Qualitative and quantitative analysis of endocytic recycling

Endocytosis, which encompasses the internalization and sorting of plasma membrane (PM) lipids and proteins to distinct membrane-bound intracellular compartments, is a highly regulated and fundamental cellular process by which eukaryotic cells dynamically regulate their PM composition. Indeed, endocytosis is implicated in crucial cellular processes that include proliferation, migration, and cell division as well as maintenance of tissue homeostasis such as apical-basal polarity. Once PM constituents have been taken up into the cell, either via clathrin-dependent endocytosis (CDE) or clathrin-independent endocytosis (CIE), they typically have two fates: degradation through the late-endosomal/lysosomal pathway or returning to the PM via endocytic recycling pathways. In this review, we will detail experimental procedures that allow for both qualitative and quantitative assessment of endocytic recycling of transmembrane proteins internalized by CDE and CIE, using the HeLa cervical cancer cell line as a model system.