PDF(Sci-hub)
Abstract：
Intracellular pH and redox states are critical for multiple processes and partly determine cell behavior. Here, we developed a genetically encoded dual-function probe, named p H and redox-sensitive fluorescent protein (pHaROS), for simultaneous real-time detection of changes in redox potential and pH in living cells. pHaROS consists of the Arabidopsis flavin mononucleotide-binding fluorescent protein iLOV and an mKATE variant, mBeRFP. Using pHaROS in Saccharomyces cerevisiae cells, we confirmed that H2O2 raises the overall redox potential of the cell and found that this increase is accompanied by a decrease in cytosolic pH. Furthermore, we observed spatiotemporal pH and redox homeostasis within the nucleus at various stages of the cell cycle in budding yeast (Saccharomyces cerevisiae) during cellular development and responses to oxidative stress. Importantly, we could tailor pHaROS to specific applications, including measurements in different organelles and cell types and the GSH/GSSG ratio, highlighting pHaROS\'s high flexibility and versatility. In summary, we have developed pHaROS as a dual-function probe that can be used for simultaneously measuring cellular pH and redox potential, representing a very promising tool for determining the cross-talk between intracellular redox- and pH-signaling processes in yeast and mammalian U87 cell.
摘要：
细胞内pH和氧化还原状态对于多个过程至关重要，并部分决定细胞行为。这里,我们开发了一种基因编码的双功能探针，命名为pH和氧化还原敏感荧光蛋白（pHaros），用于同时实时检测活细胞中氧化还原电位和pH的变化。pHaROS由拟南芥黄素单核苷酸结合荧光蛋白iLOV和mKATE变体组成，mBeRFP。在酿酒酵母细胞中使用pHaROS，我们证实H2O2会提高细胞的整体氧化还原电位，并发现这种增加伴随着细胞溶质pH值的降低。此外，在细胞发育和对氧化应激的反应过程中，我们观察了出芽酵母（酿酒酵母）细胞周期各个阶段细胞核内的时空pH和氧化还原稳态。重要的是,我们可以为特定的应用定制pharos，包括不同细胞器和细胞类型的测量以及GSH/GSSG比率，突出了pHAROS的高度灵活性和多功能性。总之,我们已经开发了pHaROS作为双功能探针，可用于同时测量细胞的pH和氧化还原电位，代表一个非常有前途的工具，用于确定酵母和哺乳动物U87细胞中细胞内氧化还原和pH信号传导过程之间的串扰。
