ROS-mediated relationships between metabolism and DAF-16 subcellular localization in Caenorhabditis elegans revealed by a novel fluorometric method.

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.