Imaging changes in the cytosolic ATP-to-ADP ratio.
Imaging changes in the cytosolic ATP-to-ADP ratio.
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DOI：
10.1016/b978-0-12-801415-8.00017-5
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发表时间：
2014
影响因子：
--
通讯作者：
Yellen, Gary
中科院分区：
文献类型：
--
作者：
Tantama, Mathew;Yellen, Gary
三磷酸腺苷（Adenosine triphosphate， ATP）是一种中心代谢物，在细胞内作为能量转移分子、磷酸供体和信号分子发挥着重要作用。ATP的磷酸基转移势为许多代谢反应提供了热力学驱动力，小代谢物和大蛋白的磷酸化都可以作为一种调节修饰。在ATP的磷酸化转移过程中，会产生二磷酸ADP，因此，ATP与ADP的比值是一个重要的生理控制参数。atp与adp的比值与细胞能量电荷和磷酸化电位成正比。此外，一些atp依赖的酶和信号蛋白受ADP调节，它们的激活谱是atp与ADP比值的函数。最后，ADP再生ATP可以作为能量代谢和线粒体功能的重要指标。因此，我们开发了一种基因编码的荧光生物传感器，用于感知健康哺乳动物细胞生理范围内atp与adp的比率。在这里，我们提出了一个协议，使用这种生物传感器可视化的能量状态使用活细胞荧光显微镜。
Adenosine triphosphate (ATP) is a central metabolite that plays fundamental roles as an energy transfer molecule, a phosphate donor, and a signaling molecule inside cells. The phosphoryl group transfer potential of ATP provides a thermodynamic driving force for many metabolic reactions, and phosphorylation of both small metabolites and large proteins can serve as a regulatory modification. In the process of phosphoryl transfer from ATP, the diphosphate ADP is produced, and as a result, the ATP-to-ADP ratio is an important physiological control parameter. The ATP-to-ADP ratio is directly proportional to cellular energy charge and phosphorylation potential. Furthermore, several ATP-dependent enzymes and signaling proteins are regulated by ADP, and their activation profiles are a function of the ATP-to-ADP ratio. Finally, regeneration of ATP from ADP can serve as an important readout of energy metabolism and mitochondrial function. We therefore developed a genetically-encoded fluorescent biosensor tuned to sense ATP-to-ADP ratios in the physiological range of healthy mammalian cells. Here we present a protocol for using this biosensor to visualize energy status using live-cell fluorescence microscopy.