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Autofluorescence Imaging to Evaluate Cellular Metabolism
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Genetically encoded fluorescent indicator for imaging NAD(+)/NADH ratio changes in different cellular compartments.

Dmitry S Bilan1, Mikhail E Matlashov1, Andrey Yu Gorokhovatsky2

  • 1Moscow Institute of Physics and Technology, 141700 Moscow Region, Russia; Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, 117997 Moscow, Russia.

Biochimica Et Biophysica Acta
|November 30, 2013
PubMed
Summary

Researchers developed RexYFP, a novel genetically encoded fluorescent probe for real-time monitoring of cellular nicotinamide adenine dinucleotide (NAD+/NADH) redox states. This tool accurately tracks NAD(H) dynamics across cellular compartments, overcoming previous limitations.

Keywords:
Fluorescent probeNAD(+)/NADH ratioRedox sensor

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Area of Science:

  • Cellular biology
  • Biochemistry
  • Biophysics

Background:

  • The cellular NAD+/NADH ratio is a critical indicator of the redox state.
  • Real-time monitoring of intracellular NAD(H) dynamics was previously limited.
  • Genetically encoded fluorescent probes offer a novel approach to study NAD(H) fluctuations.

Purpose of the Study:

  • To develop a genetically encoded fluorescent probe for real-time monitoring of NAD(H) redox state in living cells.
  • To characterize the probe's performance in vitro and in cultured mammalian cells.
  • To assess the probe's utility in different cellular compartments and under various conditions.

Main Methods:

  • Development of a genetically encoded probe (RexYFP) by inserting circularly permuted YFP into the T-REX redox sensor.
  • In vitro characterization using spectrofluorometry.
  • In vivo characterization in cultured mammalian cells using confocal fluorescent microscopy and signal normalization for pH-induced artifacts.

Main Results:

  • RexYFP successfully reports NAD+/NADH ratio changes in cytoplasm and mitochondrial matrix.
  • The probe's affinity allows comparison of NAD(H) levels in compartments with differing concentrations.
  • A method for normalizing signals to pH sensor output effectively eliminates pH-driven artifacts.

Conclusions:

  • RexYFP is a suitable tool for detecting NAD(H) redox state in various cellular compartments.
  • RexYFP offers advantages over existing sensors, including smaller size and optimal affinity.
  • Signal normalization to pH changes is a viable strategy to mitigate imaging artifacts.