The ability to monitor changes to intracellular conditions in live cells has been made possible by the development of reporter GFP molecules that change their fluorescence properties upon changes in calcium concentration or redox status.
Here we show that one such modified GFP, roGFP1-iL-KDEL, can be used to monitor changes to the redox status within the ER of mammalian cells and is suited to real-time microscopy in living cells. This reporter monitors dynamic changes to the ER redox conditions, thereby allowing the effect of either specific reagents or of modulation of the levels of individual proteins to be evaluated. The dynamic range of the reporter is sufficient to follow subtle changes in the ER redox status as well as recovery from challenges with either reducing or oxidising agents.
In contrast to the previously used roGFP probe (roGFP2) used to study changes in redox status on the ER roGFP1-iL-KDEL has a redox poise allowing more oxidising as well as more reducing changes to be monitored. The roGFP2 used previously is fully oxidised in the ER due to its more negative reduction potential than roGFP1-iL-KDEL. The disulphide bond within roGFP1 is thought to primarily equilibrate with the GSH/GSSG buffer within cells. Hence the ability to monitor the redox status of roGFP1-iL-KDEL provides a sensitive measurement of changes to the glutathione buffer within the ER.