Institute of Zoophysiology, WWU Münster, Münster, Germany.
Institute of Zoophysiology, WWU Münster, Münster, Germany.
Cell Signal. 2019 Oct;62:109330. doi: 10.1016/j.cellsig.2019.05.015. Epub 2019 May 29.
Signalling pathways provide a fine-tuned control network for catabolic and anabolic cellular processes under changing environmental conditions (e.g. changes in oxygen partial pressure, Po). These pathways frequently activate or deactivate transcription factors (TFs) in the cytoplasm, with the subsequent nuclear translocation of activated TFs constituting a prerequisite for gene control and expression. This study introduces a newly developed fluorometric method for the quantification of relationships between environmental factors and the subcellular localization of reporter-coupled TFs in Caenorhabditis elegans (and possibly other transparent organisms). We applied this method to determine and analyse the relationship between Po and the subcellular localization of the GFP-coupled transcription factor DAF-16 (FoxO) of the DAF-2 (insulin/IGF-1) signalling pathway via the DAF-16::GFP fluorescence intensity of whole worms (Po characteristic). The Po characteristic resembled the Po-specific metabolic rate of C. elegans, with a critical Po (Po) of 3.6 kPa separating two Po ranges, where either anaerobic metabolism and DAF-16::GFP nuclear occupancy strongly increased (i.e. decreasing DAF-16::GFP fluorescence intensity) (Po < Po) or aerobic metabolism and DAF-16::GFP cytoplasmic localization prevailed (Po > Po). These results and other data, which included the Po-specific mitochondrial oxidation-reduction state of whole worms (as determined using the endogenous NADH fluorescence) and the effects of higher levels of reactive oxygen species (ROS) or RNAi-mediated knockdowns of catabolic or anabolic control genes (aak-2 or let-363) on the Po characteristic, suggest that ROS play a decisive role for DAF-16 nuclear translocation due to tissue hypoxia or higher anabolic activity induced by aak-2(RNAi). As DAF-16 and its target genes are of central importance for the cellular stress resistance, ROS-mediated relationships between metabolism and DAF-16 subcellular (i.e. nuclear) localization provide protection of the cell machinery against elevated ROS formation under challenging metabolic conditions.
信号通路为细胞代谢的分解代谢和合成代谢过程提供了一个精细的调控网络，以适应不断变化的环境条件（例如，氧分压 Po 的变化）。这些途径经常在细胞质中激活或失活转录因子（TFs），随后激活的 TFs 向核内易位构成了基因控制和表达的前提。本研究介绍了一种新开发的荧光方法，用于量化环境因素与秀丽隐杆线虫（和可能其他透明生物体）中报告基因偶联 TFs 的亚细胞定位之间的关系。我们应用该方法来确定和分析 Po 与 GFP 偶联转录因子 DAF-16（FoxO）的亚细胞定位之间的关系，DAF-16 是 DAF-2（胰岛素/IGF-1）信号通路的一部分，通过整个蠕虫的 DAF-16::GFP 荧光强度（Po 特征）来进行分析。Po 特征类似于秀丽隐杆线虫的特定 Po 代谢率，临界 Po（Po）为 3.6 kPa，将两个 Po 范围分开，其中一种是无氧代谢和 DAF-16::GFP 核占据率强烈增加（即 DAF-16::GFP 荧光强度降低）（Po < Po），另一种是有氧代谢和 DAF-16::GFP 细胞质定位占主导地位（Po > Po）。这些结果和其他数据，包括整个蠕虫的特定 Po 线粒体氧化还原状态（使用内源性 NADH 荧光确定）以及更高水平的活性氧物种（ROS）或分解代谢或合成代谢控制基因（aak-2 或 let-363）的 RNAi 介导敲低对 Po 特征的影响，表明 ROS 在组织缺氧或 aak-2(RNAi) 诱导的更高的合成代谢活性下，对 DAF-16 核易位起着决定性的作用。由于 DAF-16 及其靶基因对细胞应激抵抗至关重要，因此代谢与 DAF-16 亚细胞（即核）定位之间的 ROS 介导的关系为细胞机制提供了保护，使其免受代谢条件挑战下 ROS 形成增加的影响。
