Spatio-temporal control of the cytosolic redox environment in C. elegans

Compartmentalization of redox reactions is essential to all life forms. However, it has been difficult to study the redox potential of cellular environments within intact, living organisms. It is not known how this variable changes over the life of a multicellular organism, from its conception to death. Cell-types with various energetic requirements may differentiate the redox potentials of their cytosols, as has been suggested by biochemical measurements on dissected tissues. Unfortunately, these disruptive measurements preclude the investigation of dynamical changes taking place in the organism over space and time, under normal physiological conditions or in response to stimuli. Within cellular compartments, protein activity can respond to changes in the local redox environment through the reversible oxidation of cysteine thiols. For the majority of cysteines in the proteome, this interaction takes place through equilibration with the glutathione pool; this raises the question whether this redox pool acts as a buffer, or instead as a sensitive media, transducing information from a local physiological state into protein function. To pursue this question, we measured the spatio-temporal distribution of redox potential of the cytosolic glutathione pool, EGSH, in the nematode C. elegans. We used a genetically encoded redox sensor to quantify EGSH using ratiometric fluorescence microscopy. We found the steady state of the glutathione redox potential for different tissues (-268 mV to -271 mV) to be far from its buffering regime; these values were found instead in a regime where EGSH exhibits large changes in response to slight changes in the fraction of oxidized glutathione. This supports the idea that the glutathione pool acts as a responsive media, which integrates and broadcasts a local physiological state. Our work presents an alternative to the commonly held view that glutathione is a Advisor: Professor Walter Fontana Author: Catalina Romero iv buffering system, preventing change in the oxidative state of cysteine thiols in the proteome. We also found that the cytosolic redox environment is spatially structured in vivo: in the feeding organ, the pharynx, muscles segments show distinct redox potentials. These discrete redox environments occur despite the physical and electrical coupling of their cytosols through gap junctions. The spatial pattern observed persists upon oxidative stress, with specific muscles exhibiting unique EGSH response dynamics. We identified a mechanism that controls levels of EGSH as well as its spatial differentiation: insulin signaling modulates the level of cytosol oxidation, and the FOXO/DAF-16 transcription factor contributes to the spatial patterning of cytosolic EGSH in feeding muscle. Insulin signaling also modulates the cytosolic redox environment in other tissues, and it does so in a tissue-specific manner. Furthermore, we show that remote action of the transcription factor DAF-16 in the intestine lowers the cytosolic redox environment in feeding muscles, suggesting that tissues can influence each other’s redox state in a cell non-autonomous manner. The link between redox potential and insulin signaling – a major determinant of lifespan – prompted us to investigate the effects of aging on the redox pattern. We observed the pharyngeal glutathione pool during aging, and discovered that the spatial differentiation of the cytosolic redox potential undergoes structured changes. Our results show that insulin signaling modulates those changes. Notably, while the absolute level of EGSH is uncorrelated with lifespan, we do find a correlation between the spatial differences in redox potential, and the remaining lifespan of an animal. This shows that the spatial patterning of the redox environment of the pharynx is linked to the aging process.