Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1989 Nov 1;109(5):2067–2079. doi: 10.1083/jcb.109.5.2067

Molecular trapping of a fluorescent ceramide analogue at the Golgi apparatus of fixed cells: interaction with endogenous lipids provides a trans-Golgi marker for both light and electron microscopy

PMCID: PMC2115850  PMID: 2478562

Abstract

We have previously shown that a fluorescent derivative of ceramide, N- (epsilon-7-nitrobenz-2-oxa-1,3-diazol-4-yl-aminocaproyl)-D-eryth ro- sphingosin e (C6-NBD-Cer), vitally stains the Golgi apparatus of cells (Lipsky, N. G., and R. E. Pagano. 1985. Science (Wash. DC). 228:745- 747). In the present paper we demonstrate that C6-NBD-Cer also accumulates at the Golgi apparatus of fixed cells and we explore the mechanism by which this occurs. When human skin fibroblasts were fixed with glutaraldehyde and then incubated with C6-NBD-Cer at 2 degrees C, the fluorescent lipid spontaneously transferred into the cells, labeling the Golgi apparatus as well as other intracellular membranes. Subsequent incubations with defatted BSA at 24 degrees C removed excess C6-NBD-Cer from the cells such that fluorescence was then detected only at the Golgi apparatus. Similar results were obtained using other cell types. A method for visualizing the fluorescent lipid at the electron microscopic level, based on the photoconversion of a fluorescent marker to a diaminobenzidine product (Sandell, J. H., and R. H. Masland, 1988. J. Histochem. Cytochem. 36:555-559), is described and evidence is presented that C6-NBD-Cer was localized to the trans cisternae of the Golgi apparatus. While accumulation occurred in cells fixed in various ways, it was inhibited when fixation protocols that extract or modify cellular lipids were used. In addition, Filipin, which forms complexes with cellular cholesterol, labeled the Golgi apparatus of fixed cells and inhibited accumulation of C6-NBD-Cer at the Golgi apparatus. These results are discussed in terms of a simple model based on the physical properties of C6-NBD-Cer and its interactions with endogenous lipids of the Golgi apparatus. Possible implications of these findings for metabolism and transport of (fluorescent) sphingolipids in vivo are also presented.

Full Text

The Full Text of this article is available as a PDF (6.0 MB).

Selected References

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

  1. BLIGH E. G., DYER W. J. A rapid method of total lipid extraction and purification. Can J Biochem Physiol. 1959 Aug;37(8):911–917. doi: 10.1139/o59-099. [DOI] [PubMed] [Google Scholar]
  2. Blanchette-Mackie E. J., Dwyer N. K., Amende L. M., Kruth H. S., Butler J. D., Sokol J., Comly M. E., Vanier M. T., August J. T., Brady R. O. Type-C Niemann-Pick disease: low density lipoprotein uptake is associated with premature cholesterol accumulation in the Golgi complex and excessive cholesterol storage in lysosomes. Proc Natl Acad Sci U S A. 1988 Nov;85(21):8022–8026. doi: 10.1073/pnas.85.21.8022. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Burgess T. L., Kelly R. B. Constitutive and regulated secretion of proteins. Annu Rev Cell Biol. 1987;3:243–293. doi: 10.1146/annurev.cb.03.110187.001331. [DOI] [PubMed] [Google Scholar]
  4. Griffiths G., Quinn P., Warren G. Dissection of the Golgi complex. I. Monensin inhibits the transport of viral membrane proteins from medial to trans Golgi cisternae in baby hamster kidney cells infected with Semliki Forest virus. J Cell Biol. 1983 Mar;96(3):835–850. doi: 10.1083/jcb.96.3.835. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Jamieson J. D., Palade G. E. Intracellular transport of secretory proteins in the pancreatic exocrine cell. IV. Metabolic requirements. J Cell Biol. 1968 Dec;39(3):589–603. doi: 10.1083/jcb.39.3.589. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Kobayashi T., Pagano R. E. Lipid transport during mitosis. Alternative pathways for delivery of newly synthesized lipids to the cell surface. J Biol Chem. 1989 Apr 5;264(10):5966–5973. [PubMed] [Google Scholar]
  7. Kruse N. J., Bornstein P. The metabolic requirements for transcellular movement and secretion of collagen. J Biol Chem. 1975 Jul 10;250(13):4841–4847. [PubMed] [Google Scholar]
  8. Lipsky N. G., Pagano R. E. A vital stain for the Golgi apparatus. Science. 1985 May 10;228(4700):745–747. doi: 10.1126/science.2581316. [DOI] [PubMed] [Google Scholar]
  9. Lipsky N. G., Pagano R. E. Intracellular translocation of fluorescent sphingolipids in cultured fibroblasts: endogenously synthesized sphingomyelin and glucocerebroside analogues pass through the Golgi apparatus en route to the plasma membrane. J Cell Biol. 1985 Jan;100(1):27–34. doi: 10.1083/jcb.100.1.27. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Lipsky N. G., Pagano R. E. Sphingolipid metabolism in cultured fibroblasts: microscopic and biochemical studies employing a fluorescent ceramide analogue. Proc Natl Acad Sci U S A. 1983 May;80(9):2608–2612. doi: 10.1073/pnas.80.9.2608. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Martin O. C., Pagano R. E. Transbilayer movement of fluorescent analogs of phosphatidylserine and phosphatidylethanolamine at the plasma membrane of cultured cells. Evidence for a protein-mediated and ATP-dependent process(es). J Biol Chem. 1987 Apr 25;262(12):5890–5898. [PubMed] [Google Scholar]
  12. Miller R. G. The use and abuse of filipin to localize cholesterol in membranes. Cell Biol Int Rep. 1984 Jul;8(7):519–535. doi: 10.1016/0309-1651(84)90050-x. [DOI] [PubMed] [Google Scholar]
  13. NOVIKOFF A. B., GOLDFISCHER S. Nucleosidediphosphatase activity in the Golgi apparatus and its usefulness for cytological studies. Proc Natl Acad Sci U S A. 1961 Jun 15;47:802–810. doi: 10.1073/pnas.47.6.802. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Nichols J. W., Pagano R. E. Kinetics of soluble lipid monomer diffusion between vesicles. Biochemistry. 1981 May 12;20(10):2783–2789. doi: 10.1021/bi00513a012. [DOI] [PubMed] [Google Scholar]
  15. Nichols J. W., Pagano R. E. Use of resonance energy transfer to study the kinetics of amphiphile transfer between vesicles. Biochemistry. 1982 Apr 13;21(8):1720–1726. doi: 10.1021/bi00537a003. [DOI] [PubMed] [Google Scholar]
  16. Novikoff A. B. The endoplasmic reticulum: a cytochemist's view (a review). Proc Natl Acad Sci U S A. 1976 Aug;73(8):2781–2787. doi: 10.1073/pnas.73.8.2781. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Orci L., Montesano R., Meda P., Malaisse-Lagae F., Brown D., Perrelet A., Vassalli P. Heterogeneous distribution of filipin--cholesterol complexes across the cisternae of the Golgi apparatus. Proc Natl Acad Sci U S A. 1981 Jan;78(1):293–297. doi: 10.1073/pnas.78.1.293. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Pagano R. E., Longmuir K. J. Phosphorylation, transbilayer movement, and facilitated intracellular transport of diacylglycerol are involved in the uptake of a fluorescent analog of phosphatidic acid by cultured fibroblasts. J Biol Chem. 1985 Feb 10;260(3):1909–1916. [PubMed] [Google Scholar]
  19. Pagano R. E., Martin O. C. A series of fluorescent N-acylsphingosines: synthesis, physical properties, and studies in cultured cells. Biochemistry. 1988 Jun 14;27(12):4439–4445. doi: 10.1021/bi00412a034. [DOI] [PubMed] [Google Scholar]
  20. Pagano R. E., Sleight R. G. Defining lipid transport pathways in animal cells. Science. 1985 Sep 13;229(4718):1051–1057. doi: 10.1126/science.4035344. [DOI] [PubMed] [Google Scholar]
  21. Pagano R. E. What is the fate of diacylglycerol produced at the Golgi apparatus? Trends Biochem Sci. 1988 Jun;13(6):202–205. doi: 10.1016/0968-0004(88)90082-5. [DOI] [PubMed] [Google Scholar]
  22. Quinn P., Griffiths G., Warren G. Dissection of the Golgi complex. II. Density separation of specific Golgi functions in virally infected cells treated with monensin. J Cell Biol. 1983 Mar;96(3):851–856. doi: 10.1083/jcb.96.3.851. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Sandell J. H., Masland R. H. Photoconversion of some fluorescent markers to a diaminobenzidine product. J Histochem Cytochem. 1988 May;36(5):555–559. doi: 10.1177/36.5.3356898. [DOI] [PubMed] [Google Scholar]
  24. Simons K., van Meer G. Lipid sorting in epithelial cells. Biochemistry. 1988 Aug 23;27(17):6197–6202. doi: 10.1021/bi00417a001. [DOI] [PubMed] [Google Scholar]
  25. Tartakoff A. M., Vassalli P. Plasma cell immunoglobulin secretion: arrest is accompanied by alterations of the golgi complex. J Exp Med. 1977 Nov 1;146(5):1332–1345. doi: 10.1084/jem.146.5.1332. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Tartakoff A., Vassalli P. Plasma cell immunoglobulin M molecules. Their biosynthesis, assembly, and intracellular transport. J Cell Biol. 1979 Nov;83(2 Pt 1):284–299. doi: 10.1083/jcb.83.2.284. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Weber K., Rathke P. C., Osborn M. Cytoplasmic microtubular images in glutaraldehyde-fixed tissue culture cells by electron microscopy and by immunofluorescence microscopy. Proc Natl Acad Sci U S A. 1978 Apr;75(4):1820–1824. doi: 10.1073/pnas.75.4.1820. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Yeagle P. L. Cholesterol and the cell membrane. Biochim Biophys Acta. 1985 Dec 9;822(3-4):267–287. doi: 10.1016/0304-4157(85)90011-5. [DOI] [PubMed] [Google Scholar]
  29. van Meer G., Stelzer E. H., Wijnaendts-van-Resandt R. W., Simons K. Sorting of sphingolipids in epithelial (Madin-Darby canine kidney) cells. J Cell Biol. 1987 Oct;105(4):1623–1635. doi: 10.1083/jcb.105.4.1623. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES