SUBCELLULAR IMAGING OF DYNAMIC PROTEIN INTERACTIONS BY BIOLUMINESCENCE RESONANCE ENERGY TRANSFER.

Vincent Coulon (1, 2) , Martin Audet (3) , Vincent Homburger (1) , Joël Bockaert (1) , Laurent Fagni (1) , Michel Bouvier (3) , Julie Perroy (1)

1 IGF - Institut de Génomique Fonctionnelle
2 EA3754 - Laboratoire de Dermatologie Moléculaire
3 Département de Biochimie et Médecine Moléculaire [UdeM-Montréal]

Despite the fact that numerous studies suggest the existence of receptor multiprotein complexes, visualization and monitoring of the dynamics of such protein assemblies remains a challenge. In the present study we established appropriate conditions to study spatio-temporally-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 sub-cellular 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.

Domaines: Imagerie; Neurosciences [q-bio.NC]

Vincent Coulon, Martin Audet, Vincent Homburger, Joël Bockaert, Laurent Fagni, et al.. SUBCELLULAR IMAGING OF DYNAMIC PROTEIN INTERACTIONS BY BIOLUMINESCENCE RESONANCE ENERGY TRANSFER.. Biophysical Journal, 2007, epub ahead of print. ⟨10.1529/biophysj.107.117275⟩. ⟨hal-00195266⟩