Gangliosides are glycosphingolipids mainly present at the plasma membrane (PM). After synthesis in the lumen of the Golgi apparatus, gangliosides leave the organelle via transport vesicles destined to PM. In this study, we analyzed the synthesis and membrane distribution of GD3 and GM1 gangliosides endogenously synthesized both in polarized and non-polarized Madin-Darby canine kidney (MDCK) cell lines stably expressing ganglioside glycosyltransferases. By using biochemical techniques and confocal microscopy we demonstrated that GD3 and GM1, after being synthesized at the Golgi apparatus, are sorted and accumulated mainly at the PM of non-polarized MDCK cells. Interestingly, both complex gangliosides were found enriched mainly at the apical domain when these cell lines were induced to polarize. We demonstrated that after arrival to PM, GD3 and GM1 are endocytosed by using a clathrin-independent pathway. Then, internalized GD3 is accumulated in endosomal compartments and sorted back to the PM. On the other hand, endocytosed GM1, in association with cholera toxin, is transported to endosomal compartments en route to the Golgi apparatus. The genetically modified MDCK cell lines should provide an excellent model system for studying synthesis, trafficking and polarity of glycolipids in epithelial cells as well as the biological significance of the polarized distribution of these lipids.
