This proposal is for the purchase of the Zeiss LSM 800 confocal microscope that will be shared between two NIGMS-funded laboratories at the University of Miami Miller School of Medicine: Dr. Daniel Isom in the Department of Molecular and Cellular Pharmacology, Sylvester Comprehensive Cancer Center, and Center for Computational Sciences (R35GM119518), and Dr. Emmanuel Thomas in the Department of Microbiology and Immunology and the Sylvester Comprehensive Cancer Center (R35GM124915). The ultra-sensitivity of the Zeiss LSM 800 would provide both labs with the day-to-day capability to obtain high-resolution spatiotemporal images of genetically encoded biosensors, fluorescently labeled proteins, and biomarkers of health and disease. Neither lab currently has unrestricted access to these imaging capabilities. Additional investment in the MIRA-funded projects led by Drs. Isom and Thomas would enable both research groups to improve and enhance their cellular imaging techniques to meet and exceed their stated and emergent research objectives. Dr. Isom received his MIRA support in August of 2016 to study how pH regulates G protein-coupled receptors (GPCRs), the largest and most therapeutically targeted class of cell surface receptors in humans (having more than 800 members). Using yeast and human cell models, Dr. Isom is experimentally quantifying how protons regulate a variety of GPCRs to better understand signaling from acidified organelles (such as endosomes) and acidified microenvironments (such as tumors). The long-term objectives of this project are to build a comprehensive understanding of pH sensing across all GPCRs, and to discover and design drugs that might serve as pH-selective therapeutics. The accuracy of this approach hinges partly on the ability to quantify cellular pH. That is, it is necessary to simultaneously monitor GPCR activity/localization and extracellular, cellular, and subcellular pH in real time. Currently, these measurements are done on cell populations containing genetically encoded pH biosensors quantified using a fluorescence plate reader. When Dr. Isom conceived his MIRA-funded project, the lab's pH biosensor experiments were envisioned as a means to provide the necessary controls for reporting GPCR activity at specific intracellular and extracellular pH values. As this project has evolved, Dr. Isom now realizes that his lab is in a unique position to use these pH sensors to provide much needed quantitative information on cellular pH dynamics in normal and stressful conditions. However, the Isom lab does not have a microscope capable of making these measurements. As is detailed in this proposal, the Zeiss LSM 800 confocal microscope has the resolution, speed, sensitivity, programmability, and hardware modules necessary for us to achieve this objective. The LSM 800 would also enable the Isom lab to assess the localization and trafficking of fluorescently tagged GPCRs in yeast and human cell models. This need is timely and important because GPCRs are now being found in new subcellular locations associated with highly dynamical pH characteristics (e.g. Golgi and mitochondrial membranes). Supplemental NIGMS support for the shared purchase of a customized Zeiss LSM 800 microscope system would make these measurements possible.
The parent research project related to this proposal seeks to understand how pH regulates G protein-coupled receptor signaling, the largest and most therapeutically targeted class of cell surface receptors in humans. As this project has evolved, an opportunity has emerged to quantify cellular pH dynamics and GPCR signaling. However, our laboratory does not have a microscope, nor does it have unrestricted access to a microscope with the required spatiotemporal capabilities. The purpose of this request for supplemental NIGMS support is to purchase a confocal microscope that would enable us to map subcellular pH dynamics and GPCR activity at unprecedented spatiotemporal resolution.
|Isom, Daniel G; Page, Stephani C; Collins, Leonard B et al. (2018) Coordinated regulation of intracellular pH by two glucose-sensing pathways in yeast. J Biol Chem 293:2318-2329|
|Lassuthova, Petra; Rebelo, Adriana P; Ravenscroft, Gianina et al. (2018) Mutations in ATP1A1 Cause Dominant Charcot-Marie-Tooth Type 2. Am J Hum Genet 102:505-514|