Fast and high resolution single-cell BRET imaging.
Resonance Energy Transfer (RET)-based technologies are used to report protein-protein interactions in living cells. Among them, Bioluminescence-initiated RET (BRET) provides excellent sensitivity but the low light intensity intrinsic to the bioluminescent process hampers its use for the localization of protein complexes at the sub-cellular level. Herein we have characterized the methodological conditions required to reliably perform single-cell BRET imaging using an extremely bright luciferase, Nanoluciferase (Nluc). With this, we achieved an unprecedented performance in the field of protein-protein interaction imaging in terms of temporal and spatial resolution, duration of signal stability, signal sensitivity and dynamic range. As proof-of-principle, an Nluc-containing BRET-based sensor of ERK activity enabled the detection of subtle, transient and localized variations in ERK activity in neuronal dendritic spines, induced by the activation of endogenous synaptic NMDA receptors. This development will improve our comprehension of both the spatio-temporal dynamics of protein-protein interactions and the activation patterns of specific signaling pathways.
CNRS, UMR-5203, Institut de Génomique Fonctionnelle, Montpellier, F-34094, France.|INSERM, U1191, Montpellier, F-34094, France.|Universités de Montpellier, UMR-5203, Montpellier, F-34094, France.
Elise
E
Goyet
CNRS, UMR-5203, Institut de Génomique Fonctionnelle, Montpellier, F-34094, France.|INSERM, U1191, Montpellier, F-34094, France.|Universités de Montpellier, UMR-5203, Montpellier, F-34094, France.
Nathalie
N
Bouquier
INRA, UMR866 Dynamique Musculaire et Mébabolisme, Université Montpellier, 34060 Montpellier, France.
Vincent
V
Ollendorff
CNRS, UMR-5203, Institut de Génomique Fonctionnelle, Montpellier, F-34094, France.|INSERM, U1191, Montpellier, F-34094, France.|Universités de Montpellier, UMR-5203, Montpellier, F-34094, France.
Julie
J
Perroy
D002478:Cells, Cultured; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D057809:HEK293 Cells; D006801:Humans; D008164:Luminescent Proteins / Q000378:metabolism*; D011485:Protein Binding; D015398:Signal Transduction; D059010:Single-Cell Analysis / Q000379:methods*
D008164:Luminescent Proteins