CONICET | Buscador de Institutos y Recursos Humanos

Gangliosides are glycosphingolipids mainly present at the outer leaflet of the plasma membrane of eukaryotic cells where they participate in recognition and signalling activities. The synthesis of gangliosides is carried out in the lumen of the Golgi apparatus by a complex system of glycosyltransferases. After their synthesis, gangliosides leave the Golgi apparatus via the lumenal surface of transport vesicles destined to the plasma membrane. In this study, we analyzed the synthesis and membrane distribution of GD3 and GM1 gangliosides endogenously synthesized by Madin-Darby canine kidney (MDCK) cell lines genetically modified to express appropriate ganglioside glycosyltransferases. By using biochemical techniques and confocal laser scanning microscopy analysis we demonstrated that GD3 and GM1, after being synthesized at the Golgi apparatus, are transported and accumulated mainly at the plasma membrane of non-polarized MDCK cell lines. More interestingly, both complex gangliosides were found enriched mainly at the apical domain when these cell lines were induced to polarize. In addition, we demonstrated that after arrival to plasma membrane, GD3 and GM1 gangliosides are endocytosed by using a clathrin-independent pathway. Then, internalized GD3, in association with a specific monoclonal antibody, is accumulated in endosomal compartments and is transported back to the plasma membrane. On the other hand, endocytosed GM1, in association with cholera toxin, is transported to endosomal compartments en route to the Golgi apparatus. In conclusion, our results demonstrate that complex gangliosides are apically sorted in polarized MDCK cells and that GD3 and GM1 gangliosides are internalized by clathrin-independent endocytosis to follow different intracellular destination.