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

大多数光学生物传感器报告细胞质离子或分子丰度的全局变化。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。Ca2+离子调节几乎所有的细胞活动。在大多数电不可兴奋的细胞中，Ca2+信号是由肌醇1,4,5-三磷酸受体（IP3Rs）的调控开放启动的，IP3Rs是内质网膜上的Ca2+通道。该项目的目的是使用靶向，遗传编码的荧光Ca2+指示器（GECIs）和快速，高分辨率的光学显微镜来定义Ca2+信号是如何在人类细胞中产生和解码的。许多Ca2+通道，交换器和转运体有助于Ca2+信号的时空复杂性，这允许多种过程的选择性调节。例如，IP3Rs介导内质网Ca2+的释放，而Orai通道介导Ca2+穿过质膜的进入，以响应细胞内Ca2+储存的消耗。由此产生的Ca2+信号引起不同的反应，但其机制在很大程度上仍未解决。对Ca2+具有低亲和力的GECIs可以选择性地报告开放Ca2+通道口附近[Ca2+]的大量局部增加，而有效地忽略了细胞质其余部分的更温和的变化。通过将这些传感器连接到单个蛋白质（例如IP3Rs或Orai，或它们被提议调节的靶标）上，我们可以使用先进的光学成像技术测量局部Ca2+信号。该项目将应用这些方法，首先通过修饰蛋白的异种表达，然后通过内源性对应物的基因编辑（CRISPR/cas9），探索内源性Ca2+信号蛋白、它们所唤起的Ca2+信号以及由此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.