Microscopy upgrades to study biological motility and cell signaling at surfaces

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All living things move in response to signals. At the cellular level, molecular signals interact with membrane-associated proteins in living cells. Our team of researchers mentoring 56 HQP at the University of Guelph are focused on answering fundamental questions about biological motility and signaling at surfaces, be it in living cell membranes in eukaryotes or prokaryotes or in systems reconstituted from purified proteins. We share a common challenge of obtaining high-resolution images of the molecules involved in motility and signaling as they go about their activities at cell surfaces with their interaction partners. To obtain these images, we need state of the art microscopy that provides the best resolution of molecules working at surfaces. We need a microscopy system capable of high-resolution Total Internal Reflection Fluorescence (TIRF) and Förster Resonance Energy Transfer (FRET) measurements with a heated stage to finetune for the types of interactions we are looking for, but such a system does not exist at Guelph. With TIRF microscopy, a very thin (~100 nm) layer at the slide surface is illuminated allowing analysis to be focused on surface-associated molecules. TIRF microscopy is needed by our team to image focal adhesion dynamics in motile cells, the response of transmembrane proteins to signals related to hypoxia, and calcium signaling in mammalian cells. TIRF would also be central for our team to answer fundamental questions about the dynamics of the bacterial divisome responsible for cell division and bacterial membrane protein changes responding to different growth conditions, and to study the interactions of reconstituted muscle proteins on surfaces. With FRET measurements, molecular interactions within a ~10 nm radius can be observed in living cells in real time. Within our team, FRET will be used to measure calcium signal dynamics and future second messenger signaling and kinase activity using biosensors. In this proposal, we request upgrades to an existing new Nikon Ti2-E microscope to add TIRF and FRET microscopy capabilities. Also included is an antivibration table and heated stage and objective jacket to provide needed stability and consistency. Current FRET microscopy at Guelph is outdated and there is no functional TIRF. The requested system will be housed in the Advanced Analysis Centre, a facility accessible to researchers throughout our region with a dedicated microscopist to maintain the system and provide HQP with training and guidance. High-resolution microscopy is becoming the standard for life science research, from single molecules to live cells. The need for high-resolution microscopy at the University of Guelph is critical. Funding of the proposed infrastructure will open the door to new avenues of research, offer cutting-edge technology to our research team, their HQP and collaborators, and provide the next generation of scientists, both locally and regionally, with the skills for future success.