Redox-sensitive YFP sensors monitor dynamic nuclear and cytosolic glutathione redox changes.
Redox-sensitive YFP sensors monitor dynamic nuclear and cytosolic glutathione redox changes.
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DOI：
10.1016/j.freeradbiomed.2012.04.004
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发表时间：
2012-06-01
影响因子：
7.4
通讯作者：
Huang, Meng-Er
中科院分区：
文献类型：
--
作者：
Dardalhon, Michele;Kumar, Chitranshu;Iraqui, Ismail;Vernis, Laurence;Kienda, Guy;Banach-Latapy, Agata;He, Tiantian;Chanet, Roland;Faye, Gerard;Outten, Caryn E.;Huang, Meng-Er
细胞内氧化还原稳态对于许多细胞功能是至关重要的，但是细胞区室特异性氧化还原状态的精确测量仍然具有技术挑战性。为了更好地表征细胞核中的氧化还原控制，我们将基于黄色荧光蛋白的氧化还原传感器（rxYFP）靶向酵母S.啤酒。核-rxYFP和胞质-rxYFP的氧化还原状态的平行分析使我们能够在给定条件下监测这两个隔室内的明显动态谷胱甘肽（GSH）氧化还原变化。我们观察到，在稳态条件下，细胞核GSH氧化还原环境是高度还原的，类似于细胞质。此外，这些传感器能够检测谷胱甘肽还原酶（Glr 1）和硫氧还蛋白途径（Trr 1，Trx 1，Trx 2）突变体中各自隔室的氧化还原变化，这些突变体改变了亚细胞氧化还原环境。我们的突变体氧化还原数据提供了体内证据，谷胱甘肽和硫氧还蛋白氧化还原系统在控制亚细胞氧化还原环境中发挥不同但重叠的功能。我们还监测了细胞核-rxYFP和细胞质-rxYFP对GSH耗竭和外源性低剂量和高剂量H2 O2爆发的动态响应。这些观察结果表明核-rxYFP和胞质溶胶-rxYFP两者的快速且几乎同时的氧化，突出了rxYFP传感器在测量实时区室氧化还原变化中的稳健性。总而言之，我们的数据表明，高度还原的酵母细胞核和胞质氧化还原状态在一定程度上独立维持，并且受到不同但微妙的氧化还原调节。核和胞质溶胶- rxYFP寄存器隔室特异性局部氧化还原波动，可能涉及这两个隔室之间的还原型和/或氧化型谷胱甘肽的交换。最后，我们证实，谷胱甘肽耗竭线粒体基因组的稳定性有深远的影响，但对核基因组的稳定性影响不大，从而强调在生长过程中对谷胱甘肽的关键需求是与一个依赖于谷胱甘肽的过程。
Intracellular redox homeostasis is crucial for many cellular functions but accurate measurements of cellular compartment-specific redox states remain technically challenging. To better characterize redox control in the nucleus, we targeted a yellow fluorescent protein-based redox sensor (rxYFP) to the nucleus of the yeast S. cerevisiae. Parallel analyses of the redox state of nucleus-rxYFP and cytosol-rxYFP allow us to monitor distinctively dynamic glutathione (GSH) redox changes within these two compartments in a given condition. We observed that the nuclear GSH redox environment is highly reducing and similar to the cytosol under steady state conditions. Furthermore, these sensors are able to detect redox variations specific for their respective compartments in glutathione reductase (Glr1) and thioredoxin pathway (Trr1, Trx1, Trx2) mutants that have altered subcellular redox environments. Our mutant redox data provide in vivo evidence that glutathione and the thioredoxin redox system play distinct but overlapping functions in controlling subcellular redox environments. We also monitored the dynamic response of nucleus-rxYFP and cytosol-rxYFP to GSH depletion and to exogenous low and high doses of H2O2 bursts. These observations indicate a rapid and almost simultaneous oxidation of both nucleus-rxYFP and cytosol-rxYFP, highlighting the robustness of the rxYFP sensors in measuring real-time compartmental redox changes. Taken together, our data suggest that the highly reduced yeast nuclear and cytosolic redox states are maintained independently to some extent and under distinct but subtle redox regulation. Nucleus- and cytosol- rxYFP register compartment-specific localized redox fluctuations that may involve exchange of reduced and/or oxidized glutathione between these two compartments. Finally, we confirmed that GSH depletion has profound effects on mitochondrial genome stability but little effect on nuclear genome stability, thereby emphasizing that the critical requirement for GSH during growth is linked to a mitochondria-dependent process.
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影响因子：
56.9
作者：
HWANG, C;SINSKEY, AJ;LODISH, HF
通讯作者：
LODISH, HF
线粒体膜间隙中的氧化还原环境与细胞质和基质是分开的
影响因子：
4.8
作者：
Hu, Jingjing;Dong, Lixue;Outten, Caryn E.
通讯作者：
Outten, Caryn E.
影响因子：
3.6
作者：
Grant, CM;Collinson, LP;Dawes, IW
通讯作者：
Dawes, IW
DOI：
10.1073/pnas.0509035102
发表时间：
2005-12-13
影响因子：
11.1
作者：
Austin, CD;Wen, XH;Scales, SJ
通讯作者：
Scales, SJ
影响因子：
7.4
作者：
Bonini, MG;Rota, C;Mason, RP
通讯作者：
Mason, RP