胞浆
细胞外
细胞内
三磷酸腺苷
质子泵
费斯特共振能量转移
荧光
绿色荧光蛋白
细胞生物学
生物物理学
生物化学
活体细胞成像
核苷酸
细胞
蛋白质亚单位
生物
化学
酶
基因
物理
量子力学
作者
Mark A. Lobas,Rongkun Tao,Jun Nagai,Mira Therese Kronschläger,Philip Borden,Jonathan S. Marvin,Loren L. Looger,Baljit S. Khakh
标识
DOI:10.1038/s41467-019-08441-5
摘要
Abstract 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 F 0 F 1 -ATPase from Bacillus PS3 . On the cell surface and within the cytosol, iATPSnFR 1.0 responds to relevant ATP concentrations (30 μM 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.
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