Direct Visualization of Vesicle Maturation and Plasma Membrane Protein Trafficking |
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Authors: | Douglas S Richardson and Lois M Mulligan |
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Institution: | (1) 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; |
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Abstract: | 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. |
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Keywords: | |
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