Measuring Plasma Membrane Recycling Using Microscopic and Biochemical Approaches

Abstract

The endocytic pathway has an intricate network of vesicular compartments carrying a variety of proteins referred to as cargoes. Endosomal trafficking is exclusively required to transport these cargoes through various intracellular routes for their delivery to the site of action. Among these, recycling of cargoes to the plasma membrane is a crucial pathway for the efficient functioning of the cell. Hence, endosomal cargo recycling assays are crucial to gain insight into the molecular mechanism governing recycling of the cargoes and in turn to understand their key role in maintaining cellular physiology. These assays have been efficiently utilized to study the recycling of adhesion molecules, transporters, channels, receptors, and so on to the plasma membrane. The basic methodology involves labelling of the cargo at the surface, allowing its internalization followed by direct or indirect measurement of the amount of the cargo recycled back to the plasma membrane. These microscopy-based and biochemical methods can be used as a tool to study the role of various trafficking or signaling molecules on the cell surface involved with the recycling of the membrane proteins, by altering their expression either by silencing or overexpressing the gene.

Keywords: Antibody uptake; Biotinylation; Cell surface recycling; Immunoblotting; Immunofluorescence; Recycling kinetics; TIRF microscopy.