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

大多数光学生物传感器报告胞液离子或分子丰度的全球变化。然而，细胞使用局部微域来划分生理过程。需要有针对性的传感器来检测特定蛋白质和细胞器周围的这些局部变化。钙离子调节着几乎所有的细胞活动。在大多数电不能兴奋的细胞中，钙信号是由肌醇1，4，5-三磷酸受体(IP3R)的调节开放而启动的，IP3R是驻留在内质网膜上的钙通道。该项目的目的是使用有针对性的、遗传编码的荧光钙离子指示器(GECI)和快速、高分辨率的光学显微镜来定义人类细胞中钙信号是如何产生和解码的。许多钙离子通道、交换器和转运体导致了钙离子信号的时空复杂性，这使得对不同过程的选择性调控成为可能。例如，IP3R介导内质网钙离子的释放，而ORAI通道介导钙离子跨膜进入细胞内钙储备库的耗竭。由此产生的钙信号引起了不同的反应，但其机制在很大程度上仍未解决。对钙离子亲和力低的GECI可以选择性地报告开放的钙离子通道口附近局部的大量钙离子浓度升高，而实际上忽略了细胞质其余部分较轻微的变化。通过将这些传感器与单个蛋白质(例如，IP3Rs或Orai，或它们被建议调节的靶标)捆绑在一起，我们可以使用先进的光学成像来测量局部钙信号。本项目将应用这些方法，首先通过修饰蛋白的异源表达，然后通过内源蛋白的基因编辑(CRISPR/Cas9)，来探索内源钙信号蛋白、它们引起的钙信号和所产生的生理反应之间的关系。

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.