Dataset Information

Quantitative real-time imaging of glutathione.

ABSTRACT: Glutathione plays many important roles in biological processes; however, the dynamic changes of glutathione concentrations in living cells remain largely unknown. Here, we report a reversible reaction-based fluorescent probe-designated as RealThiol (RT)-that can quantitatively monitor the real-time glutathione dynamics in living cells. Using RT, we observe enhanced antioxidant capability of activated neurons and dynamic glutathione changes during ferroptosis. RT is thus a versatile tool that can be used for both confocal microscopy and flow cytometry based high-throughput quantification of glutathione levels in single cells. We envision that this new glutathione probe will enable opportunities to study glutathione dynamics and transportation and expand our understanding of the physiological and pathological roles of glutathione in living cells.

SUBMITTER: Jiang X

PROVIDER: S-EPMC5511354 | biostudies-literature | 2017 Jul

REPOSITORIES: biostudies-literature

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Quantitative real-time imaging of glutathione.

Jiang Xiqian X Chen Jianwei J Bajić Aleksandar A Zhang Chengwei C Song Xianzhou X Carroll Shaina L SL Cai Zhao-Lin ZL Tang Meiling M Xue Mingshan M Cheng Ninghui N Schaaf Christian P CP Li Feng F MacKenzie Kevin R KR Ferreon Allan Chris M ACM Xia Fan F Wang Meng C MC Maletić-Savatić Mirjana M Wang Jin J

Nature communications 20170713

Glutathione plays many important roles in biological processes; however, the dynamic changes of glutathione concentrations in living cells remain largely unknown. Here, we report a reversible reaction-based fluorescent probe-designated as RealThiol (RT)-that can quantitatively monitor the real-time glutathione dynamics in living cells. Using RT, we observe enhanced antioxidant capability of activated neurons and dynamic glutathione changes during ferroptosis. RT is thus a versatile tool that can ...[more]

PMID: 28703127

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Project description:In the malaria parasite Plasmodium falciparum, the cellular redox potential influences signaling events, antioxidant defense, and mechanisms of drug action and resistance. Until now, the real-time determination of the redox potential in malaria parasites has been limited because conventional approaches disrupt sub-cellular integrity. Using a glutathione biosensor comprising human glutaredoxin-1 linked to a redox-sensitive green fluorescent protein (hGrx1-roGFP2), we systematically characterized basal values and drug-induced changes in the cytosolic glutathione-dependent redox potential (EGSH) of drug-sensitive (3D7) and resistant (Dd2) P. falciparum parasites. Via confocal microscopy, we demonstrated that hGrx1-roGFP2 rapidly detects EGSH changes induced by oxidative and nitrosative stress. The cytosolic basal EGSH of 3D7 and Dd2 were estimated to be -314.2±3.1 mV and -313.9±3.4 mV, respectively, which is indicative of a highly reducing compartment. We furthermore monitored short-, medium-, and long-term changes in EGSH after incubation with various redox-active compounds and antimalarial drugs. Interestingly, the redox cyclers methylene blue and pyocyanin rapidly changed the fluorescence ratio of hGrx1-roGFP2 in the cytosol of P. falciparum, which can, however, partially be explained by a direct interaction with the probe. In contrast, quinoline and artemisinin-based antimalarial drugs showed strong effects on the parasites' EGSH after longer incubation times (24 h). As tested for various conditions, these effects were accompanied by a drop in total glutathione concentrations determined in parallel with alternative methods. Notably, the effects were generally more pronounced in the chloroquine-sensitive 3D7 strain than in the resistant Dd2 strain. Based on these results hGrx1-roGFP2 can be recommended as a reliable and specific biosensor for real-time spatiotemporal monitoring of the intracellular EGSH in P. falciparum. Applying this technique in further studies will enhance our understanding of redox regulation and mechanisms of drug action and resistance in Plasmodium and might also stimulate redox research in other pathogens.

Project description:The quantitative analysis of Plasmodium development in the liver in laboratory animals in cultured cells is hampered by low parasite infection rates and the complicated methods required to monitor intracellular development. As a consequence, this important phase of the parasite's life cycle has been poorly studied compared to blood stages, for example in screening anti-malarial drugs. Here we report the use of a transgenic P. berghei parasite, PbGFP-Luc(con), expressing the bioluminescent reporter protein luciferase to visualize and quantify parasite development in liver cells both in culture and in live mice using real-time luminescence imaging. The reporter-parasite based quantification in cultured hepatocytes by real-time imaging or using a microplate reader correlates very well with established quantitative RT-PCR methods. For the first time the liver stage of Plasmodium is visualized in whole bodies of live mice and we were able to discriminate as few as 1-5 infected hepatocytes per liver in mice using 2D-imaging and to identify individual infected hepatocytes by 3D-imaging. The analysis of liver infections by whole body imaging shows a good correlation with quantitative RT-PCR analysis of extracted livers. The luminescence-based analysis of the effects of various drugs on in vitro hepatocyte infection shows that this method can effectively be used for in vitro screening of compounds targeting Plasmodium liver stages. Furthermore, by analysing the effect of primaquine and tafenoquine in vivo we demonstrate the applicability of real time imaging to assess parasite drug sensitivity in the liver. The simplicity and speed of quantitative analysis of liver-stage development by real-time imaging compared to the PCR methodologies, as well as the possibility to analyse liver development in live mice without surgery, opens up new possibilities for research on Plasmodium liver infections and for validating the effect of drugs and vaccines on the liver stage of Plasmodium.

Dataset Information

Quantitative real-time imaging of glutathione.

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Quantitative real-time imaging of glutathione.

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