Real-time in vivo imaging of extracellular ATP in the brain with a hybrid-type fluorescent sensor
Real-time in vivo imaging of extracellular ATP in the brain with a hybrid-type fluorescent sensor
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DOI：
10.7554/elife.57544
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发表时间：
2020-07-10
期刊：
影响因子：
7.7
通讯作者：
Hirose, Kenzo
中科院分区：
文献类型：
--
作者：
Kitajima, Nami;Takikawa, Kenji;Hirose, Kenzo
腺苷5'三磷酸（ATP）是一种普遍存在的细胞外信号信使。在这里，我们描述了一种方法，用于在体内成像的细胞外ATP与高时空分辨率。我们制备了一套完整的ATP结合蛋白，芽孢杆菌FoF 1 -ATP合成酶E亚基的半胱氨酸取代突变体，在引入的半胱氨酸残基上用小分子荧光团标记。筛选显示Cy 3标记的谷氨酰胺-105突变体（Q105 C-Cy 3;命名为ATPOS）在ATP存在下显示出大的荧光变化，具有亚微摩尔亲和力、pH独立性以及对ATP的高选择性超过ATP代谢物和其他核苷酸。为了实现体内验证，我们引入BoNT/C-Hc用于结合神经元质膜，并引入Alexa Fluor 488用于比率测量。由此产生的ATPOS复合物结合到活体小鼠大脑皮层的神经元上，并清楚地观察到细胞外ATP释放响应于电刺激的同心传播波。ATPOS应该是有用的，以探测细胞外ATP动力学的各种生物过程在体内。
Adenosine 5' triphosphate (ATP) is a ubiquitous extracellular signaling messenger. Here, we describe a method for in-vivo imaging of extracellular ATP with high spatiotemporal resolution. We prepared a comprehensive set of cysteine-substitution mutants of ATP-binding protein, Bacillus FoF 1 -ATP synthase E subunit, labeled with small-molecule fluorophores at the introduced cysteine residue. Screening revealed that the Cy3-labeled glutamine-105 mutant (Q105C-Cy3; designated ATPOS) shows a large fluorescence change in the presence of ATP, with submicromolar affinity, pH-independence, and high selectivity for ATP over ATP metabolites and other nucleotides. To enable in-vivo validation, we introduced BoNT/C-Hc for binding to neuronal plasma membrane and Alexa Fluor 488 for ratiometric measurement. The resulting ATPOS complex binds to neurons in cerebral cortex of living mice, and clearly visualized a concentrically propagating wave of extracellular ATP release in response to electrical stimulation. ATPOS should be useful to probe the extracellular ATP dynamics of diverse biological processes in vivo.