DOI: 10.24075/brsmu.2018.005

ORIGINAL RESEARCH

Observing the dynamics of the NAD+ to NADH ratio in Danio rerio embryo tissues using a genetically encoded biosensor

Bilan DS1,2, Shokhina AG1, Panova AS1,3, Belousov VV1,2
About authors

1 Laboratory for Molecular Technologies,
Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences, Moscow, Russia

2 Department of Brain-Computer Interfaces, Research Institute for Translational Medicine,
Pirogov Russian National Research Medical University, Moscow, Russia

3 Department of Biochemistry, Faculty of Biology,
Lomonosov Moscow State University, Moscow, Russia

Correspondence should be addressed: Vsevolod Belousov
ul. Miklukho-Maklaya, d. 16/10, Moscow, Russia, 117997; ur.hcbi@vosuoleb

About paper

Funding: this work was supported by the Russian Foundation for Basic Research (Grant 16-34-60175) and the Russian Federation Presidential Grant MK-6339.2016.4. Experiments were partially carried out using the equipment provided by the IBCH сore facility (CKP IBCH, supported by Russian Ministry of Education and Science, grant RFMEFI62117X0018).

Received: 2017-12-05 Accepted: 2017-12-25
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Fig. 1. Fluorescence excitation spectrum of the SoNar protein-based biosensor with two typical peaks at 420 nm and 490 nm. When NADH is added, fluorescence intensity increases at 420 nm and weakens at 490 nm. When NAD+ is added, signal intensity increases at 490 nm. The SoNar biosensor has one emission peak with emission maximum at 518 nm
Fig. 2. Changes in the NAD+/NADH ratio and pH in the tissues of one-day old D. rerio embryos before and after pyruvate and lactate injections into the yolk sac. (A and D) Images of a D. rerio embryo body before and after injections of 1 nl 200 mM lactate (A) and pyruvate (D) solutions. Images are shown in pseudo-colors corresponding to the SoNar signal values (F420/F490). Zones 1 and 2 circled in white in the first picture indicate regions on the embryo’s body where F420/F490 fluctuations were registered. These regions were the same for all inspected fish. (B and E) Changes in the NAD+/NADH ratio measured using the SoNar biosensor in the yolk sac (red line) and body (black line) of the D. rerio embryo before and after injections of lactate (B) and pyruvate (E) solutions. (C and F) Changes in pH registered using the SypHer2 in the yolk sac (red line) and body (black line) of a D. rerio embryo before and after injections of lactate (C) and pyruvate (F). For all graphs (B, C, E, F) the error bar corresponds to the standard deviation. Graph (C) was constructed based on the imaging of 10 fish. Graph (E) — based on the imaging of 11 fish. Graphs (C) and (F) — based on the imaging of 6 animals (per graph)