Grant: Research Tools and Instruments

Signaling events in living cells are highly dynamic. Direct microscopic observation and analysis of such events can provide a wealth of data related to molecular mechanisms regulating cellular functions. G protein-coupled receptors (GPCRs) form one of the most diverse and efficient sensors in such signaling systems and are involved in responding to various stimuli including photons, small organic molecules, hormones, neurotransmitters, bioactive peptides and physical forces. Our NSERC funded research program is focused on studying a family of GPCRs called adhesion-GPCRs (aGPCRs). These receptors are made up of an unusual two protomer structure that requires mechanical forces mediated separation to activate the receptor. Various methods have been developed to enable direct visualization of protein dynamics in living cells, including the use of genetically encoded fluorescent tags and use of genome editing techniques such as CRISPR/Cas9 to facilitate tagging of endogenously expressed proteins. Combined with protein tagging technologies, use of advanced microscopy techniques enables the simultaneous high-resolution observation of multiple proteins labelled with different spectrally separated fluorescent proteins. When studying live cells, spinning disc confocal microscopy has been widely adopted as a technique that allows rapid image acquisition that is essential for capturing cellular signaling events. Spinning disc confocal microscopy also uses low intensity excitation light, significantly reducing phototoxicity seen with other microscopy techniques. The elegant advances in techniques for labelling and imaging proteins in live cells fundamentally depends on the ability to maintain healthy cells outside of a tissue culture incubator for long periods of time while being studied using microscopy. Such carefully controlled environmental conditions are essential for maintaining cell viability and for avoiding disruption of the cellular machinery in a healthy cell. The requested equipment includes a magnetic tweezer that will be mounted on a motorized stage on an existing Nikon Ti2 microscope body. The TokaiHIT ThermoBox Package (ThermoBox Enclosure and STX Stage Top Incubator) will be added to maintain a stable temperature, humidity and gas composition for long-term culture of cells to study consequence of mechanical activation of aGPCRs. Since long term imaging also introduce the possibility of samples drifting out of focus, a hardware solution from Nikon called the Perfect Focus System (PFS) will also be acquired. Finally recent advances in image analysis software incorporating new deconvolution, artificial intelligence (AI) and deep learning methods will be incorporated in the system software. Together the requested components will create a unique instrument capable of precise mechanical stimulation of cells and a long-term live cell imaging system with broad utility in supporting multiple NSERC funded research programs.