Sub-cellular propagation of GPCR signaling; functional imaging in space and time

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Proteostasis and protein trafficking are central processes controlling the homeostasis of cells and their ability to respond to external stimuli. One of the protein families for which these processes play a central role is the G protein-coupled receptors (GPCR). These receptors form the largest family of proteins involved in the transfer of information across biological membranes. In their classical representation, GPCR detect signals at the cell surface and transmit the information across the plasma membrane, leading to the activation of heterotrimeric G proteins attached to the inner face of the plasma membrane. The G proteins then interact and regulate the activity of effectors such as enzymes and channels. Following activation, phosphorylation of the receptor promotes the recruitment of the accessory protein ßarrestin, leading to receptor endocytosis and signal arrest. This traditional view is however rapidly evolving and ligand-promoted trafficking or direct targeting of signaling complexes to lipid raft, caveolae, endosomes, nucleus, Golgi, mitochondria and endoplasmic reticulum, have been linked to non-canonical signaling as well as regulatory functions in these organelles. Yet, most of the evidence for organelle-specific GPCR activity are indirect and, in some cases, controversial. Directly monitoring organelle-specific protein trafficking and signal propagation remains a daunting challenge. The long-term objective of our NSERC-supported research program is to develop a comprehensive biophysical platform allowing the real-time monitoring of organelle-specific trafficking and activity in living cells. GPCR will be used as prototypical models to develop this platform. In the present application, we propose to: 1) develop a collection of new bioluminescence resonance energy transfer (BRET)-based biosensors detecting organelle-specific GPCR trafficking and, 2) use high-resolution and time-resolved microscopy to visualize the compartmentalized signalling activity. To reach these specific objectives, we will design and validate a collection of BRET probes engineered to restrict their localization to individual compartments. The studies will generate new fundamental knowledge about the dynamics and role of compartmentalized signaling. Because of their portability, the biophysical and imaging tools developed will be used in longer-term projects to asses a diversity of other organelle-specific biological processes.