Developing new optogenetic redox biosensors for sulfur compounds
PI : Jérémy Couturier (UMR 1136 Interactions Arbres/Micro-organismes – IAM)
Collaborations : Bruce Morgan (Univ. of Kaiserslautern, Germany), Andreas Meyer (Univ. of Bonn, Germany), Markus Schwarzländer (Univ. of Münster, Germany)
Context — A plethora of genetically-encoded/optogenetic redox fluorescent biosensors has been engineered in recent years, which opened new avenues in performing dynamic measurements of a wide range of biological substances. The roGFP2 (reduction-oxidation GFP) is one of these probes which allow non-invasive, real-time cell imaging and can be targeted to specific subcellular compartments. In recent years, the development of fluorescent probes has revolutionized our experimental access to physiological parameters in live cells. Compared to nitrogen and phosphorus metabolism, the study of plant sulfur metabolism still lags behind, although more attention has been paid to this pathway recently. At the molecular level, mechanisms of sulfur trafficking remain largely unexplored. Sulfur compounds and notably cysteine-related compounds are indispensable to plant physiology, development and stress response. Developing optogenetic biosensors that allow a better understanding of cysteine metabolism appears necessary.
Objectives — This project aims at new developing optogenetic redox biosensors for cysteine and two related sulfur compounds, thiosulfate and 3-mercaptopyruvate.
Approaches — Preliminary experiments demonstrating that some sulfurtransferase and cysteine desulfurase isoforms catalyze the oxidation of roGFP2 in presence of specific sulfur compounds, 3-mercaptopyruvate, thiosulfate and cysteine, opened up the possibility to engineer new optogenetic biosensors. We will firstly evaluate the capacity of selected enzymes to oxidize roGFP2 using either isolated recombinant proteins or fusion proteins to the N- or C-terminal part of roGFP2. Then, interesting candidates will be tested in vivo by expressing them in the adequate subcellular compartments of yeast and plant cells.
Expected results and impacts — Considering the strong impact that the glutathione- and H2O2– specific probes have had on our view of the metabolism of both molecules, developing probes assessing the levels of sulfur compounds could be of extreme importance for physiologists studying redox homeostasis, stress responses and all phenomena influencing the cysteine metabolism in plants but also in other living organisms.