Extending roGFP Emission via Förster-Type Resonance Energy Transfer Relay Enables Simultaneous Dual Compartment Ratiometric Redox Imaging in Live Cells.
Extending roGFP Emission via Förster-Type Resonance Energy Transfer Relay Enables Simultaneous Dual Compartment Ratiometric Redox Imaging in Live Cells.
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DOI：
10.1021/acssensors.7b00689
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发表时间：
2017-11-22
期刊：
影响因子：
8.9
通讯作者：
Tantama M
中科院分区：
文献类型：
--
作者：
Norcross S;Trull KJ;Snaider J;Doan S;Tat K;Huang L;Tantama M
活性氧（ROS）介导细胞间和细胞器内信号传导，并且ROS在细胞区室如线粒体和胞质溶胶之间传播氧化应激。每个细胞区室都含有自己的ROS来源以及抗氧化机制，这有助于在信号传导，代谢和应激期间发生的ROS水平的动态波动。然而，细胞室之间的氧化还原动力学的耦合还没有得到很好的研究，因为缺乏可用的传感器，同时测量一个以上的亚细胞室在同一个细胞。目前，氧化还原敏感的绿色荧光蛋白，roGFP，已被广泛用于研究区室特异性氧化还原动力学，因为它提供了一个定量的比率读数，它是服从亚细胞靶向作为一个遗传编码的传感器。在这里，我们报告了一个新的家族的遗传编码的荧光蛋白传感器，通过Förster型共振能量转移到受体红色荧光蛋白的双色活细胞显微镜延长roGFP的荧光发射。我们表征的传感器蛋白的氧化还原和光学特性，我们证明，它们可以用来同时测量活细胞中的胞质和线粒体ROS。此外，我们使用这些传感器来揭示细胞间异质性的细胞质和线粒体之间的氧化还原偶联时，神经母细胞瘤细胞暴露于还原和代谢压力。
Reactive oxygen species (ROS) mediate both intercellular and intraorganellar signaling, and ROS propagate oxidative stress between cellular compartments such as mitochondria and the cytosol. Each cellular compartment contains its own sources of ROS as well as antioxidant mechanisms, which contribute to dynamic fluctuations in ROS levels that occur during signaling, metabolism, and stress. However, the coupling of redox dynamics between cellular compartments has not been well studied because of the lack of available sensors to simultaneously measure more than one subcellular compartment in the same cell. Currently, the redox-sensitive green fluorescent protein, roGFP, has been used extensively to study compartment-specific redox dynamics because it provides a quantitative ratiometric readout and it is amenable to subcellular targeting as a genetically-encoded sensor. Here, we report a new family of genetically-encoded fluorescent protein sensors that extend the fluorescence emission of roGFP via Förster-type resonance energy transfer to an acceptor red fluorescent protein for dual-color live-cell microscopy. We characterize the redox and optical properties of the sensor proteins, and we demonstrate that they can be used to simultaneously measure cytosolic and mitochondrial ROS in living cells. Furthermore, we use these sensors to reveal cell-to-cell heterogeneity in redox coupling between the cytosol and mitochondria when neuroblastoma cells are exposed to reductive and metabolic stresses.
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DOI：
10.1073/pnas.76.5.2175
发表时间：
1979-01-01
影响因子：
11.1
作者：
DEUTSCH, C;ERECINSKA, M;SILVER, IA
通讯作者：
SILVER, IA
影响因子：
4.3
作者：
Abraham, Bobin George;Santala, Ville;Karp, Matti
通讯作者：
Karp, Matti
影响因子：
16.6
作者：
Exposito-Rodriguez M;Laissue PP;Yvon-Durocher G;Smirnoff N;Mullineaux PM
通讯作者：
Mullineaux PM
影响因子：
4.8
作者：
Li, NY;Ragheb, K;Robinson, JP
通讯作者：
Robinson, JP
影响因子：
7.4
作者：
Fan, Yichong;Chen, Zhijie;Ai, Hui-wang
通讯作者：
Ai, Hui-wang