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Red fluorescent cAMP indicator with increased affinity and expanded dynamic range
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 an...
Autores principales: | , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Nature Publishing Group UK
2018
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5789972/ https://www.ncbi.nlm.nih.gov/pubmed/29382930 http://dx.doi.org/10.1038/s41598-018-20251-1 |
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author | Ohta, Yusaku Furuta, Toshiaki Nagai, Takeharu Horikawa, Kazuki |
author_facet | Ohta, Yusaku Furuta, Toshiaki Nagai, Takeharu Horikawa, Kazuki |
author_sort | Ohta, Yusaku |
collection | PubMed |
description | 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 (K(d) = 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. |
format | Online Article Text |
id | pubmed-5789972 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2018 |
publisher | Nature Publishing Group UK |
record_format | MEDLINE/PubMed |
spelling | pubmed-57899722018-02-15 Red fluorescent cAMP indicator with increased affinity and expanded dynamic range Ohta, Yusaku Furuta, Toshiaki Nagai, Takeharu Horikawa, Kazuki Sci Rep Article 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 (K(d) = 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. Nature Publishing Group UK 2018-01-30 /pmc/articles/PMC5789972/ /pubmed/29382930 http://dx.doi.org/10.1038/s41598-018-20251-1 Text en © The Author(s) 2018 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. |
spellingShingle | Article Ohta, Yusaku Furuta, Toshiaki Nagai, Takeharu Horikawa, Kazuki Red fluorescent cAMP indicator with increased affinity and expanded dynamic range |
title | Red fluorescent cAMP indicator with increased affinity and expanded dynamic range |
title_full | Red fluorescent cAMP indicator with increased affinity and expanded dynamic range |
title_fullStr | Red fluorescent cAMP indicator with increased affinity and expanded dynamic range |
title_full_unstemmed | Red fluorescent cAMP indicator with increased affinity and expanded dynamic range |
title_short | Red fluorescent cAMP indicator with increased affinity and expanded dynamic range |
title_sort | red fluorescent camp indicator with increased affinity and expanded dynamic range |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5789972/ https://www.ncbi.nlm.nih.gov/pubmed/29382930 http://dx.doi.org/10.1038/s41598-018-20251-1 |
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