Direct visualization of vesicle maturation and plasma membrane protein trafficking

Internalization and intracellular trafficking of membrane proteins are now recognized as essential mechanisms that contribute to a number of cellular processes. Current methods lack the ability to specifically label the plasma membrane of a live cell, follow internalization of labeled membrane molecules, and conclusively differentiate newly formed membrane-derived vesicles from pre-existing endocytic or secretory structures in the cytoplasm. Here, we detail a visualization method for surface biotinylation of plasma membrane-derived vesicles that allows us to follow their progress from membrane to cytosol at specific time points. Using the transmembrane receptor RET as a model, we demonstrate how this method can be applied to identify plasma membrane-derived vesicle maturation, determine RET's presence within these structures, and monitor RET's recycling to the cell surface. This method improves on static and less discriminatory methods, providing a tool for analysis of real-time vesicle trafficking that is applicable to many systems.