Fast and high resolution single-cell BRET imaging.
Sci Rep
; 6: 28231, 2016 06 15.
Article
em En
| MEDLINE
| ID: mdl-27302735
ABSTRACT
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.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Transferência Ressonante de Energia de Fluorescência
/
Análise de Célula Única
/
Proteínas Luminescentes
Limite:
Humans
Idioma:
En
Revista:
Sci Rep
Ano de publicação:
2016
Tipo de documento:
Article
País de afiliação:
França