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Selective Visualization of Fluorescent Sterols in Caenorhabditis elegans

D. Wüstner, A.L. Larsen, J.R. Brewer, D. Sage, N.J. Færgeman

Proceedings of the Forty-Ninth International Conference on the Bioscience of Lipids (ICBL'08), Maastricht, Kingdom of the Netherlands, August 26-30, 2008, Chemistry and Physics of Lipids, vol. 154, supp. 1, pp. S42, August 2008.



Background: The nematode Caenorhabditis elegans (C. elegans) is an excellent genetically tractable model organism to investigate sterol physiology on the organism level. Fluorescence imaging of dehydroergosterol (DHE), a close cholesterol analog, has been recently introduced to characterize sterol transport in C. elegans.

Objectives: Detailed characterization of DHE distribution was hampered by C. elegans' high autofluorescence in the same spectral region as emission of DHE. We developed a new method to overcome this problem.

Methods: Using the rapid bleaching kinetics of DHE compared to cellular autofluorescence we were able to selectively detect DHE by wide field fluorescence bleach rate microscopy. Bleach kinetics of DHE were fitted on a pixel-basis to mathematical models describing the intensity decay. The developed method is compared with three-photon imaging of DHE in C. elegans and complemented with RNA interference experiments to knock-down target genes involved in sterol transport.

Results: Using this method we found enrichment of DHE in the intestine, neurons and the reproductive organs of C. elegans. We demonstrate compartmentalization of sterol in the oocytes and in intestinal cells and visualize sterol in gut granule loss (glo) mutant worms.

Conclusions: We developed a method to selectively visualize sterol distribution in vivo using fluorescence microscopy allowing for detailed investigation of sterol transport and metabolism on an organism level.


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