Despite the fact that numerous studies suggest the existence of receptor multiprotein complexes, visualization and monitoring of the dynamics of such protein assemblies remain a challenge. In this study, we established appropriate conditions to consider spatiotemporally resolved images of such protein assemblies using bioluminescence resonance energy transfer (BRET) in mammalian living cells. Using covalently linked Renilla luciferase and yellow fluorescent proteins, we depicted the time course of dynamic changes in the interaction between the V2-vasopressin receptor and beta-arrestin induced by a receptor agonist. The protein-protein interactions were resolved at the level of subcellular compartments (nucleus, plasma membrane, or endocytic vesicules) and in real time within tens-of-seconds to tens-of-minutes time frame. These studies provide a proof of principle as well as experimental parameters and controls required for high-resolution dynamic studies using BRET imaging in single cells.
Subcellular imaging of dynamic protein interactions by bioluminescence resonance energy transfer
Coulon, V.; Audet, M.; Homburger, V.; Bockaert, J.; Fagni, L.; Bouvier, M.; Perroy, J.
2008-02-01 / vol 94 / pages 1001-9
S0006-3495(08)70696-9 [pii] 10.1529/biophysj.107.117275
1542-0086 (Electronic) 0006-3495 (Linking)
Humans; Cell Line; Fluorescence Resonance Energy Transfer/*methods; Kidney/*metabolism; Luminescent Measurements; Luminescent Proteins/*metabolism; Microscopy, Fluorescence/*methods; Protein Interaction Mapping/*methods; Subcellular Fractions/*metabolism/*ultrastructure