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Abstract
BackgroundProtein-protein interactions form the basis of every organism and thus, investigating their dynamics, intracellular protein localization, trafficking and interactions of distinct proteins such as receptors and their ligand-binding are of general interest. Bioluminescence resonance energy transfer (BRET) is a powerful tool to investigate these aspects in vitro. Since in vitro approaches mostly neglect the more complex in vivo situation, we established BRET as an in vivo tool for studying protein interactions in the nematode C. elegans.ResultsWe generated worms expressing NanoBRET sensors and elucidated the interaction of two ligand-G protein-coupled receptor (GPCR) pairs, the neuropeptide receptor NPR-11 and the Adhesion GPCR LAT-1. Furthermore, we adapted the enhanced bystander BRET technology to measure subcellular protein localization. Using this approach, we traced ligand-induced internalization of NPR-11 in vivo.ConclusionsOur results indicate that in vivo NanoBRET is a tool to investigate specific protein interactions and localization in a physiological setting in real time in the living organism C. elegans.
Highlights
Protein-protein interactions form the basis of every organism and investigating their dynamics, intracellular protein localization, trafficking and interactions of distinct proteins such as receptors and their ligandbinding are of general interest
Our results indicate that in vivo NanoBRET is a tool to investigate specific protein interactions and localization in a physiological setting in real time in the living organism C. elegans
In order to establish Bioluminescence resonance energy transfer (BRET) analyses in C. elegans, we first sought to optimize the luminescence signal intensity emitted from the donor, which is highly dependent on the accessibility of the donor itself and the availability of the substrate converted by the donor
Summary
Protein-protein interactions form the basis of every organism and investigating their dynamics, intracellular protein localization, trafficking and interactions of distinct proteins such as receptors and their ligandbinding are of general interest. The donor is usually an enzyme catalyzing the reaction of a luminogenic substrate In this reaction, light is emitted and the energy is BRET has been very successfully employed to investigate general protein-protein interactions in living cells (reviewed in [6]), intracellular trafficking [7, 8], conformational (2022) 23:8 changes [9] and dimerization [10, 11] of proteins. Application of the BRET technology in vivo offers the opportunity to study protein interactions or ligand-receptor binding in a more complex cellular environment. This is especially of advantage when co-factors or other, unidentified molecules are required to e.g. stabilize ligand-binding.
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