Variable subcellular localization of glycosphingolipids

Although most glycosphingolipids (GSLs) are thought to be located in the outer leaflet of the plasma membrane, recent evidence indicates that GSLs are also associated with intracellular organelles. We now report that the subcellular localization of GSLs varies depending on the GSL structure and cell type. GSL localization was determined by indirect immunofluorescence microscopy of fixed permeabilized cells. A single GSL exhibited variable subcellular localization in different cells. For example, antibody to GalCer is localized primarily to the plasma membrane of HaCaT II-3 keratinocytes, but to intracellular organelles in other epithelial cells. GalCer is localized to small vesicles and tubulovesicular structures in MDCK cells, and to the surface of phase-dense lipid droplets in HepG2 hepatoma cells. Furthermore, within a single cell type, individual GSLs were found to exhibit different patterns of subcellular localization. In HepG2 cells, LacCer was associated with small vesicles, which differed from the phase-dense vesicles stained by anti-GalCer, and Gb4Cer was associated with the intermediate filaments of the cytoskeleton. Both anti-GalCer and monoclonal antibody A2B5, which binds polysialogangliosides, localized to mitochondria. The distinct subcellular localization patterns of GSLs raise interesting questions about their functions in different organelles. Together with published data on the enrichment of GSLs in specific organelles and in apical plasma membrane, these findings indicate the existence of specific sorting mechanisms that regulate the intracellular transport and localization of GSLs.