Real-Time Visualization of Cytosolic and Mitochondrial ATP Dynamics in Response to Metabolic Stress in Cultured Cells.
Real-Time Visualization of Cytosolic and Mitochondrial ATP Dynamics in Response to Metabolic Stress in Cultured Cells.
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5′三磷酸腺苷 (ATP) 是生命的能量货币，在线粒体（约 90%）和细胞质（少于 10%）中产生。代谢变化对细胞 ATP 动态的实时影响仍然不确定。在这里，我们报告了基因编码荧光 ATP 指示剂的设计和验证，该指示剂允许实时、同时可视化培养细胞中的胞质和线粒体 ATP。这种双 ATP 指示剂称为 smacATPi（同时线粒体和胞质 ATP 指示剂），结合了之前描述的单独胞质和线粒体 ATP 指示剂。使用 smacATPi 可以帮助回答有关活细胞中 ATP 含量和动态的生物学问题。正如预期的那样，在用 smacATPi 转染的培养 HEK293T 细胞中，2-脱氧葡萄糖（2-DG，一种糖酵解抑制剂）导致胞质 ATP 大幅减少，寡霉素（一种复合 V 抑制剂）显着减少线粒体 ATP。通过使用 smacATPi，我们还可以观察到 2-DG 处理适度减弱线粒体 ATP，寡霉素减少胞质 ATP，表明区室 ATP 的后续变化。为了评估 ATP/ADP 载体 (AAC) 在 ATP 运输中的作用，我们用 AAC 抑制剂白术苷 (ATR) 处理 HEK293T 细胞。 ATR 处理减弱了常氧条件下的细胞质和线粒体 ATP，表明 AAC 抑制减少了 ADP 从细胞质到线粒体的输入以及 ATP 从线粒体到细胞质的输出。在缺氧的 HEK293T 细胞中，ATR 处理增加了线粒体 ATP，同时减少了胞质 ATP，这表明缺氧期间 ACC 抑制可以维持线粒体 ATP，但可能不会抑制从胞质溶胶反向输入 ATP。此外，在缺氧情况下，当 ATR 与 2-DG 联合使用时，线粒体和胞质信号都会减弱。因此，使用 smacATPi 对时空 ATP 动态进行实时可视化提供了关于细胞质和线粒体 ATP 信号如何响应代谢变化的新见解，从而更好地了解健康和疾病中的细胞代谢。
Adenosine 5′ triphosphate (ATP) is the energy currency of life, which is produced in mitochondria (~90%) and cytosol (less than 10%). Real-time effects of metabolic changes on cellular ATP dynamics remain indeterminate. Here we report the design and validation of a genetically encoded fluorescent ATP indicator that allows for real-time, simultaneous visualization of cytosolic and mitochondrial ATP in cultured cells. This dual-ATP indicator, called smacATPi (simultaneous mitochondrial and cytosolic ATP indicator), combines previously described individual cytosolic and mitochondrial ATP indicators. The use of smacATPi can help answer biological questions regarding ATP contents and dynamics in living cells. As expected, 2-deoxyglucose (2-DG, a glycolytic inhibitor) led to substantially decreased cytosolic ATP, and oligomycin (a complex V inhibitor) markedly decreased mitochondrial ATP in cultured HEK293T cells transfected with smacATPi. With the use of smacATPi, we can also observe that 2-DG treatment modestly attenuates mitochondrial ATP and oligomycin reduces cytosolic ATP, indicating the subsequent changes of compartmental ATP. To evaluate the role of ATP/ADP carrier (AAC) in ATP trafficking, we treated HEK293T cells with an AAC inhibitor, Atractyloside (ATR). ATR treatment attenuated cytosolic and mitochondrial ATP in normoxia, suggesting AAC inhibition reduces ADP import from the cytosol to mitochondria and ATP export from mitochondria to cytosol. In HEK293T cells subjected to hypoxia, ATR treatment increased mitochondrial ATP along with decreased cytosolic ATP, implicating that ACC inhibition during hypoxia sustains mitochondrial ATP but may not inhibit the reversed ATP import from the cytosol. Furthermore, both mitochondrial and cytosolic signals decrease when ATR is given in conjunction with 2-DG in hypoxia. Thus, real-time visualization of spatiotemporal ATP dynamics using smacATPi provides novel insights into how cytosolic and mitochondrial ATP signals respond to metabolic changes, providing a better understanding of cellular metabolism in health and disease.
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影响因子：
8.8
作者：
Depaoli MR;Karsten F;Madreiter-Sokolowski CT;Klec C;Gottschalk B;Bischof H;Eroglu E;Waldeck-Weiermair M;Simmen T;Graier WF;Malli R
通讯作者：
Malli R
影响因子：
--
作者：
Chinopoulos C;Kiss G;Kawamata H;Starkov AA
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影响因子：
4.1
作者：
Farhat E;Cheng H;Romestaing C;Pamenter M;Weber JM
通讯作者：
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影响因子：
16.6
作者：
Lobas, Mark A.;Tao, Rongkun;Khakh, Baljit S.
通讯作者：
Khakh, Baljit S.
影响因子：
3.7
作者：
Conley JM;Radhakrishnan S;Valentino SA;Tantama M
通讯作者：
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