Investigation of the spatial distribution of glutathione redox-balance in live cells by using Fluorescence Ratio Imaging Microscopy.

The dynamics of redox elements in biologic systems is a major challenge for redox signaling and oxidative stress research. Oxidative stress or signaling events can affect sulfur switches differently, thus creating a variation in the spatial distribution of these redox states, which therefore act simultaneously as regulators and indicators of key cellular functions in both physiological and pathological settings. A gluthatione specific redox-sensitive protein (i.e. a mutant of the Yellow Fluorescent Protein (rxYFP)) has been found to equilibrate in vivo with the gluthatione/gluthatione disulfide (GSH:GSSG) redox couple. rxYFP, employed ratiometrically, allows to generate high resolution maps of the fraction of the reduced protein (R) inside a cell. Here we developed an analytical procedure able to investigate intracellular changes in the glutathione redox-balance, which can occur in live mammalian cells, based on the deconvolution of the histogram of redox maps of 293-TPhoenix human embryonic kidney cells. The intracellular spatial distributions of oxidized and reduced elements have been discriminated. Finally, by transfecting cells with human Glutaredoxin V (GRX-V), an enzyme deputed to maintain reduced the thiol groups of their partner proteins, we can disclose that the significant shift towards more reduced state, with respect to that recovered from non-transfected cells, consists, instead, in a shift towards reduced values of the high R region (reduced), while leaving unaltered the glutathione redox-balance of the intracellular side of the plasma membrane.