Evaluating the Role of Galectins in Clathrin-Independent Endocytosis.

Galectin-3 is a chimeric galectin involved in diverse intracellular and extracellular functions. Galectin-3 is synthesized in the cytoplasm and then released extracellularly by a poorly understood non-canonical secretion mechanism. As a result, it can play important roles both inside and outside the cell. One important extracellular role of galectin-3 is in modulating clathrin-independent endocytosis (CIE), a form of cellular internalization that is still not well understood. CIE, unlike clathrin-mediated endocytosis, has neither defined signaling sequences nor cytoplasmic machinery. As a result, extracellular interactions like the galectin-glycan interactions are thought to directly drive changes in CIE. This chapter discusses the methods designed to study the role of galectin-glycan interactions in CIE, which have provided us with insight into the functions of galectin-3 and cell surface glycans during CIE cargo internalization. These methods include media supplementation for metabolic glycoengineering, antibody internalization assays, lectin panels to assay changes in glycan patterns, exogenous galectin-3 supplementation, galectin-3 secretion assays, and in vitro assays to monitor the effect of galectins on CIE.