A genetically encoded single-wavelength sensor for imaging cytosolic and cell surface ATP
A genetically encoded single-wavelength sensor for imaging cytosolic and cell surface ATP
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DOI：
10.1038/s41467-019-08441-5
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发表时间：
2019-02-12
影响因子：
16.6
通讯作者：
Khakh, Baljit S.
中科院分区：
文献类型：
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作者：
Lobas, Mark A.;Tao, Rongkun;Khakh, Baljit S.
5' 三磷酸腺苷 (ATP) 是一种通用的细胞内能量来源，也是多种物种中进化古老、普遍存在的细胞外信号。在这里，我们报告了单波长基因编码荧光传感器（iATPSnFR）的生成和表征，用于通过将循环排列的超级文件夹 GFP 插入芽孢杆菌 PS3 的 F0F1-ATP 酶的 epsilon 亚基来对细胞外和胞质 ATP 进行成像。在细胞表面和细胞质内，iATPSnFR(1.0) 响应相关 ATP 浓度（30 μM 至 3 mM），荧光快速增加。 iATPSnFR 可以通过基因靶向特定的细胞类型和亚细胞区室，使用标准光学显微镜成像，不会对其他核苷酸和核苷产生反应，并且当与红色荧光蛋白融合时可用作比例指示剂。在仔细考虑其适度的 pH 敏感性后，iATPSnFR 代表了在各种环境下对细胞外空间和细胞内的 ATP 进行成像以及进一步针对特定应用进行改进的有前景的试剂。
Adenosine 5' triphosphate (ATP) is a universal intracellular energy source and an evolutionarily ancient, ubiquitous extracellular signal in diverse species. Here, we report the generation and characterization of single-wavelength genetically encoded fluorescent sensors (iATPSnFRs) for imaging extracellular and cytosolic ATP from insertion of circularly permuted superfolder GFP into the epsilon subunit of F0F1-ATPase from Bacillus PS3. On the cell surface and within the cytosol, iATPSnFR(1.0) responds to relevant ATP concentrations (30 mu M to 3 mM) with fast increases in fluorescence. iATPSnFRs can be genetically targeted to specific cell types and sub-cellular compartments, imaged with standard light microscopes, do not respond to other nucleotides and nucleosides, and when fused with a red fluorescent protein function as ratiometric indicators. After careful consideration of their modest pH sensitivity, iATPSnFRs represent promising reagents for imaging ATP in the extracellular space and within cells during a variety of settings, and for further application-specific refinements.