Blue fluorescent cGMP sensor for multiparameter fluorescence imaging.

PloS One
Yusuke NiinoKotaro Oka

Abstract

Cyclic GMP (cGMP) regulates many physiological processes by cooperating with the other signaling molecules such as cyclic AMP (cAMP) and Ca(2+). Genetically encoded sensors for cGMP have been developed based on fluorescence resonance energy transfer (FRET) between fluorescent proteins. However, to analyze the dynamic relationship among these second messengers, combined use of existing sensors in a single cell is inadequate because of the significant spectral overlaps. A single wavelength indicator is an effective alternative to avoid this problem, but color variants of a single fluorescent protein-based biosensor are limited. In this study, to construct a new color fluorescent sensor, we converted the FRET-based sensor into a single wavelength indicator using a dark FRET acceptor. We developed a blue fluorescent cGMP biosensor, which is spectrally compatible with a FRET-based cAMP sensor using cyan and yellow fluorescent proteins (CFP/YFP). We cotransfected them and loaded a red fluorescent probe for Ca(2+) into cells, and accomplished triple-parameter fluorescence imaging of these cyclic nucleotides and Ca(2+), confirming the applicability of this combination to individually monitor their dynamics in a single cell. This blue f...Continue Reading

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Citations

Mar 13, 2012·Annual Review of Plant Biology·Sakiko OkumotoWolf B Frommer
Oct 22, 2011·Nature Reviews. Molecular Cell Biology·Christopher M WelchKlaus M Hahn
Nov 12, 2013·BMC Biotechnology·Janet I WheelerHelen R Irving
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Apr 30, 2021·Biomedical Materials·Shanni HongYi Lu
May 24, 2019·The Journal of Organic Chemistry·Aleksandr S OshchepkovEvgeny A Kataev

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Methods Mentioned

BETA
biosensors
FRET
biosensor
fluorescence lifetime imaging microscopy
fluorescence imaging
transfection

Software Mentioned

MetaMorph
Cygnus

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