Fast and high resolution single-cell BRET imaging.
Sci Rep. 2016-06-15; 6(1):
DOI: 10.1038/srep28231
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1. Sci Rep. 2016 Jun 15;6:28231. doi: 10.1038/srep28231.
Fast and high resolution single-cell BRET imaging.
Goyet E(1)(2)(3), Bouquier N(1)(2)(3), Ollendorff V(4), Perroy J(1)(2)(3).
Author information:
(1)CNRS, UMR-5203, Institut de Génomique Fonctionnelle, Montpellier, F-34094,
France.
(2)INSERM, U1191, Montpellier, F-34094, France.
(3)Universités de Montpellier, UMR-5203, Montpellier, F-34094, France.
(4)INRA, UMR866 Dynamique Musculaire et Mébabolisme, Université Montpellier,
34060 Montpellier, France.
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.
DOI: 10.1038/srep28231
PMCID: PMC4908377
PMID: 27302735 [Indexed for MEDLINE]