Minimizing ATP depletion by oxygen scavengers for single-molecule fluorescence imaging in live cells
Minimizing ATP depletion by oxygen scavengers for single-molecule fluorescence imaging in live cells
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DOI：
10.1073/pnas.1717724115
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发表时间：
2018-02
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影响因子：
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通讯作者：
Seung-Ryoung Jung;Yi Deng;C. Kushmerick;C. Asbury;B. Hille;D. Koh
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作者：
Seung-Ryoung Jung;Yi Deng;C. Kushmerick;C. Asbury;B. Hille;D. Koh
意义 单分子活细胞成像可以回答许多生物学问题，但一个重大障碍是溶解氧对染料分子的光漂白。提高光稳定性的一个极好方法是使用葡萄糖氧化酶（一种除氧剂）来降低溶液中的氧含量。然而，对于活细胞实验来说，清除剂的一个关键缺点是，随着线粒体缺氧，ATP 水平会下降。我们发现，某些代谢补充剂可显着减缓除氧剂对 ATP 的消耗，并显着恢复 ATP 依赖性功能，如脂质合成和受体内吞作用。这种方法对于需要控制活细胞中氧张力和细胞内 ATP 水平的科学研究很有价值。有机染料抗光漂白的稳定性在单分子追踪和定位显微镜中至关重要。由于氧气会加速大多数有机染料的光漂白，因此葡萄糖氧化酶通常用于通过消耗氧气来减缓染料的光漂白。如此处所示，吡喃糖 2-氧化酶可将 Alexa647 染料的漂白速度减慢约 20 倍。然而，缺氧可能会减少线粒体氧化磷酸化和 ATP 产生，从而给活细胞带来严重问题。我们制定了一种在氧清除剂存在的情况下维持细胞内 ATP 水平的方法。尽管氧水平降低，但补充代谢中间体（包括甘油醛、谷氨酰胺和 α-酮异己酸）可通过 FADH2 和 NADH 之间的平衡维持细胞内 ATP 水平至少 10 分钟。此外，这些代谢物支持 ATP 依赖性磷脂酰肌醇 4,5-二磷酸的合成和 PAR2 受体的内化。我们的方法可能与涉及氧气水平急剧下降的其他情况相关，例如大脑或心脏或移植组织的缺血性损伤。
Significance Single-molecule live-cell imaging can answer many biological questions, but a significant obstacle is photobleaching of the dye molecules by dissolved oxygen. An excellent way to improve photostability is to reduce the oxygen content of the solutions using glucose oxidase, an oxygen scavenger. However, for live-cell experiments a critical downside of the scavengers is that ATP levels fall as mitochondria become oxygen-deprived. We show that certain metabolic supplements significantly slow the depletion of ATP by oxygen scavengers and substantially restore ATP-dependent functions like lipid synthesis and receptor endocytosis. This method could be valuable for scientific studies that need to control oxygen tension and intracellular ATP levels in live cells. The stability of organic dyes against photobleaching is critical in single-molecule tracking and localization microscopy. Since oxygen accelerates photobleaching of most organic dyes, glucose oxidase is commonly used to slow dye photobleaching by depleting oxygen. As demonstrated here, pyranose-2-oxidase slows bleaching of Alexa647 dye by ∼20-fold. However, oxygen deprivation may pose severe problems for live cells by reducing mitochondrial oxidative phosphorylation and ATP production. We formulate a method to sustain intracellular ATP levels in the presence of oxygen scavengers. Supplementation with metabolic intermediates including glyceraldehyde, glutamine, and α-ketoisocaproate maintained the intracellular ATP level for at least 10 min by balancing between FADH2 and NADH despite reduced oxygen levels. Furthermore, those metabolites supported ATP-dependent synthesis of phosphatidylinositol 4,5-bisphosphate and internalization of PAR2 receptors. Our method is potentially relevant to other circumstances that involve acute drops of oxygen levels, such as ischemic damage in the brain or heart or tissues for transplantation.