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, 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 (Pco2) 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 < Pco2) or aerobic metabolism and DAF-16::GFP cytoplasmic localization prevailed (Po2 > Pco2). 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.

PMK-1 p38 MAPK promotes cadmium stress resistance, the expression of SKN-1/Nrf and DAF-16 target genes, and protein biosynthesis in Caenorhabditis elegans.

The mechanisms of cadmium (Cd) resistance are complex and not sufficiently understood. The present study, therefore, aimed at assessing the roles of important components of stress-signaling pathways and of ABC transporters under severe Cd stress in Caenorhabditis elegans. Survival assays on mutant and control animals revealed a significant promotion of Cd resistance by the PMK-1 p38 MAP kinase, the transcription factor DAF-16/FoxO, and the ABC transporter MRP-1. Transcriptome profiling by RNA-Seq on wild type and a pmk-1 mutant under control and Cd stress conditions revealed, inter alia, a PMK-1-dependent promotion of gene expression for the translational machinery. PMK-1 also promoted the expression of target genes of the transcription factors SKN-1/Nrf and DAF-16 in Cd-stressed animals, which included genes for molecular chaperones or immune proteins. Gene expression studies by qRT-PCR confirmed the positive effects of PMK-1 on DAF-16 activity under Cd stress and revealed negative effects of DAF-16 on the expression of genes for MRP-1 and DAF-15/raptor. Additional studies on pmk-1 RNAi-treated wild type and mutant strains provided further information on the effects of PMK-1 on SKN-1 and DAF-16, which resulted in a model of these relationships. The results of this study demonstrate a central role of PMK-1 for the processing of cellular responses to abiotic and biotic stressors, with the promoting effects of PMK-1 on Cd resistance mostly mediated by the transcription factors SKN-1 and DAF-16.

Detoxification and sensing mechanisms are of similar importance for Cd resistance in Caenorhabditis elegans.

The present study employed mass spectrometry (ICP-MS) to measure the internal cadmium concentrations (Cdint) in Caenorhabditis elegans to determine Cd uptake from a Cd-containing environment as well as Cd release under Cd-free conditions. To analyze the functional role of several ATP binding cassette (ABC) transporters (e.g., HMT-1 and MRP-1) and phytochelatin synthase (PCS), we compared wild-type (WT) and different mutant strains of C. elegans. As a pre-test on selected mutant strains, several time-resolved experiments were performed to determine the survival rate and avoidance behavior of C. elegans under Cd stress, which confirmed the already known Cd sensitivity of the deletion mutants mrp-1Δ, pcs-1Δ, and hmt-1Δ. In addition, these experiments revealed flight reactions under Cd stress to be almost completely absent in mrp-1Δ mutants. The ICP-MS studies showed Cd uptake to be significantly higher in mrp-1Δ and WT than in hmt-1Δ. As Cd is ingested with food, food refusal due to very early Cd stress and its perception was likely the reason for the reduced Cd uptake of hmt-1Δ. Cd release (detoxification) was found to be maximal in mrp-1Δ, minimal in hmt-1Δ, and intermediate in WT. High mortality under Cd stress, food refusal, and minimal Cd release in the case of hmt-1Δ suggest a vital importance of the HMT-1/PCS-1 detoxification system for the survival of C. elegans under Cd stress. High mortality under Cd stress, absence of an avoidance behavior, missing food refusal, and maximal Cd release in the case of mrp-1Δ indicate that MRP-1 is less important for Cd detoxification under severe stress, but is probably important for Cd perception. Accordingly, our results suggest that the survival of WT under Cd stress (or possibly other forms of metal stress) primarily depends on the function of the HMT-1/PCS-1 detoxification system and the presence of a sensing mechanism to control the uptake of Cd (or other metals), which keeps internal Cd (or metal) concentrations under control, to some extent, for the timely mobilization of protection and repair systems.