Imaging extracellular ATP with a genetically-encoded, ratiometric fluorescent sensor.
Imaging extracellular ATP with a genetically-encoded, ratiometric fluorescent sensor.
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DOI：
10.1371/journal.pone.0187481
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发表时间：
2017
期刊：
影响因子：
3.7
通讯作者：
Tantama M
中科院分区：
文献类型：
--
作者：
Conley JM;Radhakrishnan S;Valentino SA;Tantama M
细胞外三磷酸腺苷（ATP）是一个关键的嘌呤能信号，介导器官系统内部和之间的细胞间通讯。我们解决了需要一个强大的和微创的方法来测量细胞外ATP通过重新设计ATP传感器在细胞表面表达。使用这种方法，我们利用完全遗传编码的传感器对细胞外ATP水平的实时变化进行成像，不需要外源底物。此外，该传感器是比例计量，允许可靠的定量细胞外ATP通量。利用活细胞显微镜，我们表征了传感器在培养的Neuro2A细胞上表达时的性能，并测量了ATP的刺激释放和外核苷酶对其的清除。因此，这一原理证明了第一代传感器可以报告细胞外ATP动力学，这可能对研究活标本中的嘌呤能信号传导有用。
Extracellular adenosine triphosphate (ATP) is a key purinergic signal that mediates cell-to-cell communication both within and between organ systems. We address the need for a robust and minimally invasive approach to measuring extracellular ATP by re-engineering the ATeam ATP sensor to be expressed on the cell surface. Using this approach, we image real-time changes in extracellular ATP levels with a sensor that is fully genetically-encoded and does not require an exogenous substrate. In addition, the sensor is ratiometric to allow for reliable quantitation of extracellular ATP fluxes. Using live-cell microscopy, we characterize sensor performance when expressed on cultured Neuro2A cells, and we measure both stimulated release of ATP and its clearance by ectonucleotidases. Thus, this proof-of-principle demonstrates a first-generation sensor to report extracellular ATP dynamics that may be useful for studying purinergic signaling in living specimens.
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