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Imaging cytosolic NADH-NAD(+) redox state with a genetically encoded fluorescent biosensor
Yin Pun Hung1, John G Albeck, Mathew Tantama
1Department of Neurobiology, Harvard Medical School, Boston, MA 02115, USA.
Cell Metabolism
|October 11, 2011
Summary
Researchers developed Peredox, a novel biosensor for accurately measuring the NADH:NAD(+) redox state in live cells. This tool helps understand cellular metabolism and bioenergetics by visualizing NADH levels crucial for metabolic pathways.
Area of Science:
- Cellular and Molecular Biology
- Biochemistry
- Metabolic Engineering
Background:
- Accurate detection of cytosolic NADH (Nicotinamide adenine dinucleotide) in live cells is crucial for understanding cellular metabolism but remains challenging.
- Existing methods often lack the sensitivity, specificity, or real-time monitoring capabilities required for dynamic cellular processes.
Purpose of the Study:
- To engineer a sensitive and specific fluorescent biosensor for quantifying the cytosolic NADH to NAD(+) redox state in live cells.
- To validate the biosensor's utility in various cell types and under different metabolic conditions.
- To investigate cellular bioenergetics and metabolic pathway responses using the developed biosensor.
Main Methods:
- Construction of a genetically encoded fluorescent biosensor by fusing a circularly permuted GFP variant (T-Sapphire) with a bacterial NADH-binding protein (Rex).
- Site-directed mutagenesis to eliminate pH sensitivity and optimize the biosensor for reporting NADH:NAD(+) ratios.
- Calibration using exogenous lactate and pyruvate, and application in cultured and primary cell types, including high-content image analysis.
Main Results:
- Development of the engineered biosensor, Peredox, which accurately reports cytosolic NADH:NAD(+) ratios.
- Demonstration of Peredox's utility in diverse cell types, revealing distinct redox states in primary mouse astrocytes and neurons.
- Observation that glycolysis promotes a reduced cytosolic NADH:NAD(+) redox state by opposing the lactate dehydrogenase equilibrium.
- Monitoring of NADH responses to PI3K pathway inhibition in live cells.
Conclusions:
- The Peredox biosensor provides a robust tool for real-time monitoring of the cytosolic NADH:NAD(+) redox state in live cells.
- The findings offer insights into metabolic differences between cell types and the impact of metabolic pathways like glycolysis on cellular redox balance.
- Peredox is expected to significantly advance the study of cellular bioenergetics and metabolic regulation.
