ROS-mediated relationships between metabolism and DAF-16 subcellular localization in Caenorhabditis elegans revealed...

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, Po₂). 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 Po₂ 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 (Po₂ characteristic). The Po₂ characteristic resembled the Po₂-specific metabolic rate of C. elegans, with a critical Po₂ (P_co₂) of 3.6 kPa separating two Po₂ ranges, where either anaerobic metabolism and DAF-16::GFP nuclear occupancy strongly increased (i.e. decreasing DAF-16::GFP fluorescence intensity) (Po₂ < P_co₂) or aerobic metabolism and DAF-16::GFP cytoplasmic localization prevailed (Po₂ > P_co₂). These results and other data, which included the Po₂-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 Po₂ 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.