Springer Nature is making SARS-CoV-2 and COVID-19 research free. View research | View latest news | Sign up for updates

Fluorescence lifetimes of diphenylhexatriene-containing probes reflect local probe concentrations: Application to the measurement of membrane fusion

  • 67 Accesses

  • 3 Citations

Abstract

An important process in the life of a cell is fusion between cellular membranes. This is the process by which two cellular compartments surrounded by different membranes join to become a single compartment surrounded by a single membrane, without significant loss of compartment contents. To demonstrate fusion, the cell biophysicist must demonstrate all three critical aspects of the process: (1) mixing of membrane components, (2) mixing of compartment contents; and (3) retention of compartment contents. Most commonly, accomplishing this involves the use of fluorescence probes. The general theme to the methods described involves some form of concentration-dependent quenching. An unique method developed in our laboratory utilizes the concentration dependence of the fluorescence lifetime of a phosphatidylcholine containing carboxyethyl diphenylhexatriene at position 2 and palmitic acid at position 1 of glycerol (DPHpPC). The fluorescence lifetime of this molecule and that of its parent fluorophore diphenylhexatriene (DPH) shorten dramatically as their two-dimensional concentrations in a membrane increase. This “lifetime quenching” can be described by dimer formation that reduces the symmetry of the DPH excited state. This phenomenon allows one to use the fluorescence lifetime to gain insight into the local concentration of probe in microscopic regions of a membrane. One application of this is in distinguishing lipid transfer between the outer leaflets of two contacting membrane bilayers from fusion between these membranes that leads to mixing of lipids in both the inner and outer leaflets of the membrane bilayers. This allows a single measurement to demonstrate fusion between membrane pairs.

This is a preview of subscription content, log in to check access.

Abbreviations

PEG:

poly(ethylene glycol)

Na2EDTA:

ethyiene-diamine-tetraacedic acid, disodium salt

LUV:

large, unilamellar vesicles made by rapid extrusion technique

DPH:

1,6-diphenyl-trans-1,3,5-hexatriene

DPHpPC:

1-palmitoyl-2-[[[2-[4- (phenyl-trans-1,3,5-hexatrienyl)phenyl]ethyl]oxy]carbonyl]-3-sn-phosphatidylcholine

DPPC:

1,2-dipalmitoyl-3-sn-phosphatidylcholine

PA:

palmitic acid

NBD-PE:

N-(7-nitro-2,1,3-benzoxadiazol-4-yl)-PE

Rh-PE:

N-(lissamine Rhodamine B sulfoyl)-PE

R18 :

octadecyl Rhodamine B chloride

ANTS:

1-aminonaphthalene-3,6,8-trisulfonic acid

DPX:

N,N′-p-xylylene-bis(pyradinium bromide)

References

  1. 1.

    S. W. Burgess, T. J. McIntosh, and B. R. Lentz (1992)Biochemistry 31, 2653–2661.

  2. 2.

    J. R. Monck and J. M. Fernandez (1992)J. Cell Biol. 119, 1395–1404.

  3. 3.

    M. M. Kozlov, S. L. Leikin, L. V. Chernomordik, V. S. Markin, and Y. A. Chizmedzhev (1989)Eur. Biophys. J. 17, 121–129.

  4. 4.

    J. Zimmerberg, S. S. Vogel, and L. V. Chernomordik (1993)Annu. Rev. Biomol. Struct. 22, 433–466.

  5. 5.

    A. E. Spruce, A. Iwata, and W. Almers (1991)Proc. Natl. Acad. Sci. USA 88, 3623–3627.

  6. 6.

    C. Nanavati, V. S. Markin, A. F. Oberhauser, and J. M. Fernandez (1992)Biophys. J. 63, 1118–1132.

  7. 7.

    N. Düzgüneş and J. Bentz (1988) in L. M. Lowe (Ed.),Spectroscopic Membrane Probes, CRC Press, Boca Raton, Florida, Chapter 6.

  8. 8.

    D. K. Struck, D. Hoekstra, and R. E. Pagano (1981)Biochemistry 20, 4093–4099.

  9. 9.

    J. C. McIntyre and R. G. Sleight (1991)Biochemistry 30, 11819–11827.

  10. 10.

    D. Hoekstra, T. de Boer, K. Klappe, and J. Wilshuit (1984)Biochemistry 23, 5675–5681.

  11. 11.

    R. I. MacDonald (1990)J. Biol. Chem. 265, 13533–13539.

  12. 12.

    H. Ellens, J. Bentz, and F. C. Szoka (1985)Biochemistry 24, 3099–3106.

  13. 13.

    R. A. Parente and B. R. Lentz (1985)Biochemistry 24, 6178–6185.

  14. 14.

    D. A. Barrow and B. R. Lentz (1985)Biophys. J. 48, 221–234.

  15. 15.

    R. A. Parente and B. R. Lentz (1986)Biochemistry 25, 1021–1026.

  16. 16.

    S. W. Burgess and B. R. Lentz (1993)Meth. Enzymol. 220, 42–50.

  17. 17.

    E. D. Cehelnik, R. B. Cundall, J. R. Lockwood, and T. F. Palmer (1975)J. Phys. Chem. 79, 1369–1376.

  18. 18.

    B. E. Kohler and T. Itoh (1988)J. Chem. Phys. 92, 5120–5122.

  19. 19.

    D. Papahadjopoulos (1978) G. Poste and G. L. Nicolson (Eds.),Membrane Fusion Elsevier/North-Holland Biomedical Press, Amsterdam, pp. 765–790.

  20. 20.

    J. R. Wu and B. R. Lentz (1991)Biochemistry 30, 6780–6787.

  21. 21.

    J. R. Wu and B. R. Lentz (1994)J. Fluorescence 4, 153–163.

  22. 22.

    S. Nir, J. Bentz, J. Wilschut, and N. Düzgüneş. (1983)Prog. Surf. Sci. 13, 1–124.

  23. 23.

    J. Bentz, S. Nir, and J. Wilschut (1983)Colloids Surfaces 6, 333–363.

  24. 24.

    B. R. Lentz and S. W. Burgess (1989)Biophys. J. 56, 723–733.

  25. 25.

    B. R. Lentz, G. F. McIntyre, D. J. Parks, J. C. Yates, and D. Massenburg (1992)Biochemistry 31, 2643–2652.

  26. 26.

    D. M. Massenburg and B. R. Lentz (1993)Biochemistry 32, 9172–9180.

Download references

Author information

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Lentz, B.R. Fluorescence lifetimes of diphenylhexatriene-containing probes reflect local probe concentrations: Application to the measurement of membrane fusion. J Fluoresc 5, 29–38 (1995). https://doi.org/10.1007/BF00718780

Download citation

Key words

  • fluorescence
  • DPH
  • fusion
  • poly(ethylene glycol)