Exploration into Galectin-3 Driven Endocytosis and Lattices

Essentially all plasma membrane proteins are glycosylated, and their activity is regulated by tuning their cell surface dynamics. This is achieved by glycan-binding proteins of the galectin family that either retain glycoproteins within lattices or drive their endocytic uptake via the clathrin-independent glycolipid-lectin (GL-Lect) mechanism. Here, we have used immunofluorescence-based assays to analyze how lattice and GL-Lect mechanisms affect the internalization of the cell adhesion and migration glycoprotein α₅β₁ integrin. In retinal pigment epithelial (RPE-1) cells, internalized α₅β₁ integrin is found in small peripheral endosomes under unperturbed conditions. Pharmacological compounds were used to competitively inhibit one of the galectin family members, galectin-3 (Gal3), or to inhibit the expression of glycosphingolipids, both of which are the fabric of the GL-Lect mechanism. We found that under acute inhibition conditions, endocytic uptake of α₅β₁ integrin was strongly reduced, in agreement with previous studies on the GL-Lect driven internalization of the protein. In contrast, upon prolonged inhibitor treatment, the uptake of α₅β₁ integrin was increased, and the protein was now internalized by alternative pathways into large perinuclear endosomes. Our findings suggest that under these prolonged inhibitor treatment conditions, α₅β₁ integrin containing galectin lattices are dissociated, leading to an altered endocytic compartmentalization.