RRC ID 82131
Author Ohta Y, Furuta T, Nagai T, Horikawa K.
Title Red fluorescent cAMP indicator with increased affinity and expanded dynamic range.
Journal Sci Rep
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.
Volume 8(1)
Pages 1866
Published 2018-1-30
DOI 10.1038/s41598-018-20251-1
PII 10.1038/s41598-018-20251-1
PMID 29382930
PMC PMC5789972
MeSH Calcium / metabolism Cells, Cultured Cyclic AMP / metabolism* Dictyostelium / metabolism* Fluorescent Dyes / chemistry* Humans Insulin-Secreting Cells / metabolism* Luminescent Proteins / metabolism* Molecular Imaging / methods* Red Fluorescent Protein Spectrometry, Fluorescence
Resource
Human and Animal Cells 293T(RCB2202)