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Red fluorescent cAMP indicator with increased affinity and expanded dynamic range
- Source :
- Scientific Reports, Vol 8, Iss 1, Pp 1-9 (2018), Scientific Reports
- Publication Year :
- 2018
- Publisher :
- Nature Publishing Group, 2018.
-
Abstract
- cAMP is one of the most important second messengers in biological processes. Cellular dynamics of cAMP have been investigated using a series of fluorescent indicators; however, their sensitivity was sub-optimal for detecting cAMP dynamics at a low concentration range, due to a low ligand affinity and/or poor dynamic range. Seeking an indicator with improved detection sensitivity, we performed insertion screening of circularly permuted mApple, a red fluorescent protein, into the cAMP-binding motif of PKA regulatory subunit Iα and developed an improved cAMP indicator named R-FlincA (Red Fluorescent indicator for cAMP). Its increased affinity (Kd = 0.3 μM) and expanded dynamic range (860% at pH 7.2) allowed the detection of subtle changes in the cellular cAMP dynamics at sub-μM concentrations, which could not be easily observed with existing indicators. Increased detection sensitivity also strengthened the advantages of using R-FlincA as a red fluorescent indicator, as it permits a series of applications, including multi-channel/function imaging of multiple second messengers and combinatorial imaging with photo-manipulation. These results strongly suggest that R-FlincA is a promising tool that accelerates cAMP research by revealing unobserved cAMP dynamics at a low concentration range.
- Subjects :
- 0301 basic medicine
Protein subunit
lcsh:Medicine
Article
Green fluorescent protein
03 medical and health sciences
Insulin-Secreting Cells
Cyclic AMP
Humans
Dictyostelium
Cellular dynamics
lcsh:Science
Cells, Cultured
Fluorescent Dyes
Multidisciplinary
Chemistry
Dynamic range
lcsh:R
Ligand (biochemistry)
Fluorescence
Molecular Imaging
Luminescent Proteins
Spectrometry, Fluorescence
030104 developmental biology
Second messenger system
Biophysics
Calcium
lcsh:Q
Function (biology)
Subjects
Details
- Language :
- English
- ISSN :
- 20452322
- Volume :
- 8
- Issue :
- 1
- Database :
- OpenAIRE
- Journal :
- Scientific Reports
- Accession number :
- edsair.doi.dedup.....af4f31609fd0f58b3d30590b0fdde454