Homeostatic Regulation of PIP2-Calcium Signaling

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Abstract This is an administrative supplement application for funding to upgrade an integrated spinning-disk confocal and total internal reflection fluorescence (TIRF) microscope system in the PI’s lab. This microscope system is required for carrying out all live-cell imaging experiments proposed in the parent project that is focused on defining mechanisms regulating PIP2 homeostasis by Nir2 protein at endoplasmic reticulum (ER)-plasma membrane (PM) junctions during receptor-induced Ca2+ signaling. This integrated confocal-TIRF microscope system is uniquely set up for monitoring dynamic changes of PM PIP2 levels and translocation of proteins to ER-PM junctions in the same cells during receptor stimulation. Such a system is not available at the Imaging Core Facility of our institute. Recently, several components of this microscope system that was set up in 2009 are losing functionality at a fast pace. An upgrade of this integrated microscope system is urgently needed to prevent disruptions of the parent project due to failure of the system. In addition, this upgrade will enable integration of new technology into the microscope system to meet our imaging needs for the next five to ten years. The Physiology Department at UT Southwestern will support this upgrade by providing funds to cover the balance exceeding the budget, and by providing space for the upgraded microscope system. By replacing the failing and outdated parts with new state-of-the-art microscope components, this upgrade will significantly increase the capability and throughput of our integrated confocal-TIRF microscope system to obtain high-quality quantitative imaging results for years to come. This is an investment that will greatly facilitate the progress and enhance the impact of the parent project.