Red fluorescent cAMP indicator with increased affinity and expanded dynamic range

Yusaku Ohta,Toshiaki Furuta,Kazuki Horikawa,Takeharu Nagai

doi:10.1038/s41598-018-20251-1

Yusaku Ohta, Toshiaki Furuta + Show 2 more

Open Access

https://doi.org/10.1038/s41598-018-20251-1

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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.

Highlights

3′,5′-cyclic adenosine monophosphate is one of the major signalling mediators that regulates a variety of cellular functions, including synaptic plasticity of neurons[1], T cell immune response[2], insulin secretion from pancreatic β-cells[3], and excitation–contraction coupling in cardiac myocytes[4]
Its increased affinity (Kd = 0.3 μM) and expanded D.R. (860% at pH 7.2) allowed for high-sensitivity detection of faint cyclic adenosine monophosphate (cAMP) dynamics associated with spontaneous cellular signalling, which could not be achieved by other 1-FP cAMP indicators
Increased detection sensitivity strengthened the advantages of R-FlincA as a red fluorescent indicator, as it provided new insights regarding the cAMP dynamics in the insulin secretion of pancreatic β-cells and in the development of Dictyostelium discoideum cells by multi-channel/function imaging with spectrally distinct indicators

Summary

Introduction

3′,5′-cyclic adenosine monophosphate (cAMP) is one of the major signalling mediators that regulates a variety of cellular functions, including synaptic plasticity of neurons[1], T cell immune response[2], insulin secretion from pancreatic β-cells[3], and excitation–contraction coupling in cardiac myocytes[4]. Increased detection sensitivity strengthened the advantages of R-FlincA as a red fluorescent indicator, as it provided new insights regarding the cAMP dynamics in the insulin secretion of pancreatic β-cells and in the development of Dictyostelium discoideum cells by multi-channel/function imaging with spectrally distinct (blue to yellow) indicators. These results suggest that R-FlincA is a promising tool for the acceleration of cAMP research

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