Centre National de la Recherche Scientifique, UMR3348, Genotoxic Stress and Cancer, Orsay, 91405, France; Institut Curie, Centre de Recherche, Orsay, 91405, France.
Centre National de la Recherche Scientifique, UMR3348, Genotoxic Stress and Cancer, Orsay, 91405, France; Institut Curie, Centre de Recherche, Orsay, 91405, France.
Free Radic Biol Med. 2013 Dec;65:436-445. doi: 10.1016/j.freeradbiomed.2013.07.033. Epub 2013 Jul 25.
Intracellular redox homeostasis is crucial for many cellular functions but accurate measurements of cellular compartment-specific redox states remain technically challenging. Genetically encoded biosensors including the glutathione-specific redox-sensitive yellow fluorescent protein (rxYFP) may provide an alternative way to overcome the limitations of conventional glutathione/glutathione disulfide (GSH/GSSG) redox measurements. This study describes the use of rxYFP sensors for investigating compartment-specific steady redox state and their dynamics in response to stress in human cells. RxYFP expressed in the cytosol, nucleus, or mitochondrial matrix of HeLa cells was responsive to the intracellular redox state changes induced by reducing as well as oxidizing agents. Compartment-targeted rxYFP sensors were able to detect different steady-state redox conditions among the cytosol, nucleus, and mitochondrial matrix. These sensors expressed in human epidermal keratinocytes HEK001 responded to stress induced by ultraviolet A radiation in a dose-dependent manner. Furthermore, rxYFP sensors were able to sense dynamic and compartment-specific redox changes caused by 100 μM hydrogen peroxide (H2O2). Mitochondrial matrix-targeted rxYFP displayed a greater dynamics of oxidation in response to a H2O2 challenge than the cytosol- and nucleus-targeted sensors, largely due to a more alkaline local pH environment. These observations support the view that mitochondrial glutathione redox state is maintained and regulated independently from that of the cytosol and nucleus. Taken together, our data show the robustness of the rxYFP sensors to measure compartmental redox changes in human cells. Complementary to existing redox sensors and conventional redox measurements, compartment-targeted rxYFP sensors provide a novel tool for examining mammalian cell redox homeostasis, permitting high-resolution readout of steady glutathione state and dynamics of redox changes.
细胞内氧化还原稳态对于许多细胞功能至关重要，但准确测量细胞区室特异性氧化还原状态在技术上仍然具有挑战性。包括谷胱甘肽特异性氧化还原敏感黄色荧光蛋白（rxYFP）在内的基因编码生物传感器可能提供了一种克服传统谷胱甘肽/谷胱甘肽二硫化物（GSH/GSSG）氧化还原测量限制的替代方法。本研究描述了 rxYFP 传感器在研究人类细胞中特定区室的稳态氧化还原状态及其对应激的动力学反应中的应用。在 HeLa 细胞的细胞质、核或线粒体基质中表达的 rxYFP 对还原剂和氧化剂诱导的细胞内氧化还原状态变化有反应。靶向区室的 rxYFP 传感器能够检测细胞质、核和线粒体基质之间不同的稳态氧化还原条件。这些在人表皮角质形成细胞 HEK001 中表达的传感器能够以剂量依赖的方式响应紫外线 A 辐射诱导的应激。此外，rxYFP 传感器能够感知 100μM 过氧化氢（H2O2）引起的动态和区室特异性氧化还原变化。与靶向细胞质和核的传感器相比，靶向线粒体基质的 rxYFP 显示出对 H2O2 挑战更大的氧化动力学，这主要是由于局部 pH 值更碱性。这些观察结果支持这样的观点，即线粒体谷胱甘肽氧化还原状态的维持和调节独立于细胞质和核。总之，我们的数据表明 rxYFP 传感器能够在人类细胞中测量区室氧化还原变化的稳健性。与现有的氧化还原传感器和传统的氧化还原测量方法互补，靶向区室的 rxYFP 传感器为研究哺乳动物细胞氧化还原稳态提供了一种新工具，能够对谷胱甘肽稳态和氧化还原变化的动力学进行高分辨率读出。
