New approaches to the study of sphingolipid enriched membrane domains: Theuse of electron microscopic autoradiography to reveal metabolically tritium labeled sphingolipids in cell cultures

This paper is the first report on the use of the electron microscopy autora diography technique to detect metabolically tritium labeled sphingolipids i n intact cells in culture. To label cell sphingolipids, human fibroblasts i n culture were fed by a 24 hours pulse, repeated 5 times, of 3 x 10(-7) M [ 1-H-3]Sphingosine. [1-H-3]sphingosine was efficently taken up by the cells and very rapidly used for the biosynthesis of complex sphingolipids, includ ing neutral glycolipids, gangliosides, ceramide and sphingomyelin. The trea tment with [1-H-3]sphingosine did not induce any morphological alteration o f cell structures, and well preserved cells, plasma membranes, and intracel lular organelles could be observed by microscopy. Ultrathin sections from m etabolic radiolabeled cells were coated with autoradiographic emulsion. One to four weeks of exposition resulted in pictures where the location of rad ioactive sphingolipids was evidenced by the characteristic appearance of si lver grains as irregular coiled ribbons of metallic silver. Radioactive sph ingolipids were found at the level of the plasma membranes, on the endoplas mic reticulum and inside of cytoplasmic vesicles. Thus, electron microscopy autoradiography is a very useful technique to study sphingolipid-enriched membrane domain organization and biosynthesis.