Real-time monitoring of NADPH levels in living mammalian cells using fluorescence-enhancing protein bound to NADPHs.
Biosens Bioelectron
; 146: 111753, 2019 Dec 15.
Article
em En
| MEDLINE
| ID: mdl-31600627
ABSTRACT
Nicotinamide adenine nucleotide phosphate (NADPH) has been known to be involved in the multiple pathways of cell metabolism. However, conventional quantification assays for NADPH have required breaking down the cell membranes of around one million cells per assay, and monitoring NADPH flux in living cells has been limited by a few available tools. Here, we visualized NADPH levels in human cervical cancer cells HeLa using metagenome-derived blue fluorescent protein (mBFP), which specifically binds to NADPH and enhances the intrinsic fluorescence of NADPH up to 10-fold when imaged by two-photon microscopy to reduce photodamage. Adding an oxidizing agent such as diamide to HeLa cells that expressed mBFP led to an immediate decrease of intracellular NADPH depending on glucose availability in culture media. Furthermore, inhibiting glucose-6-phosphate dehydrogenase (G6PD) in the pentose phosphate pathway with dehydroandrosterone (DHEA) and knockdown of G6PD transcripts gradually decreased NADPH when diamide was added to living cells. These results demonstrate that introducing a bacterial mBFP gene into mammalian cells is a straightforward approach to monitoring intracellular NADPH flux in real time at the single-cell level. Moreover, this strategy can be expanded to tracking the spatio-temporal changes in NADPH even in single-cell organelles such as mitochondria and chloroplasts, which will allow us to more precisely assess the efficacy of biochemically or biophysically metabolic perturbations in animal and plant cells.
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Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Técnicas Biossensoriais
/
Corantes Fluorescentes
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Proteínas Luminescentes
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NADP
Limite:
Humans
Idioma:
En
Revista:
Biosens Bioelectron
Assunto da revista:
BIOTECNOLOGIA
Ano de publicação:
2019
Tipo de documento:
Article