Access

You are not currently logged in.

Access your personal account or get JSTOR access through your library or other institution:

login

Log in to your personal account or through your institution.

If You Use a Screen Reader

This content is available through Read Online (Free) program, which relies on page scans. Since scans are not currently available to screen readers, please contact JSTOR User Support for access. We'll provide a PDF copy for your screen reader.

Sphingolipid Metabolism in Cultured Fibroblasts: Microscopic and Biochemical Studies Employing a Fluorescent Ceramide Analogue

Naomi G. Lipsky and Richard E. Pagano
Proceedings of the National Academy of Sciences of the United States of America
Vol. 80, No. 9, [Part 1: Biological Sciences] (May 1, 1983), pp. 2608-2612
Stable URL: http://www.jstor.org/stable/13949
Page Count: 5
  • Read Online (Free)
  • Subscribe ($19.50)
  • Cite this Item
Since scans are not currently available to screen readers, please contact JSTOR User Support for access. We'll provide a PDF copy for your screen reader.
Sphingolipid Metabolism in Cultured Fibroblasts: Microscopic and Biochemical Studies Employing a Fluorescent Ceramide Analogue
Preview not available

Abstract

A fluorescent analogue of ceramide, N-[7-(4-nitrobenzo-2-oxa-1,3-diazole)]-ε -aminocaproyl sphingosine (C6-NBD-ceramide), was used to investigate sphingolipid metabolism in Chinese hamster fibroblasts. C6-NBD-ceramide was incorporated into small unilamellar dioleoyl phosphatidylcholine vesicles and incubated with cells in monolayer culture at 2 degrees C, resulting in rapid and preferential transfer of the labeled ceramide from vesicles to cells. The cells were then washed and subsequently incubated at 37 degrees C for various intervals. The metabolism of C6-NBD-ceramide was monitored by lipid extraction and analysis, and the intracellular distribution of the labeled molecule was followed by fluorescence microscopy. Intially, fluorescence was detected almost exclusively in mitochondria, with over 90% of the extractable lipid fluorescence due to C6-NBD-ceramide. After 30 min at 37 degrees C, intense fluorescence appeared in the Golgi apparatus. This organelle was identified by colocalization of NBD fluorescence with a Golgi-apparatus-specific stain. At later times the plasma membrane became visibly labeled as well, at which point 90% of the cell-associated fluorescence was recovered as NBD-labeled sphingomyelin and NBD-labeled cerebroside. These metabolites were identified by enzymatic and biochemical analysis and by thin-layer chromatography of the fluorescent lipid extracts. The finding that C6-NBD-ceramide is used by these cells in standard pathways of sphingolipid biosynthesis suggests that this fluorescent precursor will be a valuable tool for correlating the metabolism of sphingolipids with their intracellular distribution and translocation. In addition, during its metabolism by Chinese hamster fibroblasts, this compound acts as a vital stain for the Golgi apparatus.

Page Thumbnails

  • Thumbnail: Page 
2608
    2608
  • Thumbnail: Page 
2609
    2609
  • Thumbnail: Page 
2610
    2610
  • Thumbnail: Page 
2611
    2611
  • Thumbnail: Page 
2612
    2612