Green fluorescent protein-based monitoring of endoplasmic reticulum redox poise.
Green fluorescent protein-based monitoring of endoplasmic reticulum redox poise.
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DOI：
10.3389/fgene.2013.00108
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发表时间：
2013
影响因子：
3.7
通讯作者：
Appenzeller-Herzog C
中科院分区：
文献类型：
--
作者：
Birk J;Ramming T;Odermatt A;Appenzeller-Herzog C
病理性内质网应激与活性氧的积累密切相关，而活性氧可能是内质网应激的上游和下游。因此，有害的细胞内应激信号通过建立一个恶性循环被放大。越来越多的人类疾病以内质网应激和氧化损伤引起的组织萎缩为特征。因此，实验监测应力诱导的内质网还原-氧化（氧化还原）状态的时间分辨变化是重要的。基因编码的荧光探针的出现促进了氧化还原变化的细胞器特异性检查，荧光探针可以通过特定的氨基酸延伸来靶向不同的亚细胞位置。这些探针包括氧化还原敏感的绿色荧光蛋白（roGFPs）和基于黄色荧光蛋白的氧化还原生物传感器HyPer。在rogfp的情况下，已知的特异性的变体定义氧化还原对现在是可用的。在这里，我们回顾了利用内质网靶向荧光生物传感器测量内质网氧化还原变化的实验框架。讨论了平板阅读器和基于显微镜的测量的优点和缺点，并在选择的内质网应激细胞培养模型的背景下证明了这些技术的力量。
Pathological endoplasmic reticulum (ER) stress is tightly linked to the accumulation of reactive oxidants, which can be both upstream and downstream of ER stress. Accordingly, detrimental intracellular stress signals are amplified through establishment of a vicious cycle. An increasing number of human diseases are characterized by tissue atrophy in response to ER stress and oxidative injury. Experimental monitoring of stress-induced, time-resolved changes in ER reduction-oxidation (redox) states is therefore important. Organelle-specific examination of redox changes has been facilitated by the advent of genetically encoded, fluorescent probes, which can be targeted to different subcellular locations by means of specific amino acid extensions. These probes include redox-sensitive green fluorescent proteins (roGFPs) and the yellow fluorescent protein-based redox biosensor HyPer. In the case of roGFPs, variants with known specificity toward defined redox couples are now available. Here, we review the experimental framework to measure ER redox changes using ER-targeted fluorescent biosensors. Advantages and drawbacks of plate-reader and microscopy-based measurements are discussed, and the power of these techniques demonstrated in the context of selected cell culture models for ER stress.