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.
Highlights
Resonance Energy Transfer (RET)-based technologies are used to report protein-protein interactions in living cells
To distinguish the transfer of energy signal from the signal resulting from an overflow of the energy donor output into the energy acceptor detection channel, we measured the Em535/Em480 ratios from the same microscopic field of cells expressing either the Bioluminescence-initiated RET (BRET) donor alone (Rluc[8] or Nluc co-transfected with DsRed as a transfection reporter, basal ratio) or expressing the donor genetically fused to the Venus acceptor (Rluc8-Venus or Nluc-Venus, BRETpositive fusions) (Fig. 1)
We compared the temporal resolution, kinetics and sensitivity of single-cell BRET imaging in cells expressing Nluc-Venus versus Rluc8-Venus
Summary
Resonance Energy Transfer (RET)-based technologies are used to report protein-protein interactions in living cells. 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. To illustrate the benefits of using Nluc in BRET imaging, we designed an Nluc-optimized BRET-based sensor of ERK activity This reporter displayed a higher sensitivity and improved spatio-temporal resolution, enabling the detection of subtle, transient and localized variations in ERK activity in the dendritic spines of hippocampal neurons
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