min after reneutralization. A transient (1-min) exposure to 0.1 mM CPZ induced substantial amounts of CF to transfer to almost all the WT cells (E). There was additional spread of R18 during the time between A and E (not shown). In contrast, treatment with 1 mM M-CPZ did not lead to full fusion, although washing it out induced some CF transfer (F, arrow) by an unknown mechanism.
Figure 6
Figure 6
The extent of transfer of small (CF) and large (RD) aqueous probes between RBCs and WT cells. Within 5–10 min after fusion was triggered, a few cells were stained by CF (A), whereas only a rare cell was stained by RD (B). Even 30 min after acidification, a substantial fraction (about one-half) of the WT cells were stained with CF, whereas virtually all the RD was still confined to RBCs (not shown). A brief (1-min) exposure to 0.1 mM CPZ dramatically increased the transfer of CF (C), but only slightly facilitated the spread of RD (D, arrowhead). Subsequent transient treatment of these cells with 0.5 mM CPZ resulted in virtually complete spread of CF (E) and significantly higher transfer of RD (F). The fluorescence of CF was brighter after CF spread into WT cells (compare C and E to A): RD probably quenched CF fluorescence within RBCs by resonance energy transfer.
Figure 7
Figure 7
Transfer of a small (CF, filled symbols) and large (RD, open symbols) probe from RBCs to GPI (A, circles) and WT cells (B, squares) as a function of CPZ concentration 5 min after reneutralizaion. These results are replotted as the ratio of WT (squares) and GPI (circles) cells stained with RD to those stained with CF vs CPZ concentration (C). Error bars represent standard errors of three to seven independent experiments.
Figure 8
Figure 8
LPC incorporated into external leaflets of hemifused cells inhibits CPZ-induced fusion. The fraction of cells stained with NBD-t after exposure to 0.3 mM CPZ is shown (lefthand bar). Preincubation of hemifused cell pairs with 5 μM S-LPC for 2 min followed by washing out unincorporated S-LPC resulted in a strong inhibition of complete fusion induced by a subsequent application of 0.3 mM CPZ (middle bar). When S-LPC was removed from the external leaflets by incubating the cells with 4 mg/ml of delipidated BSA for 5 min before CPZ treatment, the extent of fusion was restored (righthand bar). Standard errors are shown for three experiments.
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References

    1. Abidor IG, Arakelian VB, Chernomordik LV, Yu, Chizmadzhev A, Pastushenko VF, Tarasevich MR. Electrical breakdown of BLM: main experimental facts and their qualitative discussion. Bioelectrochem Bioenerg. 1979;6:37–52.
    1. Binford JS, Jr, Palm WH. Adsorption of surfactants by membranes: erythrocytes versus synthetic vesicles. Biophys J. 1994;66:2024–2028. - PMC - PubMed
    1. Browning JL, Nelson DL. Amphipathic amines affect membrane excitability in Paramecium: role of bilayer couple. Proc Natl Acad Sci USA. 1976;73:452–456. - PMC - PubMed
    1. Chernomordik LV, Melikyan GB, Yu, Chizmadzhev A. Biomembrane fusion: a new concept derived from model studies using two interacting planar lipid bilayers. Biochim Biophys Acta. 1987;906:309–352. - PubMed
    1. Chernomordik LV, Vogel SS, Leikina EA, Sokoloff A, Onoran HO, Zimmerberg J. Lysolipids reversibly inhibit Ca2+-, GTP- and pH- dependent fusion of biological membranes. FEBS Lett. 1993;318:71–76. - PubMed

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