Skip to main content
NCBI home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1988 Dec 1;107(6):2511–2521. doi: 10.1083/jcb.107.6.2511

Fusion of liposomes with the plasma membrane of epithelial cells: fate of incorporated lipids as followed by freeze fracture and autoradiography of plastic sections

PMCID: PMC2115627  PMID: 3204118

Abstract

The fusion of liposomes with the plasma membrane of influenza virus- infected monolayers of an epithelial cell line, Madin-Darby canine kidney cells (van Meer et al., 1985. Biochemistry. 24:3593-3602), has been analyzed by morphological techniques. The distribution of liposomal lipids over the apical and basolateral plasma membrane domains after fusion was assessed by autoradiography of liposomal [3H]dipalmitoylphosphatidylcholine after rapid freezing or chemical fixation and further processing by freeze substitution and low temperature embedding. Before fusion, radioactivity was solely detected on the apical cell surface, indicating the absence of redistribution artifacts and demonstrating the reliability of lipid autoradiography on both a light and electron microscopical level. After induction of fusion by a low pH treatment, the basolateral plasma membrane domain became progressively labeled, indicative of rapid lateral diffusion of [3H]dipalmitoylphosphatidylcholine in the plasma membrane. Analysis of individual fusion events by freeze fracture after rapid freezing confirmed the rapid diffusion of the liposomal lipids into the plasma membrane, as intramembrane particle-free lipid patches were never observed. After the induction of liposome-cell fusion, well-defined intramembrane particles were present on the otherwise smooth liposomal fracture faces and on the fracture faces of the plasma membrane. Morphological evidence thus was obtained in favor of a local point fusion mechanism with an intramembrane particle as a specific structural fusion intermediate.

Full Text

The Full Text of this article is available as a PDF (4.6 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bachmann L., Salpeter M. M. Absolute sensitivity of electron microscope radioautography. J Cell Biol. 1967 May;33(2):299–305. doi: 10.1083/jcb.33.2.299. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Branton D., Bullivant S., Gilula N. B., Karnovsky M. J., Moor H., Mühlethaler K., Northcote D. H., Packer L., Satir B., Satir P. Freeze-etching nomenclature. Science. 1975 Oct 3;190(4209):54–56. doi: 10.1126/science.1166299. [DOI] [PubMed] [Google Scholar]
  3. Burger K. N., Knoll G., Verkleij A. J. Influenza virus-model membrane interaction. A morphological approach using modern cryotechniques. Biochim Biophys Acta. 1988 Mar 22;939(1):89–101. doi: 10.1016/0005-2736(88)90050-8. [DOI] [PubMed] [Google Scholar]
  4. Chandler D. E. Comparison of quick-frozen and chemically fixed sea-urchin eggs: structural evidence that cortical granule exocytosis is preceded by a local increase in membrane mobility. J Cell Sci. 1984 Dec;72:23–36. doi: 10.1242/jcs.72.1.23. [DOI] [PubMed] [Google Scholar]
  5. Chandler D. E., Heuser J. E. Arrest of membrane fusion events in mast cells by quick-freezing. J Cell Biol. 1980 Aug;86(2):666–674. doi: 10.1083/jcb.86.2.666. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Costello M. J., Fetter R., Höchli M. Simple procedures for evaluating the cryofixation of biological samples. J Microsc. 1982 Feb;125(Pt 2):125–136. doi: 10.1111/j.1365-2818.1982.tb00330.x. [DOI] [PubMed] [Google Scholar]
  7. Davoust J., Gruenberg J., Howell K. E. Two threshold values of low pH block endocytosis at different stages. EMBO J. 1987 Dec 1;6(12):3601–3609. doi: 10.1002/j.1460-2075.1987.tb02691.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Düzgüneş N. Membrane fusion. Subcell Biochem. 1985;11:195–286. doi: 10.1007/978-1-4899-1698-3_5. [DOI] [PubMed] [Google Scholar]
  9. Futaesaku Y., Mizuhira V. Negative-staining autoradiography: a new technique for ultracryotomy utilizing an interposed film. J Histochem Cytochem. 1986 Aug;34(8):1085–1094. doi: 10.1177/34.8.2426334. [DOI] [PubMed] [Google Scholar]
  10. Hansson G. C., Simons K., van Meer G. Two strains of the Madin-Darby canine kidney (MDCK) cell line have distinct glycosphingolipid compositions. EMBO J. 1986 Mar;5(3):483–489. doi: 10.1002/j.1460-2075.1986.tb04237.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Heuser J. E., Reese T. S., Dennis M. J., Jan Y., Jan L., Evans L. Synaptic vesicle exocytosis captured by quick freezing and correlated with quantal transmitter release. J Cell Biol. 1979 May;81(2):275–300. doi: 10.1083/jcb.81.2.275. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Jacobson K., Hou Y., Derzko Z., Wojcieszyn J., Organisciak D. Lipid lateral diffusion in the surface membrane of cells and in multibilayers formed from plasma membrane lipids. Biochemistry. 1981 Sep 1;20(18):5268–5275. doi: 10.1021/bi00521a027. [DOI] [PubMed] [Google Scholar]
  13. Jost P., Brooks U. J., Griffith O. H. Fluidity of phospholipid bilayers and membranes after exposure to osmium tetroxide and gluteraldehyde. J Mol Biol. 1973 May 15;76(2):313–318. doi: 10.1016/0022-2836(73)90394-x. [DOI] [PubMed] [Google Scholar]
  14. Kachar B., Serrano J. A., da Silva P. P. Particle displacement in epithelial cell membranes of rat prostate and pancreas induced by routine low temperature fixation. Cell Biol Int Rep. 1980 Apr;4(4):347–356. doi: 10.1016/0309-1651(80)90216-7. [DOI] [PubMed] [Google Scholar]
  15. Miller R. G. Do 'lipidic particles' represent intermembrane attachment sites? Nature. 1980 Sep 11;287(5778):166–167. doi: 10.1038/287166a0. [DOI] [PubMed] [Google Scholar]
  16. Ornberg R. L., Reese T. S. Beginning of exocytosis captured by rapid-freezing of Limulus amebocytes. J Cell Biol. 1981 Jul;90(1):40–54. doi: 10.1083/jcb.90.1.40. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Papahadjopoulos D., Mayhew E., Poste G., Smith S., Vail W. J. Incorporation of lipid vesicles by mammalian cells provides a potential method for modifying cell behaviour. Nature. 1974 Nov 8;252(5479):163–166. doi: 10.1038/252163a0. [DOI] [PubMed] [Google Scholar]
  18. Poste G., Papahadjopoulos D. The influence of vesicle membrane properties on the interaction of lipid vesicles with cultured cells. Ann N Y Acad Sci. 1978;308:164–184. doi: 10.1111/j.1749-6632.1978.tb22021.x. [DOI] [PubMed] [Google Scholar]
  19. Poste G., Porter C. W., Papahadjopoulos D. Identification of a potential artifact in the use of electron microscope autoradiography to localize saturated phospholipids in cells. Biochim Biophys Acta. 1978 Jul 4;510(2):256–263. doi: 10.1016/0005-2736(78)90025-1. [DOI] [PubMed] [Google Scholar]
  20. Pscheid P., Schudt C., Plattner H. Cryofixation of monolayer cell cultures for freeze-fracturing without chemical pre-treatments. J Microsc. 1981 Feb;121(Pt 2):149–167. doi: 10.1111/j.1365-2818.1981.tb01208.x. [DOI] [PubMed] [Google Scholar]
  21. Saffitz J. E., Gross R. W., Williamson J. R., Sobel B. E. Autoradiography of phosphatidyl choline. J Histochem Cytochem. 1981 Mar;29(3):371–378. doi: 10.1177/29.3.7240719. [DOI] [PubMed] [Google Scholar]
  22. Siegel D. P. Inverted micellar intermediates and the transitions between lamellar, cubic, and inverted hexagonal lipid phases. I. Mechanism of the L alpha----HII phase transitions. Biophys J. 1986 Jun;49(6):1155–1170. doi: 10.1016/S0006-3495(86)83744-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Siegel D. P. Inverted micellar intermediates and the transitions between lamellar, cubic, and inverted hexagonal lipid phases. II. Implications for membrane-membrane interactions and membrane fusion. Biophys J. 1986 Jun;49(6):1171–1183. doi: 10.1016/S0006-3495(86)83745-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Stein O., Stein Y. Light and electron microscopic radioautography of lipids: techniques and biological applications. Adv Lipid Res. 1971;9:1–72. doi: 10.1016/b978-0-12-024909-1.50008-9. [DOI] [PubMed] [Google Scholar]
  25. Verkleij A. J. Lipidic intramembranous particles. Biochim Biophys Acta. 1984 Jan 27;779(1):43–63. doi: 10.1016/0304-4157(84)90003-0. [DOI] [PubMed] [Google Scholar]
  26. Weibull C., Villiger W., Carlemalm E. Extraction of lipids during freeze-substitution of Acholeplasma laidlawii-cells for electron microscopy. J Microsc. 1984 May;134(Pt 2):213–216. doi: 10.1111/j.1365-2818.1984.tb02513.x. [DOI] [PubMed] [Google Scholar]
  27. White J., Kielian M., Helenius A. Membrane fusion proteins of enveloped animal viruses. Q Rev Biophys. 1983 May;16(2):151–195. doi: 10.1017/s0033583500005072. [DOI] [PubMed] [Google Scholar]
  28. Wiley D. C., Skehel J. J. The structure and function of the hemagglutinin membrane glycoprotein of influenza virus. Annu Rev Biochem. 1987;56:365–394. doi: 10.1146/annurev.bi.56.070187.002053. [DOI] [PubMed] [Google Scholar]
  29. da Silva P. P., Kachar B. Quick freezing vs. chemical fixation: capture and identification of membrane fusion intermediates. Cell Biol Int Rep. 1980 Jul;4(7):625–640. doi: 10.1016/0309-1651(80)90201-5. [DOI] [PubMed] [Google Scholar]
  30. van Meer G., Davoust J., Simons K. Parameters affecting low-pH-mediated fusion of liposomes with the plasma membrane of cells infected with influenza virus. Biochemistry. 1985 Jul 2;24(14):3593–3602. doi: 10.1021/bi00335a030. [DOI] [PubMed] [Google Scholar]
  31. van Meer G., Gumbiner B., Simons K. The tight junction does not allow lipid molecules to diffuse from one epithelial cell to the next. Nature. 1986 Aug 14;322(6080):639–641. doi: 10.1038/322639a0. [DOI] [PubMed] [Google Scholar]
  32. van Meer G., Simons K. An efficient method for introducing defined lipids into the plasma membrane of mammalian cells. J Cell Biol. 1983 Nov;97(5 Pt 1):1365–1374. doi: 10.1083/jcb.97.5.1365. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. van Meer G., Simons K. The function of tight junctions in maintaining differences in lipid composition between the apical and the basolateral cell surface domains of MDCK cells. EMBO J. 1986 Jul;5(7):1455–1464. doi: 10.1002/j.1460-2075.1986.tb04382.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. von Bonsdorff C. H., Fuller S. D., Simons K. Apical and basolateral endocytosis in Madin-Darby canine kidney (MDCK) cells grown on nitrocellulose filters. EMBO J. 1985 Nov;4(11):2781–2792. doi: 10.1002/j.1460-2075.1985.tb04004.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES