Compartment‐specific neuron activity‐dependent redox dynamics

Abstract

Reactive oxygen species (ROS), redox active proteins, and molecules such as glutathione are central to the propagation of redox signals and oxidative stress. Propagation can occur between cells in the tissue environment, and it can also occur across organelles and cellular compartments within a single cell. However, simultaneous measurements of compartment‐specific redox dynamics at the single‐cell level have remained a challenge. We are therefore developing genetically‐encoded red fluorescent redox sensors that can be used together with currently available green fluorescent redox sensors in multiplexed live‐cell imaging experiments. Our protein engineering strategy employs Förster‐type resonance energy transfer (FRET) between the redox‐sensitive green fluorescent protein (roGFP) as the energy donor tethered to various red fluorescent proteins acceptors. This FRET‐based design generates a spectral relay which will ideally shift the green emission from roGFP to orange and red emission wavelengths. We have validated a set of second‐generation “rogRFP” sensors, and we have measured activity‐dependent redox changes in the cytosol and mitochondria of individual neurons that differ in compartment‐specific phenotypes. Our experiments with cultured primary mouse hippocampal neurons demonstrates that our sensors will be effective tools for studying compartment‐specific redox dynamics and signaling.Support or Funding InformationNIH/NINDS R21 NS106319This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.