Bioluminescence resonance energy transfer detection system for the monitoring of protein-protein interactions, post-translational modifications and cell signalling.

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This application to the Research Tools and Instruments Grants program is for the acquisition of a multimodal plate reader adapted for high sensitivity and medium throughput bioluminescence resonance energy transfer (BRET) measurements. Manipulation of the naturally occurring BRET has enabled the design of sensors detecting various cellular events in real time. The laboratory of the principal applicant pioneered the biological use of BRET and continues to develop BRET-based sensors (over 70 distinct sensors/assays have been designed and validated so far), probing protein-protein interactions, as well as protein conformational changes, trafficking and signaling. The BRET-compatible plate reader requested will be used in many NSERC-supported (Discovery Grants) projects that include the use of BRET. We present four of these projects in the current application, which illustrates the wide spectrum of possible applications of the instrument. Project 1 aims at developing and using new BRET-based approaches to monitor the spatio-temporal propagation of signal transmission by G protein-coupled receptors (GPCR). The translocation and activity of GPCR effectors will be monitored at subcellular resolution in real time. In project 2, BRET-based assays will be used to investigate the mechanisms controlling the changes in the cell surface proteome during mitosis by monitoring the cell cycle-regulated subcellular trafficking of proteins between various organelles. Structural determinants controlling the activity of the T cell receptor-associated Lck kinase will be assessed, in project 3, with the use of intramolecular BRET-based sensors monitoring the conformational rearrangement of the kinase upon binding to CD3. Project 4 aims at characterizing the role of SUMOylation in controlling protein-protein interactions associated with the senescence and migration of cancer cells. For this purpose, a BRET-based assay will be developed to monitor the effects of small molecules on the SUMOylation state of vimentin and UBC9, and their impact on protein-protein interactions and activity. Students, post-doctoral fellows and highly qualified personnel will be involved in each project, allowing them to learn how to design, perform and interpret BRET results in addition to gaining insights into the biological questions that they address. Because the BRET principles are similar to those of fluorescence resonance energy transfer (FRET) and other proximity-based assays, such as protein-complementation, protein-ligation and proximity-dependent biotin identification (BioID), the trainees will be exposed to these innovative and highly sought after approaches. Given that the existing instrument is already used at its maximal capacity, the additional NSERC supported projects necessitate the acquisition of a new BRET reader. The greater sensitivity and throughput of the new generation instruments will allow the efficient realization of these projects.