Grant: Discovery Grants Program - Individual

While Gas subunit is classically known to relay extracellular signals from G-protein coupled receptors (GPCRs) located on plasma membrane (PM) to downstream effectors, it has also been recently demonstrated to be localized on endosomes. The long-term objective of this research program, funded 5 years ago, and long-time interest of my lab, is to characterize the non-conventional role of Gas on this intracellular compartment. In the last few years, a plethora of data and tools highlighted the endosomal functions of Gas in receptor signaling and trafficking. It is now accepted that canonical GPCR-Gas signalling occurs from endosome as well as the PM. Furthermore, our group has pointed out a novel role of Gas on the endosomal sorting of GPCRs and single transmembrane receptor to lysosome. Over the next 5 years, we plan to outline the mechanism of endosomal translocation of Gas and its molecular organization and regulation on endosomal microdomains. New tools will also be developed in order to answer these questions. AIM1. To investigate Gas recruitment on endosomes Although Gas localization on endosome is now well accepted, much less is understood about the trafficking of Gas to and from endosomes. Under this aim, live-cell microscopy of Gas tagged to a photoactivatable green-to red fluorescent proteins will be used to monitor the intracellular dynamic of a specific pool of Gas (photactivated at the PM or endosome). CRISPR/Cas9 gene editing will also be used to tag the endogenous Gas for better functional analysis. Gas recruitment on endosome will be examined following stimulation of various receptors that are coupled or not to Gas in their signaling pathway. Gas trafficking pathway will be determines using specific inhibitors/siRNA and colocalization various intracellular markers. Using a specific biosensor, the distribution of the active form of Gas will be examined by confocal microscopy imaging and Biolumiescence Resonance Energy Transfer (BRET) assays. AIM2. To characterize Gas distribution and function on endosome microdomains Endosomes are compartmentalized into distinct functional subdomains such as signaling, recycling and sorting into intraluminal vesicles for lysosomal degradation. Under this aim, live-cell and super-resolution microscopy (STED) microsopy will be used to characterize the distribution, dynamics and molecular organization of Gas in endosomal subdomains following activation and endocytosis of various receptors. Significance: In the past decade, we have learned much about Gas organization at the plasma membrane, but much still remains to be learned both with regards to the nature of its endosomal recruitment, organization, regulation, and the physiological function. Our work will lead to novel insights into these fundamental questions that will impact our understanding of receptor signaling.