A Redox-Sensitive Yellow Fluorescent Protein Sensor for Monitoring Nuclear Glutathione Redox Dynamics

Abstract

Intracellular redox homeostasis is crucial for many cellular functions, but accurate measurements of cellular compartment-specific redox states remain technically challenging. Genetically encoded biosensors, including the glutathione-specific redox-sensitive yellow fluorescent protein (rxYFP), provide an alternative approach to overcome the limitations of conventional glutathione/glutathione disulfide (GSH/GSSG) redox measurements. In this chapter we describe methods to measure the nuclear rxYFP redox state in human cells by a redox Western blot technique. A nucleus-targeted rxYFP sensor can be used to sense nuclear steady-state and dynamic redox changes in response to oxidative stress. Complementary to existing redox sensors and conventional redox measurements, nucleus-targeted rxYFP sensors provide a novel tool for examining nuclear redox homeostasis in mammalian cells, permitting high-resolution readout of steady glutathione state and dynamics of redox changes. The technique described may be used with minimal variations to study the effects of stress conditions which lead to glutathione redox changes.

Discerning the Chemistry in Individual Organelles with Small‐Molecule Fluorescent Probes

“…Therefore, the above problems should be taken into consideration to develop excellent NuTAFPs. Other than small‐molecule probes, we should also notice that researchers have made substantial progress in nucleus‐targeting fluorescent proteins and nanomaterials –…”

Wahrnehmung der chemischen Prozesse in einzelnen Organellen mit niedermolekularen Fluoreszenzsonden

“…Daher sollten die zuvor genannten Schwierigkeiten bei der Entwicklung hervorragender NuTAFPs berücksichtigt werden. Außerdem sind nicht nur bei den niedermolekularen Sonden, sondern auch bei auf den Zellkern zielenden Fluoreszenzproteinen und Nanomaterialien wesentliche Fortschritte erzielt worden –…”