Subcellular dynamic imaging of protein-protein interactions in live cells by bioluminescence resonance energy transfer.
Protein functions rely on their ability to engage in specific protein-protein interactions and form complexes that are dynamically regulated by stimuli. Bioluminescence resonance energy transfer (BRET) is a highly sensitive technique, which allows monitoring of interaction between two proteins: one tagged with the luminescent donor Renilla luciferase, the other with a fluorescent acceptor such as YFP. We adapted this method to single-cell imaging. To this aim, we tag proteins of interest, transfect cells with these fusions, and use the high-sensitivity microscopy, combined with electron multiplying cooled charge-coupled device (EMCCD) cameras and improved bioluminescence probes. We thus achieve rapid acquisition of high-resolution BRET images and study the localization and dynamics of protein-protein interactions in individual live cells.
Perroy
Julie
J
Department of Neurobiology, Functional Genomic Institute Unité mixte de recherche 5203 Centre National de la Recherche Scientifique, Unité 661 Institut National de la Santé et de la Recherche Médicale, Université Montpellier I & II, Montpellier, France.
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002460:Cell Line; D002470:Cell Survival; D006801:Humans; D007091:Image Processing, Computer-Assisted; D042541:Intracellular Space / Q000378:metabolism*; D049410:Luciferases, Renilla / Q000737:chemistry / Q000378:metabolism; D008163:Luminescent Measurements; D057054:Molecular Imaging / Q000379:methods*; D008969:Molecular Sequence Data; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D014162:Transfection
D011993:Recombinant Fusion Proteins; D049410:Luciferases, Renilla