scholarly journals A Fluorescence Resonance Energy Transfer-Based Analytical Tool for Nitrate Quantification in Living Cells

ACS Omega ◽  
2020 ◽  
Vol 5 (46) ◽  
pp. 30306-30314
Author(s):  
Urooj Fatima ◽  
Fuad Ameen ◽  
Neha Soleja ◽  
Parvez Khan ◽  
Abobakr Almansob ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Fluorescence Resonance Energy Transfer ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Living Cells ◽  
Analytical Tool ◽  
Fluorescence Resonance

scholarly journals Visualization of ATP levels inside single living cells with fluorescence resonance energy transfer-based genetically encoded indicators

2009 ◽  
Vol 106 (37) ◽  
pp. 15651-15656 ◽  
Author(s):  
Hiromi Imamura ◽  
Kim P. Huynh Nhat ◽  
Hiroko Togawa ◽  
Kenta Saito ◽  
Ryota Iino ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Fluorescence Resonance Energy Transfer ◽  
Atp Synthase ◽  
Hela Cells ◽  
Dissociation Constants ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Living Cells ◽  
Atp Levels ◽  
Fluorescence Resonance

Adenosine 5′-triphosphate (ATP) is the major energy currency of cells and is involved in many cellular processes. However, there is no method for real-time monitoring of ATP levels inside individual living cells. To visualize ATP levels, we generated a series of fluorescence resonance energy transfer (FRET)-based indicators for ATP that were composed of the ε subunit of the bacterial FoF1-ATP synthase sandwiched by the cyan- and yellow-fluorescent proteins. The indicators, named ATeams, had apparent dissociation constants for ATP ranging from 7.4 μM to 3.3 mM. By targeting ATeams to different subcellular compartments, we unexpectedly found that ATP levels in the mitochondrial matrix of HeLa cells are significantly lower than those of cytoplasm and nucleus. We also succeeded in measuring changes in the ATP level inside single HeLa cells after treatment with inhibitors of glycolysis and/or oxidative phosphorylation, revealing that glycolysis is the major ATP-generating pathway of the cells grown in glucose-rich medium. This was also confirmed by an experiment using oligomycin A, an inhibitor of FoF1-ATP synthase. In addition, it was demonstrated that HeLa cells change ATP-generating pathway in response to changes of nutrition in the environment.

scholarly journals Design of fluorescence resonance energy transfer (FRET)-based cGMP indicators: a systematic approach

2007 ◽  
Vol 407 (1) ◽  
pp. 69-77 ◽  
Author(s):  
Michael Russwurm ◽  
Florian Mullershausen ◽  
Andreas Friebe ◽  
Ronald Jäger ◽  
Corina Russwurm ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Fluorescence Resonance Energy Transfer ◽  
Systematic Approach ◽  
Dynamic Range ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Living Cells ◽  
Binding Domains ◽  
Fluorescence Resonance

The intracellular signalling molecule cGMP regulates a variety of physiological processes, and so the ability to monitor cGMP dynamics in living cells is highly desirable. Here, we report a systematic approach to create FRET (fluorescence resonance energy transfer)-based cGMP indicators from two known types of cGMP-binding domains which are found in cGMP-dependent protein kinase and phosphodiesterase 5, cNMP-BD [cyclic nucleotide monophosphate-binding domain and GAF [cGMP-specific and -stimulated phosphodiesterases, Anabaena adenylate cyclases and Escherichia coli FhlA] respectively. Interestingly, only cGMP-binding domains arranged in tandem configuration as in their parent proteins were cGMP-responsive. However, the GAF-derived sensors were unable to be used to study cGMP dynamics because of slow response kinetics to cGMP. Out of 24 cGMP-responsive constructs derived from cNMP-BDs, three were selected to cover a range of cGMP affinities with an EC50 between 500 nM and 6 μM. These indicators possess excellent specifity for cGMP, fast binding kinetics and twice the dynamic range of existing cGMP sensors. The in vivo performance of these new indicators is demonstrated in living cells and validated by comparison with cGMP dynamics as measured by radioimmunoassays.

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