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.
Richardson
Douglas S
DS
Department of Pathology and Molecular Medicine, Division of Cancer Biology and Genetics, Cancer Research Institute, Queen's University, Rm A315 Botterell Hall, Kingston, ON, K7L 3N6, Canada.
Mulligan
Lois M
LM
D019921:Biotinylation; D002462:Cell Membrane / Q000378:metabolism*; D022162:Cytoplasmic Vesicles / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D004705:Endocytosis; D006367:HeLa Cells; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism*; D057054:Molecular Imaging / Q000379:methods*; D021381:Protein Transport; D051096:Proto-Oncogene Proteins c-ret / Q000378:metabolism; D022142:Secretory Vesicles / Q000378:metabolism; D013194:Staining and Labeling
D008565:Membrane Proteins; D051096:Proto-Oncogene Proteins c-ret