Live Visualization of Endoplasmic Reticulum Redox Potential in Zebrafish Embryos Reveals Region-Specific...

Objective: Redox homeostasis is an integral part of many cellular processes, and its perturbation is associated with conditions such as diabetes, aging, and neurodegenerative disorders. Redox homeostasis or redox potential in organelles is maintained within a particular range to facilitate the organelle-specific cellular redox reactions. Previous studies using yeast, cell systems, and nematodes have demonstrated that the Endoplasmic Reticulum (ER) has a more oxidizing environment, while the cytosol exhibits a reducing redox potential. However, we know very little about how universal this phenomenon is. Methods: We created transgenic zebrafish (Danio rerio) lines with roGFP sensors targeted to the ER and cytosol for studying physiological redox potential at the systems level. In the process, we also characterized the ER-targeting signal sequence in D. rerio for the first time. Results: Measurements of the redox state in live embryos found that the endoplasmic reticulum exhibits consistent deviations from its expected oxidizing redox state in multiple regions of the developing embryos. The ER is far more reduced than expected in certain tissues of the embryo, including certain regions of the brain. We confirmed this heterogeneity using another transgenic line expressing ER-targeted roGFPiE, a redox-sensitive GFP better suited to measuring changes in ER redox potential. We also observed the resilient nature of the ER redox state following tunicamycin (Tm) and Azetidine-2-carboxylic acid-induced proteostasis perturbations and only mild changes with Tm. Conclusions: While our study provides a first glimpse of the diversity in ER redox homeostasis, these unanticipated redox states of the ER will require new biological definitions.