Advertisement

Protoplasma

, Volume 160, Issue 2–3, pp 159–166 | Cite as

Colocalization of luciferin binding protein and luciferase to the scintillons ofGonyaulax polyedra revealed by double immunolabeling after fast-freeze fixation

  • Marie -Thérèse Nicolas
  • D. Morse
  • Jean -Marie Bassot
  • J. Woodland Hastings
Original Papers

Summary

Two proteins,Gonyaulax luciferase and the luciferin binding protein, are involved in the bioluminescent reaction of the unicellular marine algaGonyaulax polyedra. Using antibodies raised separately against the purified proteins, their ultrastructural localizations were visualized by double immunogold labeling on sections after fast-freeze fixation, freeze-substitution and embedding in Epon or in LR White. Gold particles of two sizes attached to the secondary antibodies allowed the two primary antibodies to be distinguished. The two colocalized to cytoplasmic densifications (scintillons), which occurred in close association with the vacuolar membrane near the periphery of the cell. They also occurred in the cytoplasm of the Golgi area, either over densifications without associated membranes (prescintillons), or as very small colocalizations not associated with any evident cytoplasmic differentiation. No other site of colocalization was observed, thus unambiguously establishing the ultrastructural identity of the bioluminescent organelles.

Keywords

Bioluminescence Dinoflagellates Fast-freeze fixation Gonyaulax Immunogold labeling 

Abbreviations

FFF

fast-freeze fixation

FS

freeze-substitution

IGS

immunogold staining

LBP

luciferin binding protein

PBS

phosphate buffered saline

TBS

tris-buffered saline

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Afzelius BA, Halvarson M (1964) The fine structure of the photogenic granules in Dinoflagellates. In: Third European Regional Conference on Electron Microscopy. Czechoslovak Academy of Sciences, Prague, pp 175–176Google Scholar
  2. De Mey J (1983) Colloidal gold probes in immunocytochemistry. In: Polak JM, Van Noorden S (eds) Immunocytochemistry. Wright-PSG, Bristol, pp 82–112Google Scholar
  3. De Sa R, Hastings JW, Vatter AE (1963) Luminescent “crystalline” particles: an organized subcellular bioluminescent system. Science 41: 1269–1270Google Scholar
  4. Dunlap JC, Hastings JW (1981) The biological clock inGonyaulax controls luciferase acitivity by regulating turnover. J Biol Chem 256: 10509–10518PubMedGoogle Scholar
  5. Eckert R (1966) Subcellular sources of luminescence inNoctiluca. Science 151: 349–352PubMedGoogle Scholar
  6. —, Sibaoka T (1968) The flash triggering action potential of the luminescent dinoflagellateNoctiluca. J Gen Physiol 52: 258–282PubMedGoogle Scholar
  7. Escaig J (1982) New instruments which facilitate rapid freezing at 83 K and 6 K. J Microsc 126: 221–229Google Scholar
  8. Fogel M, Hastings JW (1971) A substrate binding protein in theGonyaulax bioluminescence reaction. Arch Biochem Biophys 142: 310–321PubMedGoogle Scholar
  9. — —, (1972) Bioluminescence: mechanism and mode of action of scintillon activity. Proc Natl Acad Sci USA 69: 690–693PubMedGoogle Scholar
  10. —, Schmitter R, Hastings JW (1972) On the physical identity of scintillons: bioluminescent particles inGonyaulax polyedra. J Cell Sci 11: 305–317PubMedGoogle Scholar
  11. Gautier A, Fakan J (1980) Les trichocystes d'un Dinoflagellé continendraient-ils de 1'ADN? Un défi à la cytochimie ultrastructurale. Biol Cell 39: 257–260Google Scholar
  12. Guillard RRL, Ryther JH (1962) Studies on marine planktonic diatoms. I.Cyclotella nana Husted andDetonula conf. vacae(Clsve.). Can J Microbiol 8: 229–239PubMedGoogle Scholar
  13. Hastings JW (1986) Bioluminescence in bacteria and dinoflagellates. In: Govindjee, Amsez J, Fork DC (eds) Light emission by plants. Academic Press, New York, pp 363–398Google Scholar
  14. —, Dunlap JC (1986) Cell free components in dinoflagellate bioluminescence. The particulate activity: scintillons; the soluble components: luciferase, luciferin binding protein. Methods Enzymol 133: 307–327Google Scholar
  15. Hausmann K, Fok AK, Allen R (1988) Immunocytochemical analysis of trichocyst structure and development inParamecium. J Ultrastruct Mol Struct Res 99: 213–225Google Scholar
  16. Ichikawa M, Sasaki K, Ichikawa A (1989) Immunocytochemical localization of amylase in gerbil salivary gland acinar cells processed by rapid freezing and freeze substitution fixation. J Histochem Cytochem 37: 185–194PubMedGoogle Scholar
  17. Johnson CH, Hastings JW (1986) The elusive mechanism of the circadian clock. Amer Sci 74: 29–36Google Scholar
  18. —, Inoue S, Flint A, Hastings JW (1985) Compartmentalization of algal bioluminescence: autofluorescence of bioluminescent particles in the dinoflagellateGonyaulax as studied with imageintensified video microscopy and flow cytometry. J Cell Biol 100: 1435–1446PubMedGoogle Scholar
  19. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685PubMedGoogle Scholar
  20. Morse D, Pappenheimer AM, Hastings JW (1989 a) Role of luciferin binding protein in the circadian bioluminescent reaction ofGonyaulax polyedra. J Biol Chem 264: 11822–11826PubMedGoogle Scholar
  21. —, Milos PM, Roux E, Hastings JW (1989 b) Circadian regulation of bioluminescence involves translational control. Proc Natl Acad Sci USA 86: 172–176PubMedGoogle Scholar
  22. Nakamura H, Kishi Y, Shimomura O, Morse D, Hastings JW (1989) Structure of dinoflagellate luciferin and its enzymatic and nonenzymatic air-oxidation products. J Am Chem Soc 111: 7607–7611Google Scholar
  23. Nawata T, Siboaka T (1979) Coupling between action potential and bioluminescence inNoctiluca: effects of inorganic ions and pH in vacuolar sap. J Comp Physiol 134: 137–149Google Scholar
  24. Newman GR, Jasani B, Williams ED (1982) The preservation of ultrastructure and antigenicity. J Microsc 127: 5–6Google Scholar
  25. Nicolas M-T, Johnson CH, Bassot J-M, Hastings JW (1985) Immunogold labeling of organelles in the bioluminescent dinoflagellateGonyaulax polyedra with anti-luciferase antibody. Cell Biol Int Rep 9: 797–802PubMedGoogle Scholar
  26. —, Nicolas G, Johnson CH, Bassot J-M, Hastings JW(1987 a) Characterization of the bioluminescent organelles inGonyaulax polyedra (dinoflagellates) after fast-freeze fixation and anti-luciferase immunogold staining. J Cell Biol 105: 723–735PubMedGoogle Scholar
  27. —, Sweeney BM, Hastings JW (1987 b) The ultrastructural localization of luciferase in three bioluminescent dinoflagellates, two species ofPyrocystis, andNoctiluca, using antiluciferase and immunogold labeling. J Cell Sci 87: 187–196Google Scholar
  28. —, Bassot J-M, Nicolas G (1989) Immunogold labeling of luciferase in the luminous bacteriumVibrio harveyiafter fast freeze fixation and different freeze-substitution and embedding procedures. J Histochem Cytochem 37: 663–674Google Scholar
  29. Olmstead JB (1981) Affinity purification of antibodies from diazotized paper blots of heterogenous protein samples. J Biol Chem 256: 11955–11957PubMedGoogle Scholar
  30. Towbin H, Stahelin T, Gordon J (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76: 4350–4354PubMedGoogle Scholar

Copyright information

© Springer-Verlag 1991

Authors and Affiliations

  • Marie -Thérèse Nicolas
    • 1
    • 2
  • D. Morse
    • 1
  • Jean -Marie Bassot
    • 2
  • J. Woodland Hastings
    • 1
  1. 1.Department of Cellular and Developmental BiologyHarvard UniversityCambridgeUSA
  2. 2.Laboratoire de BioluminescenceCNRSParis

Personalised recommendations