A genetically-encoded sensor for imaging extracellular ATP onto astrocytes and other cells

项目说明 星形胶质细胞遍布哺乳动物的大脑，它们在空间和空间上相互作用 在功能上与神经元、血管和其他胶质细胞有关。它们提供多种动态平衡 并参与突触的形成、移除和调节。一个长期存在的和 主要悬而未决的问题涉及星形胶质细胞如何与神经元等其他细胞沟通， 小胶质细胞和星形胶质细胞。从这个角度来看，很多注意力都集中在细胞外 ATP是由神经元、星形胶质细胞和多个其他细胞通过几个 机械装置。一旦释放，ATP就会激活一系列电离型和代谢型ATP 受体及其降解产物激活腺苷受体。然而，事实证明， 直接测量细胞外ATP水平和我们的大部分知识具有极大的挑战性 关于大脑中的三磷酸腺苷信号是基于药物和遗传干预 以ATP受体为靶标。因此，ATP在体内的释放、浓度、动态和扩散 活的脑组织几乎没有被研究过，尽管它被广泛地牵连到 星形胶质细胞与胶质细胞和星形胶质细胞与神经元的相互作用。受到设计上的重大进步的鼓舞 使用基因编码的谷氨酸传感器，我们着手设计和表征一种 细胞外ATP的遗传编码传感器。在本申请的初步数据中，我们 报道了一种基于强度的ATP敏感荧光报告(IATPSnFR)。IATPSnFR为 在细胞表面表达，并对预期的细胞外ATP浓度做出反应 适当连接的环形置换超折叠的荧光强度增加 GFP(CpSFGFP)。IATPSnFR可以在基因上针对特定类型的细胞进行成像 标准的荧光和共聚焦显微镜。IATPsNFR的使用将揭示 细胞释放ATP，揭示星形胶质细胞在何时、何地和如何接收ATP信号 生理和病理生理过程。我们预计iATPSnFR将允许 首次直接测量和跟踪细胞外ATP动态。虽然我们 聚焦于星形胶质细胞，iATPsNFR可以应用于任何类型的细胞。在这项提案中，我们有两个 我们寻求发展我们的新方法并提供重要、新的、 星形胶质细胞和三磷酸腺苷信号研究的广泛适用和急需的资源。

Project description Astrocytes are found throughout the mammalian brain and interact spatially and functionally with neurons, blood vessels and other glia. They serve multiple homeostatic functions and are involved in synapse formation, removal and regulation. One long standing and major open question concerns how astrocytes communicate with other cells such as neurons, microglia and astrocytes. From this perspective, much attention has focussed on extracellular ATP, which is released from neurons, astrocytes and multiple other cells by several mechanisms. Once released, ATP activates a family of ionotropic and metabotropic ATP receptors, and its degradation product activates adenosine receptors. However, it has proven extremely challenging to measure extracellular ATP levels directly and much of our knowledge about ATP signaling in the brain is based on pharmacological and genetic interventions targeting ATP receptors. Hence, the release, concentration, dynamics and spread of ATP in living brain tissue has hardly been explored, despite the fact that it is implicated widely in astrocyte-glial and astrocyte-neuron interactions. Buoyed by significant advances in the design and use of genetically-encoded glutamate sensors, we set out to design and characterise a genetically-encoded sensor for extracellular ATP. In the preliminary data of this application we report an intensity-based ATP-sensing fluorescent reporter (iATPSnFR). iATPSnFR is expressed on cell surfaces and responds to expected extracellular ATP concentrations with an increase in fluorescence intensity of an appropriately attached circularly permuted super folder GFP (cpSFGFP). iATPSnFR can be genetically targeted to specific cell types and imaged with standard epifluoresence and confocal microscopes. The use of iATPsNFR will shed light on the cells releasing ATP and reveal when, where and how astrocytes receive ATP signals during physiological and pathophysiological processes. We expect that iATPSnFR will permit the measurement and tracking of extracellular ATP dynamics directly for the first time. Although we focus on astrocytes, iATPsNFR can be applied to any cell type. In this proposal we have two specific aims with which we seek to develop our new approach and provide important, new, broadly applicable and much needed resources for astrocyte and ATP signalling research.