Genetically encoded fluorescent sensors reveal dynamic regulation of NADPH metabolism

Tao, Rongkun; Zhao, Yuzheng; Chu, Huanyu; Wang, Aoxue; Zhu, Jiahuan; Chen, Xianjun; Zou, Yejun; Shi, M.; Liu, Renmei; Su, Ni; Du, Jiulin; Zhou, Hai‐Meng; Zhu, Linyong; Qian, Xuhong; Liu, Haiyan; Loscalzo, Joseph; Yang, Yi

doi:10.1038/nmeth.4306

Cited by 261 publications

(286 citation statements)

References 48 publications

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“…[83] PercevalHRbinds ATPand ADP with similar,micromolar affinities. [87] pH: pHluorin and pHred are protein-based pH sensors. [84] As in the Queen sensors, ac ircularp ermutated FP allowsr atiometric readout.…”

Section: Atpmentioning

confidence: 99%

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Cell Fuelling and Metabolic Energy Conservation in Synthetic Cells

et al. 2019

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We are aiming for a blue print for synthesizing (moderately complex) subcellular systems from molecular components and ultimately for constructing life. However, without comprehensive instructions and design principles, we rely on simple reaction routes to operate the essential functions of life. The first forms of synthetic life will not make every building block for polymers de novo according to complex pathways, rather they will be fed with amino acids, fatty acids and nucleotides. Controlled energy supply is crucial for any synthetic cell, no matter how complex. Herein, we describe the simplest pathways for the efficient generation of ATP and electrochemical ion gradients. We have estimated the demand for ATP by polymer synthesis and maintenance processes in small cell‐like systems, and we describe circuits to control the need for ATP. We also present fluorescence‐based sensors for pH, ionic strength, excluded volume, ATP/ADP, and viscosity, which allow the major physicochemical conditions inside cells to be monitored and tuned.

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Section: Atpmentioning

confidence: 99%

“…[86] iNap is ad erivativeo fS oNar and reports the NADPH concentration instead of the ratio between NADP + and NADPH. [87] pH: pHluorin and pHred are protein-based pH sensors. [88,89] pHluorini sb ased on GFP and has spectral properties in the yellow and green region.…”

Section: Atpmentioning

confidence: 99%

Cell Fuelling and Metabolic Energy Conservation in Synthetic Cells

et al. 2019

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“…ATP and ADP binding increase its fluorescence excited at 500 nm and 420 nm, respectively; there is a fixed isosbestic point at ~455 nm (25). NADPH concentration was imaged with the sensor iNap1, which increases its fluorescence excited at 420 nm and decreases its fluorescence excited at 485 nm upon NADPH binding (26). H2O2 levels were compared using the sensor HyPer-3 which increases its fluorescence ratio F500/F420 upon excitation at 500 and 420 nm (27).…”

Section: Test Of Toxic Effects Caused By the Expression Of Co Luxmentioning

confidence: 99%

Autonomous bioluminescence imaging of single mammalian cells with the bacterial bioluminescence system

Gregor

Pape

Gwosch

et al. 2019

Preprint

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Bioluminescence based imaging of living cells has become an important tool in biological and medical research. However, many bioluminescence imaging applications are limited by the requirement of an externally provided luciferin substrate and the low bioluminescence signal which restricts the sensitivity and spatiotemporal resolution. The bacterial bioluminescence system is fully genetically encodable and hence produces autonomous bioluminescence without an external luciferin, but its brightness in cell types other than bacteria has so far not been sufficient for imaging single cells. We coexpressed codon-optimized forms of the bacterial luxCDABE and frp genes from multiple plasmids in different mammalian cell lines. Our approach produces high luminescence levels that are comparable to firefly luciferase, thus enabling autonomous bioluminescence microscopy of mammalian cells. Significance statementBioluminescence is generated by luciferases that oxidize a specific luciferin. The enzymes involved in the synthesis of the luciferin from widespread cellular metabolites have so far been identified for only two bioluminescence systems, those of bacteria and fungi. In these cases, the complete reaction cascade is genetically encodable, meaning that heterologous expression of the corresponding genes can potentially produce autonomous bioluminescence in cell types other than the bacterial or fungal host cells. However, the light levels achieved in mammalian cells so far are not sufficient for single-cell applications. Here we present, for the first time, autonomous bioluminescence images of single mammalian cells by coexpression of the genes encoding the six enzymes from the bacterial bioluminescence system.

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“…5,6 NADH and NADPH, generally referred to as NAD(P)H, participate in two different types of electron transfer reactions. [15][16][17] Its mechanism of sensing depends on the conformational variation of the protein and requires intracellular gene expression. Conversely, NADPH reacts primarily with enzymes that catalyze the anabolic reactions that provide the high-energy electrons needed to synthesize biologically energetic molecules.…”

Section: Introductionmentioning

confidence: 99%

A Quencher‐Fluorophore‐Type Probe for Detection and Imaging of NADPH in Human Breast Cancer Cells

Kim

Bae

et al. 2019

Bulletin Korean Chem Soc

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A new fluorogenic trimethyl lock quinone (TLQ) derivative, designated as the quencher-TLQ-fluorophore-type probe (Q-TLQ-F), was developed for selective detection of nicotinamide adenine dinucleotide phosphate (NADPH). By taking advantage of the well-known facile intramolecular ring cyclization reaction (δ-lactonization) of TLQ that can release a reporter molecule upon reduction, Q-TLQ-F was successfully applied for the detection of physiological NADPH generated by glucose-6-phosphate dehydrogenase in human breast cancer MDA-MB-231 cells.

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Genetically encoded fluorescent sensors reveal dynamic regulation of NADPH metabolism

Cited by 261 publications

References 48 publications

Cell Fuelling and Metabolic Energy Conservation in Synthetic Cells

Cell Fuelling and Metabolic Energy Conservation in Synthetic Cells

Autonomous bioluminescence imaging of single mammalian cells with the bacterial bioluminescence system

A Quencher‐Fluorophore‐Type Probe for Detection and Imaging of NADPH in Human Breast Cancer Cells

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