Expanding the genetic toolbox to explore redox signalling
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Over the last decade, the importance of redox signalling and reactive oxygen species (ROS) for maintaining physiological signal transduction have been discovered. ROS, and in particular hydrogen peroxide (H2O2), have previously been seen as harmful to cells, leading to oxidative stress and disease. However, in physiological concentrations, H2O2 acts as a second messenger in multiple signalling pathways. Research in redox signalling requires physiological environments and requires tools that can measure and model the dynamic nature of oxidation reactions. Here, we set-up two genetic tools in different model systems, to help uncover the role of the redox environment in cells. HyPer7 is a H2O2 sensor, that enables visualisation of the net rate of H2O2 production and scavenging with subcellular resolution. We set-up and characterised HyPer7 in 3D systems, to explore the use of HyPer7 in future experiments on the importance of ROS in cell processes like polarity and differentiation. We deployed HyPer7 to visualise whether there are changes in the H2O2-dependent redox environment after treating cells with the anti-cancer drug 5-FU, that is suspected to induce increase ROS production in cells. However, we detect no increase in HyPer7 signal upon 5-FU treatment. Besides HyPer7, we used D-amino acid oxidase (DAAO), a genetically encoded enzyme that, in the presence of D-amino acids, can produce H2O2 inside cells. We set-up the DAAO system in haploid cells, where DAAO proves to be a simple way to model physiological H2O2 production in cells. Haploid cells are often used in genetic knock-out screens, as knock-out of only one allele is sufficient for a full knock-out phenotype. DAAO expression in haploid cells creates an opportunity to use these cells in haploid genetic knock-out screens to help discover the mechanism of redox-related cell cycle arrest. Characterisation of DAAO in haploid cells showed that there is no induction of redox-related cell cycle arrest. However, ROS-induced cell death is seen in haploid cells, and can be used in future genetic screens. All in all, HyPer7 and DAAO both are useful tools and create new opportunities for exploring redox signalling.