Abstract
Hemifusion, the linkage of contacting lipid monolayers of two membranes before the opening of a fusion pore, is hypothesized to proceed through the formation of a stalk intermediate, a local and strongly bent connection between membranes. When the monolayers' propensity to bend does not support the stalk (e.g., as it is when lysophosphatidylcholine is added), hemifusion is inhibited. In contrast, short-chain alcohols, reported to affect monolayer bending in a manner similar to that of lysophosphatidylcholine, were here found to promote hemifusion between fluorescently labeled liposomes and planar lipid bilayers. Single hemifusion events were detected by fluorescence microscopy. Methanol or ethanol (1.2-1.6 w/w %) added to the same compartment of the planar bilayer chamber as liposomes caused a 5-50 times increase in the number of hemifusion events. Alcohol-induced hemifusion was inhibited by lysophosphatidylcholine. Promotion of membrane hemifusion by short-chain alcohol was also observed for cell-cell fusion mediated by influenza virus hemagglutinin (HA). Alcohol promoted a fusion stage subsequent to the low pH-dependent activation of HA. We propose that binding of short-chain alcohol to the surface of membranes promotes hemifusion by facilitating the transient breakage of the continuity of each of the contacting monolayers, which is required for their subsequent merger in the stalk intermediate.
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- Aldwinckle T. J., Ahkong Q. F., Bangham A. D., Fisher D., Lucy J. A. Effects of poly(ethylene glycol) on liposomes and erythrocytes. Permeability changes and membrane fusion. Biochim Biophys Acta. 1982 Aug 12;689(3):548–560. doi: 10.1016/0005-2736(82)90313-3. [DOI] [PubMed] [Google Scholar]
- Barry J. A., Gawrisch K. Direct NMR evidence for ethanol binding to the lipid-water interface of phospholipid bilayers. Biochemistry. 1994 Jul 5;33(26):8082–8088. doi: 10.1021/bi00192a013. [DOI] [PubMed] [Google Scholar]
- Basáez G., Goñi F. M., Alonso A. Effect of single chain lipids on phospholipase C-promoted vesicle fusion. A test for the stalk hypothesis of membrane fusion. Biochemistry. 1998 Mar 17;37(11):3901–3908. doi: 10.1021/bi9728497. [DOI] [PubMed] [Google Scholar]
- Brahm J. Permeability of human red cells to a homologous series of aliphatic alcohols. Limitations of the continuous flow-tube method. J Gen Physiol. 1983 Feb;81(2):283–304. doi: 10.1085/jgp.81.2.283. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Chanturiya A., Chernomordik L. V., Zimmerberg J. Flickering fusion pores comparable with initial exocytotic pores occur in protein-free phospholipid bilayers. Proc Natl Acad Sci U S A. 1997 Dec 23;94(26):14423–14428. doi: 10.1073/pnas.94.26.14423. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Chen Z., Rand R. P. Comparative study of the effects of several n-alkanes on phospholipid hexagonal phases. Biophys J. 1998 Feb;74(2 Pt 1):944–952. doi: 10.1016/S0006-3495(98)74017-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Chernomordik L. V., Frolov V. A., Leikina E., Bronk P., Zimmerberg J. The pathway of membrane fusion catalyzed by influenza hemagglutinin: restriction of lipids, hemifusion, and lipidic fusion pore formation. J Cell Biol. 1998 Mar 23;140(6):1369–1382. doi: 10.1083/jcb.140.6.1369. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Chernomordik L. V., Leikina E., Frolov V., Bronk P., Zimmerberg J. An early stage of membrane fusion mediated by the low pH conformation of influenza hemagglutinin depends upon membrane lipids. J Cell Biol. 1997 Jan 13;136(1):81–93. doi: 10.1083/jcb.136.1.81. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Chernomordik L. V., Melikyan G. B., Chizmadzhev Y. A. Biomembrane fusion: a new concept derived from model studies using two interacting planar lipid bilayers. Biochim Biophys Acta. 1987 Oct 5;906(3):309–352. doi: 10.1016/0304-4157(87)90016-5. [DOI] [PubMed] [Google Scholar]
- Chernomordik L., Chanturiya A., Green J., Zimmerberg J. The hemifusion intermediate and its conversion to complete fusion: regulation by membrane composition. Biophys J. 1995 Sep;69(3):922–929. doi: 10.1016/S0006-3495(95)79966-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Chernomordik L., Kozlov M. M., Zimmerberg J. Lipids in biological membrane fusion. J Membr Biol. 1995 Jul;146(1):1–14. doi: 10.1007/BF00232676. [DOI] [PubMed] [Google Scholar]
- Cohen F. S., Akabas M. H., Zimmerberg J., Finkelstein A. Parameters affecting the fusion of unilamellar phospholipid vesicles with planar bilayer membranes. J Cell Biol. 1984 Mar;98(3):1054–1062. doi: 10.1083/jcb.98.3.1054. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Cohen F. S., Zimmerberg J., Finkelstein A. Fusion of phospholipid vesicles with planar phospholipid bilayer membranes. II. Incorporation of a vesicular membrane marker into the planar membrane. J Gen Physiol. 1980 Mar;75(3):251–270. doi: 10.1085/jgp.75.3.251. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Cunningham B. A., Midmore L., Kucuk O., Lis L. J., Westerman M. P., Bras W., Wolfe D. H., Quinn P. J., Qadri S. B. Sterols stabilize the ripple phase structure in dihexadecylphosphatidylcholine. Biochim Biophys Acta. 1995 Jan 26;1233(1):75–83. doi: 10.1016/0005-2736(94)00240-p. [DOI] [PubMed] [Google Scholar]
- Düzgüneş N., Wilschut J., Fraley R., Papahadjopoulos D. Studies on the mechanism of membrane fusion. Role of head-group composition in calcium- and magnesium-induced fusion of mixed phospholipid vesicles. Biochim Biophys Acta. 1981 Mar 20;642(1):182–195. doi: 10.1016/0005-2736(81)90148-6. [DOI] [PubMed] [Google Scholar]
- Epand R. M. Diacylglycerols, lysolecithin, or hydrocarbons markedly alter the bilayer to hexagonal phase transition temperature of phosphatidylethanolamines. Biochemistry. 1985 Dec 3;24(25):7092–7095. doi: 10.1021/bi00346a011. [DOI] [PubMed] [Google Scholar]
- Epand R. M. Hydrophobicity curvature and membrane fusion. Trends Biochem Sci. 1993 Mar;18(3):81–81. doi: 10.1016/0968-0004(93)90157-i. [DOI] [PubMed] [Google Scholar]
- Helfrich W. Elastic properties of lipid bilayers: theory and possible experiments. Z Naturforsch C. 1973 Nov-Dec;28(11):693–703. doi: 10.1515/znc-1973-11-1209. [DOI] [PubMed] [Google Scholar]
- Helm C. A., Israelachvili J. N., McGuiggan P. M. Role of hydrophobic forces in bilayer adhesion and fusion. Biochemistry. 1992 Feb 18;31(6):1794–1805. doi: 10.1021/bi00121a030. [DOI] [PubMed] [Google Scholar]
- Ho C., Stubbs C. D. Effect of n-alkanols on lipid bilayer hydration. Biochemistry. 1997 Sep 2;36(35):10630–10637. doi: 10.1021/bi9703150. [DOI] [PubMed] [Google Scholar]
- Holte L. L., Gawrisch K. Determining ethanol distribution in phospholipid multilayers with MAS-NOESY spectra. Biochemistry. 1997 Apr 15;36(15):4669–4674. doi: 10.1021/bi9626416. [DOI] [PubMed] [Google Scholar]
- Hornby A. P., Cullis P. R. Influence of local and neutral anaesthetics on the polymorphic phase preferences of egg yolk phosphatidylethanolamine. Biochim Biophys Acta. 1981 Oct 2;647(2):285–292. doi: 10.1016/0005-2736(81)90256-x. [DOI] [PubMed] [Google Scholar]
- Kemble G. W., Danieli T., White J. M. Lipid-anchored influenza hemagglutinin promotes hemifusion, not complete fusion. Cell. 1994 Jan 28;76(2):383–391. doi: 10.1016/0092-8674(94)90344-1. [DOI] [PubMed] [Google Scholar]
- Kim S., Martin G. M. Preparation of cell-size unilamellar liposomes with high captured volume and defined size distribution. Biochim Biophys Acta. 1981 Aug 6;646(1):1–9. doi: 10.1016/0005-2736(81)90264-9. [DOI] [PubMed] [Google Scholar]
- Komatsu H., Okada S. Ethanol-induced aggregation and fusion of small phosphatidylcholine liposome: participation of interdigitated membrane formation in their processes. Biochim Biophys Acta. 1995 May 4;1235(2):270–280. doi: 10.1016/0005-2736(95)80014-7. [DOI] [PubMed] [Google Scholar]
- Komatsu H., Rowe E. S. Effect of cholesterol on the ethanol-induced interdigitated gel phase in phosphatidylcholine: use of fluorophore pyrene-labeled phosphatidylcholine. Biochemistry. 1991 Mar 5;30(9):2463–2470. doi: 10.1021/bi00223a024. [DOI] [PubMed] [Google Scholar]
- Korte T., Ludwig K., Krumbiegel M., Zirwer D., Damaschun G., Herrmann A. Transient changes of the conformation of hemagglutinin of influenza virus at low pH detected by time-resolved circular dichroism spectroscopy. J Biol Chem. 1997 Apr 11;272(15):9764–9770. doi: 10.1074/jbc.272.15.9764. [DOI] [PubMed] [Google Scholar]
- Kozlov M. M., Leikin S., Rand R. P. Bending, hydration and interstitial energies quantitatively account for the hexagonal-lamellar-hexagonal reentrant phase transition in dioleoylphosphatidylethanolamine. Biophys J. 1994 Oct;67(4):1603–1611. doi: 10.1016/S0006-3495(94)80633-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Leckband D. E., Helm C. A., Israelachvili J. Role of calcium in the adhesion and fusion of bilayers. Biochemistry. 1993 Feb 2;32(4):1127–1140. doi: 10.1021/bi00055a019. [DOI] [PubMed] [Google Scholar]
- Lee J., Lentz B. R. Evolution of lipidic structures during model membrane fusion and the relation of this process to cell membrane fusion. Biochemistry. 1997 May 27;36(21):6251–6259. doi: 10.1021/bi970404c. [DOI] [PubMed] [Google Scholar]
- Lee J., Lentz B. R. Outer leaflet-packing defects promote poly(ethylene glycol)-mediated fusion of large unilamellar vesicles. Biochemistry. 1997 Jan 14;36(2):421–431. doi: 10.1021/bi9622332. [DOI] [PubMed] [Google Scholar]
- Lee J., Lentz B. R. Secretory and viral fusion may share mechanistic events with fusion between curved lipid bilayers. Proc Natl Acad Sci U S A. 1998 Aug 4;95(16):9274–9279. doi: 10.1073/pnas.95.16.9274. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Leikin S. L., Kozlov M. M., Chernomordik L. V., Markin V. S., Chizmadzhev Y. A. Membrane fusion: overcoming of the hydration barrier and local restructuring. J Theor Biol. 1987 Dec 21;129(4):411–425. doi: 10.1016/s0022-5193(87)80021-8. [DOI] [PubMed] [Google Scholar]
- Leikin S., Parsegian V. A., Rau D. C., Rand R. P. Hydration forces. Annu Rev Phys Chem. 1993;44:369–395. doi: 10.1146/annurev.pc.44.100193.002101. [DOI] [PubMed] [Google Scholar]
- Lentz B. R. Polymer-induced membrane fusion: potential mechanism and relation to cell fusion events. Chem Phys Lipids. 1994 Sep 6;73(1-2):91–106. doi: 10.1016/0009-3084(94)90176-7. [DOI] [PubMed] [Google Scholar]
- McIntosh T. J., Kulkarni K. G., Simon S. A. Membrane fusion promoters and inhibitors have contrasting effects on lipid bilayer structure and undulations. Biophys J. 1999 Apr;76(4):2090–2098. doi: 10.1016/S0006-3495(99)77365-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
- McIntosh T. J., Magid A. D., Simon S. A. Range of the solvation pressure between lipid membranes: dependence on the packing density of solvent molecules. Biochemistry. 1989 Sep 19;28(19):7904–7912. doi: 10.1021/bi00445a053. [DOI] [PubMed] [Google Scholar]
- Melikyan G. B., Niles W. D., Cohen F. S. The fusion kinetics of influenza hemagglutinin expressing cells to planar bilayer membranes is affected by HA density and host cell surface. J Gen Physiol. 1995 Nov;106(5):783–802. doi: 10.1085/jgp.106.5.783. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Melikyan G. B., White J. M., Cohen F. S. GPI-anchored influenza hemagglutinin induces hemifusion to both red blood cell and planar bilayer membranes. J Cell Biol. 1995 Nov;131(3):679–691. doi: 10.1083/jcb.131.3.679. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Monck J. R., Fernandez J. M. The fusion pore and mechanisms of biological membrane fusion. Curr Opin Cell Biol. 1996 Aug;8(4):524–533. doi: 10.1016/s0955-0674(96)80031-7. [DOI] [PubMed] [Google Scholar]
- Mou J., Yang J., Huang C., Shao Z. Alcohol induces interdigitated domains in unilamellar phosphatidylcholine bilayers. Biochemistry. 1994 Aug 23;33(33):9981–9985. doi: 10.1021/bi00199a022. [DOI] [PubMed] [Google Scholar]
- Niles W. D., Cohen F. S. Video fluorescence microscopy studies of phospholipid vesicle fusion with a planar phospholipid membrane. Nature of membrane-membrane interactions and detection of release of contents. J Gen Physiol. 1987 Nov;90(5):703–735. doi: 10.1085/jgp.90.5.703. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ohki S. Effects of divalent cations, temperature, osmotic pressure gradient, and vesicle curvature on phosphatidylserine vesicle fusion. J Membr Biol. 1984;77(3):265–275. doi: 10.1007/BF01870574. [DOI] [PubMed] [Google Scholar]
- Powell G. L., Marsh D. Polymorphic phase behavior of cardiolipin derivatives studied by 31P NMR and X-ray diffraction. Biochemistry. 1985 Jun 4;24(12):2902–2908. doi: 10.1021/bi00333a013. [DOI] [PubMed] [Google Scholar]
- Rand R. P., Fuller N. L., Gruner S. M., Parsegian V. A. Membrane curvature, lipid segregation, and structural transitions for phospholipids under dual-solvent stress. Biochemistry. 1990 Jan 9;29(1):76–87. doi: 10.1021/bi00453a010. [DOI] [PubMed] [Google Scholar]
- Safinya CR, Sirota EB, Roux D, Smith GS. Universality in interacting membranes: The effect of cosurfactants on the interfacial rigidity. Phys Rev Lett. 1989 Mar 6;62(10):1134–1137. doi: 10.1103/PhysRevLett.62.1134. [DOI] [PubMed] [Google Scholar]
- Sarkar D. P., Morris S. J., Eidelman O., Zimmerberg J., Blumenthal R. Initial stages of influenza hemagglutinin-induced cell fusion monitored simultaneously by two fluorescent events: cytoplasmic continuity and lipid mixing. J Cell Biol. 1989 Jul;109(1):113–122. doi: 10.1083/jcb.109.1.113. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Schwichtenhövel C., Deuticke B., Haest C. W. Alcohols produce reversible and irreversible acceleration of phospholipid flip-flop in the human erythrocyte membrane. Biochim Biophys Acta. 1992 Oct 19;1111(1):35–44. doi: 10.1016/0005-2736(92)90271-m. [DOI] [PubMed] [Google Scholar]
- Siegel D. P. Energetics of intermediates in membrane fusion: comparison of stalk and inverted micellar intermediate mechanisms. Biophys J. 1993 Nov;65(5):2124–2140. doi: 10.1016/S0006-3495(93)81256-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Siegel D. P., Epand R. M. The mechanism of lamellar-to-inverted hexagonal phase transitions in phosphatidylethanolamine: implications for membrane fusion mechanisms. Biophys J. 1997 Dec;73(6):3089–3111. doi: 10.1016/S0006-3495(97)78336-X. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Simon S. A., McIntosh T. J. Interdigitated hydrocarbon chain packing causes the biphasic transition behavior in lipid/alcohol suspensions. Biochim Biophys Acta. 1984 Jun 13;773(1):169–172. doi: 10.1016/0005-2736(84)90562-5. [DOI] [PubMed] [Google Scholar]
- Slater J. L., Huang C. H. Interdigitated bilayer membranes. Prog Lipid Res. 1988;27(4):325–359. doi: 10.1016/0163-7827(88)90010-0. [DOI] [PubMed] [Google Scholar]
- Smaal E. B., Schreuder C., van Baal J. B., Tijburg P. N., Mandersloot J. G., de Kruijff B., de Gier J. Calcium-induced changes in permeability of dioleoylphosphatidylcholine model membranes containing bovine heart cardiolipin. Biochim Biophys Acta. 1987 Feb 12;897(1):191–196. doi: 10.1016/0005-2736(87)90327-0. [DOI] [PubMed] [Google Scholar]
- Veiro J. A., Khalifah R. G., Rowe E. S. The polymorphic phase behavior of dielaidoylphosphatidylethanolamine. Effect of n-alkanols. Biochim Biophys Acta. 1989 Feb 27;979(2):251–256. doi: 10.1016/0005-2736(89)90441-0. [DOI] [PubMed] [Google Scholar]
- Walter A., Yeagle P. L., Siegel D. P. Diacylglycerol and hexadecane increase divalent cation-induced lipid mixing rates between phosphatidylserine large unilamellar vesicles. Biophys J. 1994 Feb;66(2 Pt 1):366–376. doi: 10.1016/s0006-3495(94)80786-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
- White J. M. Membrane fusion: the influenza paradigm. Cold Spring Harb Symp Quant Biol. 1995;60:581–588. doi: 10.1101/sqb.1995.060.01.062. [DOI] [PubMed] [Google Scholar]
- White J. M., Wilson I. A. Anti-peptide antibodies detect steps in a protein conformational change: low-pH activation of the influenza virus hemagglutinin. J Cell Biol. 1987 Dec;105(6 Pt 2):2887–2896. doi: 10.1083/jcb.105.6.2887. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wilschut J., Hoekstra D. Membrane fusion: lipid vesicles as a model system. Chem Phys Lipids. 1986 Jun-Jul;40(2-4):145–166. doi: 10.1016/0009-3084(86)90068-x. [DOI] [PubMed] [Google Scholar]
- Zeng J., Smith K. E., Chong P. L. Effects of alcohol-induced lipid interdigitation on proton permeability in L-alpha-dipalmitoylphosphatidylcholine vesicles. Biophys J. 1993 Oct;65(4):1404–1414. doi: 10.1016/S0006-3495(93)81204-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Zimmerberg J., Cohen F. S., Finkelstein A. Fusion of phospholipid vesicles with planar phospholipid bilayer membranes. I. Discharge of vesicular contents across the planar membrane. J Gen Physiol. 1980 Mar;75(3):241–250. doi: 10.1085/jgp.75.3.241. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Zimmerberg J., Curran M., Cohen F. S., Brodwick M. Simultaneous electrical and optical measurements show that membrane fusion precedes secretory granule swelling during exocytosis of beige mouse mast cells. Proc Natl Acad Sci U S A. 1987 Mar;84(6):1585–1589. doi: 10.1073/pnas.84.6.1585. [DOI] [PMC free article] [PubMed] [Google Scholar]
