Targeted fluorescent sensors to determine mechanisms of Ca2+ signalling in live cells

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Most optical biosensors report global changes in the abundance of cytosolic ions or molecules. However, cells use local micro-domains to compartmentalize physiological processes. There is a need for targeted sensors that can detect these local changes around specific proteins and organelles. Ca2+ ions regulate almost every cellular activity. In most electrically inexcitable cells, Ca2+ signals are initiated by regulated opening of inositol 1,4,5-trisphosphate receptors (IP3Rs), which are Ca2+ channels resident in ER membranes. The aim of this project is to use targeted, genetically-encoded fluorescent Ca2+ indicators (GECIs) and fast, high-resolution optical microscopy to define how Ca2+ signals are generated and decoded in human cells. Many Ca2+ channels, exchangers and transporters contribute to the spatiotemporal complexity of Ca2+ signals, which allows selective regulation of diverse processes. For example, IP3Rs mediaterelease of Ca2+ from the ER, while Orai channels mediate entry of Ca2+ across the plasma membrane in response to depletion of intracellular Ca2+ stores. The resulting Ca2+ signals evoke different responses, but the mechanisms remain largely unresolved. GECIs with low-affinity for Ca2+ can selectively report the massive local increases in [Ca2+] near the mouths of open Ca2+ channels, while effectively ignoring the more modest changes in the rest of the cytosol. By tethering these sensors to individual proteins (eg, IP3Rs or Orai, or the targets they are proposed to regulate), we can measure local Ca2+ signals using advanced optical imaging. This project will apply these methods, initially by heterologous expression of the modified proteins and then by gene-editing (CRISPR/cas9) of their endogenous counterparts, to explore the the relationships between endeogenous Ca2+ signalling proteins, the Ca2+ signals they evoke, and the physiological responses that result.