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

用于确定活细胞中 Ca2 信号传导机制的靶向荧光传感器

基本信息

批准号：
2119934
负责人：
金额：
--
依托单位：
University of Cambridge
依托单位国家：
英国
项目类别：
Studentship
财政年份：
2018
资助国家：
英国
起止时间：
2018 至无数据
项目状态：
已结题

来源：
https://gtr.ukri.org/projects?ref=studentship-2119934
关键词：
Targeted fluorescent sensors determine mechanisms

项目摘要

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.

大多数光学生物传感器报告细胞溶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，可以检测特定蛋白质和细胞器周围的这些局部变化。Ca 2+离子调节几乎所有的细胞活动。在大多数电不兴奋细胞中，Ca 2+信号通过调节1，4，5-三磷酸肌醇受体（IP 3R）的开放而启动，IP 3R是驻留在ER膜上的Ca 2+通道。该项目的目的是使用有针对性的遗传编码荧光Ca 2+指示剂（GECIs）和快速，高分辨率的光学显微镜来定义Ca 2+信号如何在人类细胞中产生和解码。许多Ca 2+通道，交换器和转运体有助于Ca 2+信号的时空复杂性，这使得不同过程的选择性调节成为可能。例如，IP 3受体介导Ca 2+从内质网释放，而奥赖通道介导Ca 2+穿过质膜进入细胞内，以响应细胞内Ca 2+储存的耗尽。由此产生的Ca 2+信号引起不同的反应，但机制在很大程度上仍然没有解决。对Ca 2+亲和力低的GECIs可以选择性地报告开放Ca 2+通道口附近[Ca 2 +]的大量局部增加，同时有效地忽略胞质溶胶其余部分的更适度的变化。通过将这些传感器与单个蛋白质（例如，IP 3R或奥赖，或它们被提议调节的靶标）连接，我们可以使用先进的光学成像来测量局部Ca 2+信号。该项目将应用这些方法，首先通过修饰蛋白的异源表达，然后通过内源性对应物的基因编辑（CRISPR/cas9），来探索内源性Ca 2+信号蛋白，它们引起的Ca 2+信号和生理反应之间的关系。