Optogenetic Control of ROS Production in C. elegans

Alicia Wei, Keith Nehrke and Andrew Wojtovich 

Reactive Oxygen Species (ROS) can be detrimental or may lead to beneficial adaptive responses. The factors that distinguish between these outcomes are not readily determined using existing techniques. Here, we have developed a novel C. elegans model to study the effects of ROS in a physiologic context using a combination of CRISPR/Cas9 gene editing and optogenetics. Mitochondria are a main source of ROS and are central to cell death and adaptation to stress. We used cutting edge genetic techniques to fuse components of the mitochondrial respiratory chain to proteins that can produce ROS in response to light. MiniSOG produces singlet oxygen, which has the singular capacity to illicit damage, while SuperNova creates superoxide, which we predict may be beneficial in limited amounts. We fused miniSOG to complex II of the mitochondrial respiratory chain in C. elegans. The strain exhibited light-sensitive loss of complex II activity and the worms exhibited adverse reactions to light under conditions of mild stress such as, paraquat, an ROS generator or FCCP, a protonophore. In contrast, the strain expressing the SuperNova fusion had no adverse reaction to light and the conditions of mild stress when coupled with light. Future experiments will be necessary to determine whether light is in fact beneficial in this strain, as we hypothesize. With these constructs, we will be able to study the complex II of the mitochondrial respiratory chain ROS microdomains.

Advertisements