Imaging Erg and Jun transcription factor interaction in living cells using fluorescence resonance energy transfer analyses

Abstract The pituitary-specific transcription factor Pit-1 forms dimers when interacting with specific DNA elements and has been shown to associate with several other nuclear proteins. Recently, techniques have become available that allow visualization of protein-protein interactions as they occur in single living cells. In this study, the technique of fluorescence resonance energy transfer (FRET) microscopy was used to visualize the physical interactions of Pit-1 proteins fused to spectral variants of the jellyfish green fluorescent protein (GFP) that emit green or blue light [blue fluorescent protein (BFP)]. An optimized imaging system was used to discriminate fluorescence signals from single cells coexpressing the BFP- and GFP-fusion proteins, and the contribution of spectral overlap to background fluorescence detected in the FRET images was established. Energy transfer signals from living cells expressing a fusion protein in which GFP was tethered to BFP by short protein linker was used to demonstrate acquisition of FRET signals. Genetic vectors encoding GFP- and BFP-Pit-1 proteins were prepared, and biological function of the fusion proteins was confirmed. FRET microscopy of HeLa cells coexpressing the GFP- and BFP-Pit-1 demonstrated energy transfer, which required the two fluorophores to be separated by less than 100 Å. Biochemical studies previously demonstrated that Pit-1 physically interacts with both c-Ets-1 and the estrogen receptor. FRET imaging of cells coexpressing BFP-Pit-1 and GFP-Ets-1 demonstrated energy transfer between these fusion proteins, a result consistent with their association in the nucleus of these living cells. In contrast, there was no evidence for energy transfer between the BFP-Pit-1 and an estrogen receptor-GFP fusion proteins. It is likely that the FRET imaging approach described here can be applied to many different protein-partner pairs in a variety of cellular contexts.

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

ACS Omega ◽

10.1021/acsomega.0c04868 ◽

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

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

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.0904764106 ◽

2009 ◽

Vol 106 (37) ◽

pp. 15651-15656 ◽

Cited By ~ 586

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.

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Detection of constitutive heterodimerization of the integrin Mac-1 subunits by fluorescence resonance energy transfer in living cells

Biochemical and Biophysical Research Communications ◽

10.1016/j.bbrc.2006.06.015 ◽

2006 ◽

Vol 346 (3) ◽

pp. 986-991 ◽

Cited By ~ 17

Author(s):

Guo Fu ◽

Hua-yan Yang ◽

Chen Wang ◽

Feng Zhang ◽

Zhen-dong You ◽

...

Keyword(s):

Energy Transfer ◽

Fluorescence Resonance Energy Transfer ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Living Cells ◽

Fluorescence Resonance

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

Biochemical Journal ◽

10.1042/bj20070348 ◽

2007 ◽

Vol 407 (1) ◽

pp. 69-77 ◽

Cited By ~ 95

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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