Sorting of sphingolipids in epithelial (Madin-Darby canine kidney) cells.

• DOI: 10.1083/jcb.105.4.1623
• Publication Date: 2004-05-15T00:18:20Z
• Authors: G van Meer; E H Stelzer; R W Wijnaendts-va...; K Simons
• Keywords: Sphingolipid

To study the intracellular transport of newly synthesized sphingolipids in epithelial cells we have used a fluorescent ceramide analog, N-6[7-nitro-2,1,3-benzoxadiazol-4-yl] aminocaproyl sphingosine (C6-NBD-ceramide; Lipsky, N. G., and R. E. Pagano, 1983, Proc. Natl. Acad. Sci. USA, 80:2608-2612) as probe. This was readily taken up by filter-grown Madin-Darby canine kidney (MDCK) from liposomes at 0 degrees C. After penetration into cell, probe accumulated Golgi area temperatures between 20 Chemical analysis showed that C6-NBD-ceramide being converted C6-NBD-sphingomyelin C6-NBD-glucosyl-ceramide. An fluorescence pattern after 1 h C means confocal scanning laser microscope revealed marker most likely concentrated complex itself. Little observed plasma membrane. Raising temperature to 37 for resulted intense membrane staining loss complex. Addition BSA apical medium cleared but not basolateral domain. The could be depleted only adding basal side monolayer MDCK grown on polycarbonate filters. We conclude sphingomyelin glucosylceramide were delivered where they external leaflet bilayer. results also demonstrated fatty acyl labeled lipids unable pass tight junctions either direction. Quantitation amount NBD-lipids membranes during incubation C6-NBD-glucosylceramide two- fourfold enriched compared side, while about equally distributed. Since surface is much smaller than membrane, both achieved higher concentration surface. Altogether, our suggest are sorted way similar their natural counterparts.