Complex gangliosides are apically sorted in polarized MDCK cells and internalized by clathrin-independent endocytosis

• Published in: European journal of biochemistry 2008-12, Vol.275 (23), p.6043-6056
• Main Authors: Crespo, Pilar M, von Muhlinen, Natalia, Iglesias-Bartolomé, Ramiro, Daniotti, Jose L
• Format: Article
• Language: English
• Subjects: Animals; Biochemistry; Biological Transport; Cell Line; Cell Membrane - metabolism; Cellular biology; Chemical synthesis; Cholera Toxin - metabolism; Clathrin-Coated Vesicles - metabolism; Dogs; Endocytosis - physiology; Epithelial Cells - cytology; Epithelial Cells - metabolism; G(M1) Ganglioside - metabolism; gangliosides; Gangliosides - metabolism; glycolipids; Intracellular Signaling Peptides and Proteins - genetics; Intracellular Signaling Peptides and Proteins - metabolism; intracellular trafficking; Kidneys; MDCK cells; Membranes; Microscopy, Confocal; N-Acetylgalactosaminyltransferases - genetics; N-Acetylgalactosaminyltransferases - metabolism; polarized cells; rab GTP-Binding Proteins - metabolism; rab5 GTP-Binding Proteins - metabolism; Sialyltransferases - genetics; Sialyltransferases - metabolism; Transfection; Transport Vesicles - metabolism
• ISSN: 1742-464X; 0014-2956; 1742-4658; 1432-1033
• DOI: 10.1111/j.1742-4658.2008.06732.x

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 synthesis, gangliosides leave the Golgi apparatus via the lumenal surface of transport vesicles destined to the plasma membrane. In this study, we analysed 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. Using biochemical techniques and confocal laser scanning microscopy analysis, we demonstrated that GD3 and GM1, after being synthesized at the Golgi apparatus, were transported and accumulated mainly at the plasma membrane of nonpolarized MDCK cell lines. More interestingly, both complex gangliosides were found to be enriched mainly at the apical domain when these cell lines were induced to polarize. In addition, we demonstrated that, after arrival at the plasma membrane, GD3 and GM1 gangliosides were endocytosed using a clathrin-independent pathway. Then, internalized GD3, in association with a specific monoclonal antibody, was accumulated in endosomal compartments and transported back to the plasma membrane. In contrast, endocytosed GM1, in association with cholera toxin, was 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 destinations.