Membrane Compartmentalisation and Endocytosis by Galectin-3 in Mammalian Cells

Abstract

Galectin-3 is a carbohydrate binding protein that is widely expressed and can be found in tissues and cells of the immune system. At the cell surface, galectin-3 interacts with various cell surface glycoproteins through its carbohydrate recognition domain (CRD) and exhibits multivalent binding properties by assembling into pentamers through its N-terminal domain. Galectin-3 molecules have been implicated in the formation of molecular lattices at plasma membranes and in endocytosis. The aim of this study was to examine the molecular arrangement of galectin-3 on the surface of fibroblasts and Hela cells as well resting and activated T cells, and investigate whether and how galectin-3 is internalized in T cells to generate signalling endosomes. To map the molecular organisation of galectin-3 on the cell surface, direct stochastic optical reconstruction microscopy (dSTORM) and quantitative analysis of galectin-3 and galectin-3 ligands was established. It was found that galectin-3 clustering depended on glycosphingolipids in the plasma membrane and that galectin-3-dependent clustering of known galectin-3 binding partners was sensitive to branched N-acetylglucosamine saccharides that were absent in β-1,6-N-acetylglycosaminyltransferase V (Mgat5)-deficient mouse embryonic fibroblasts (Mgat5-/- MEF). These data supports the concept that galectin-3 compartmentalises the plasma membrane. Next, it was demonstrated with confocal microscopy and flow cytometry that the binding of galectin-3 to the plasma membrane of T cells occurred in a carbohydrate-dependent fashion and led to internalisation. Further it was shown that uptake of galectin-3 was facilitated by different endocytic mechanisms suggesting that galectin-3 participates in various endocytic routes in T cells. Finally, in activated T cells, data is presented to show that galectin-3- positive vesicles were positioned at or near the immunological synapse and co-localised with proteins involved in signalling processes, suggesting that galectin-3 functions in the regulation of T cell signalling. In conclusion, the data presented in this PhD thesis suggest that galectin-3 binding to the cell surface creates distinct membrane domains in a glycosphingolipid- and branched N-glycosylation-dependent manner. In T cells, galectin-3 domains lead to the formation of galectin-3 vesicles that may function as signaling endosomes.