Analysis of galectin-3 induced plasma membrane reorganization in clathrin-independent endocytosis
Final Report Abstract
One mechanism that facilitates communication between the exterior and the interior of a eukaryotic cell is endocytosis. Best characterized is clathrin-dependent uptake. Here, the cytosolic clathrin coat concentrates endocytic cargo in a clathrin-coated pit and generates membrane curvature, resulting in the formation of an endocytic carrier. In recent years it became increasingly clear that clathrin-independent endocytosis processes are also indispensable for cell function (signaling, migration, membrane homeostasis). It is not understood how proteins are sorted into clathrin-independent carriers and what drives membrane curvature changes during carrier formation, as cytosolic coats could not be detected at sites of membrane invagination. Previously, my host lab investigated the biophysical basis of clathrin-independent endocytosis of bacterial Shiga toxin and Simian Virus 40. These pathogenic factors multivalently bind plasma membrane glycosphingolipids and the resulting clustering of GSLs in the outer leaflet of the plasma membrane is thought to drive membrane bending. Here we found, that not only glycosphingolipid-binding pathogenic factors are able to induce endocytic vesicle formation, but that also the endogenous lectin galectin-3 triggered the glycosphingolipid-dependent biogenesis of clathrin-independent carriers. Galectin-3 interacted with a defined set of glycosylated plasma membrane proteins, among them several putative and established cargo proteins of clathrin-independent endocytosis, including CD44. Endocytosis of CD44 was reliant on its N-glycosylation, glycosphingolipids and on plasma membrane bound galectin-3. Superresolution microscopy revealed the presence of galectin-3 and CD44 clusters on the plasma membrane. Clustering of galectin-3 was glycosphingolipid-dependent. The average size and density of CD44 clusters was influenced by its N-glycosylation, suggesting that clustering is driven by N-glycan-lectin interaction. Galectin-3, which was recruited to protein-free model membranes induced membrane tubulation in a glycosphingolipid-dependent manner. Membrane deformation required galectin-3 oligomerization and its capacity to bind carbohydrates. These results suggest that the interaction of galectin-3 oligomers with the carbohydrate glycosphingolipid head groups is needed for its membrane bending capacity. Based on our results we propose the first mechanistic model for cargo sorting and membrane bending in clathrin-independent carrier formation: Monomeric galectin-3 is recruited to the plasma membrane by binding of glycosylated membrane proteins like CD44. Galectin-3 oligomerizes on the membrane and becomes capable to bind glycosphingolipid head groups in the outer plasma membrane leaflet. This could happen due to an avidity effect based on the binding of the galectin-3 oligomer to multiple GSL receptors. Galectin-3 oligomerization triggers the formation of a membrane domain in which galectin-3, its interacting proteins and glycosphingolipids are coclustert. Coclustering generates a mechanical strain, which results in membrane bending and endocytic pit formation. Hence galectins serve as an adaptor between glycosylated endocytic cargo and glycosphingolipids for the clathrin-independent formation of endocytic carriers.
Publications
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Galectin-3 drives glycosphingolipid-dependent biogenesis of clathrin-independent carriers. Nat Cell Biol. 2014 Jun: 16(6):595-606
Lakshminarayan, Ramya; Wunder, Christian; Becken, Ulrike; Howes, Mark T.; Benzing, Carola; Arumugam, Senthil; Sales, Susanne; Ariotti, Nicholas; Chambon, Valérie; Lamaze, Christophe; Loew, Damarys; Shevchenko, Andrej; Gaus, Katharina; Parton, Robert G. & Johannes, Ludger
