Red fluorescent cAMP indicator with increased affinity and expanded dynamic range

• Ohta, Yusaku

• Furuta, Toshiaki

• Nagai, Takeharu

• Horikawa, Kazuki

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.

Journal

• Scientific Reports

  Scientific Reports 8 1866-, 2018-01-30

  Springer Nature

Keywords

• Multidisciplinary

Details

• CRID: 1050845762732983552
• NII Article ID: 120006535055
• ISSN: 20452322
• DOI: 10.1038/s41598-018-20251-1
• Web Site: http://repo.lib.tokushima-u.ac.jp/112438
• Web Site: https://www.nature.com/articles/s41598-018-20251-1
• Web Site: https://www.nature.com/articles/s41598-018-20251-1.pdf
• Text Lang: en
• Article Type: journal article
• Data Source:
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  • Crossref
  • CiNii Articles
  • KAKEN