1b), but it could isomerize to its zwitterionic form upon binding to the self-labelling protein HaloTag (HT) 14, 15, 16, switching both its fluorescence and reactivity against GSH from an ‘off’ to an ‘on’ state. We hypothesized that a SiR dye in a spirocyclic form would be non-reactive against GSH (Fig. We based our design on the reactivity of SiR dyes, which in their zwitterionic form can serve as electrophiles that react with GSH (Fig. With this limitation in mind, we set out to develop a GSH sensor that is locally activated in the compartment of interest, thus providing a reliable measure of intra-organelle GSH concentrations. In fact, a similar question could be asked of any other targeted small-molecule fluorescent sensor. Accordingly, sensors could react with GSH in the cytosol, and it is difficult to assess whether small molecules actually sense the real concentrations of GSH in their target organelle or shuttle GSH from the cytosol. In the case of GSH sensing, the problem is even more difficult, because, to reach its intended organelle, the small molecule must cross the cytosol, where the concentration of GSH is in the range of 10 −3 M. Targeting small molecules to specific subcellular compartments, however, is challenging. The latter are particularly useful because of their redshifted excitation, selectivity, reversibility and fast kinetics. Notable examples include coumarin 9, 10, 11, 12 and silicon rhodamine (SiR) 13 dyes. Most of these probes are π-conjugated electrophiles that change their fluorescence upon reaction with GSH. Small-molecule sensors have been a popular choice to measure total GSH concentrations. Despite their usefulness, these proteins operate at short wavelengths (<450 nm), display modest brightness and have limited dynamic ranges 8. The GSH/GSSG ratio can be measured in specific organelles using redox-sensitive green fluorescent proteins (roGFPs), particularly those fused to the GSH-specific Grx1 protein 7. There is thus a need for robust tools that can quantify this critical redox modulator with subcellular specificity. A detailed understanding of the mechanisms of intracellular redox homeostasis and their connection to disease depends on our ability to quantify, among other parameters, changes in GSH concentration in specific organelles. Consequently, disruption of intracellular GSH homeostasis is linked to many pathologies, including cancer 4, diabetes 5 and neurodegenerative disorders 6. Furthermore, the concentration of GSH and the GSH/GSSG ratio vary between intracellular organelles, and this compartmentalization is actively regulated in the cell 3. Owing to its high concentration in the cell (~10 −3 M) 1, the GSH/GSSG pair acts as the main intracellular redox buffer, plays a key role in the detoxification of reactive oxygen species (ROS), and participates in redox signalling 2. Upon oxidation, it forms a disulfide-bonded dimer (GSSG). Glutathione (GSH) is a small peptide ( l-γ-glutamyl- l-cysteinyl glycine) that can react as a nucleophile or a reducing agent. Finally, by exchanging the fluorescent protein, we created a near-infrared, targetable and quantitative GSH sensor. This sensor was used in combination with a redox-sensitive fluorescent protein to quantify redox potential and GSH concentration simultaneously in the endoplasmic reticulum. Using TRaQ-G fused to a redox-insensitive fluorescent protein, we demonstrate that the nuclear and cytosolic GSH pools are independently regulated during cell proliferation. Furthermore, TRaQ-G can be fused to a fluorescent protein to give a ratiometric response. This chemogenetic sensor possesses a unique reactivity turn-on mechanism, ensuring that the small molecule is only sensitive to GSH in a desired location. Here we present a GSH-sensing platform for live-cell imaging, termed targetable ratiometric quantitative GSH (TRaQ-G). Achieving a detailed understanding of intracellular GSH homeostasis depends on the development of tools to map GSH compartmentalization and intra-organelle fluctuations. Glutathione (GSH) is the main determinant of intracellular redox potential and participates in multiple cellular signalling pathways.
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