A high-performance genetically encoded fluorescent indicator for in vivo cAMP imaging

Liang Wang; Chunling Wu; Wanling Peng; Ziliang Zhou; Jianzhi Zeng; Xuelin Li; Yini Yang; Shuguang Yu; Ye Zou; Mian Huang; Chang Liu; Yefei Chen; Yi Li; Panpan Ti; Wenfeng Liu; Yufeng Gao; Wei Zheng; Haining Zhong; Shangbang Gao; Zhonghua Lu; Pei Gen Ren; Ho Leung Ng; Jie He; Shoudeng Chen; Min Xu; Yulong Li; Jun Chu

doi:10.1038/s41467-022-32994-7

A high-performance genetically encoded fluorescent indicator for in vivo cAMP imaging

Liang Wang, Chunling Wu, Wanling Peng, Ziliang Zhou, Jianzhi Zeng, Xuelin Li, Yini Yang, Shuguang Yu, Ye Zou, Mian Huang, Chang Liu, Yefei Chen, Yi Li, Panpan Ti, Wenfeng Liu, Yufeng Gao, Wei Zheng, Haining Zhong, Shangbang Gao, Zhonghua LuPei Gen Ren, Ho Leung Ng, Jie He, Shoudeng Chen, Min Xu, Yulong Li, Jun Chu

Vollum Institute

Research output: Contribution to journal › Article › peer-review

17 Scopus citations

Abstract

cAMP is a key second messenger that regulates diverse cellular functions including neural plasticity. However, the spatiotemporal dynamics of intracellular cAMP in intact organisms are largely unknown due to low sensitivity and/or brightness of current genetically encoded fluorescent cAMP indicators. Here, we report the development of the new circularly permuted GFP (cpGFP)-based cAMP indicator G-Flamp1, which exhibits a large fluorescence increase (a maximum ΔF/F₀ of 1100% in HEK293T cells), decent brightness, appropriate affinity (a K_d of 2.17 μM) and fast response kinetics (an association and dissociation half-time of 0.20 and 0.087 s, respectively). Furthermore, the crystal structure of the cAMP-bound G-Flamp1 reveals one linker connecting the cAMP-binding domain to cpGFP adopts a distorted β-strand conformation that may serve as a fluorescence modulation switch. We demonstrate that G-Flamp1 enables sensitive monitoring of endogenous cAMP signals in brain regions that are implicated in learning and motor control in living organisms such as fruit flies and mice.

Original language	English (US)
Article number	5363
Journal	Nature communications
Volume	13
Issue number	1
DOIs	https://doi.org/10.1038/s41467-022-32994-7
State	Published - Dec 2022

ASJC Scopus subject areas

General Chemistry
General Biochemistry, Genetics and Molecular Biology
General Physics and Astronomy

Access to Document

10.1038/s41467-022-32994-7

Cite this

Wang, L., Wu, C., Peng, W., Zhou, Z., Zeng, J., Li, X., Yang, Y., Yu, S., Zou, Y., Huang, M., Liu, C., Chen, Y., Li, Y., Ti, P., Liu, W., Gao, Y., Zheng, W., Zhong, H., Gao, S., ... Chu, J. (2022). A high-performance genetically encoded fluorescent indicator for in vivo cAMP imaging. Nature communications, 13(1), Article 5363. https://doi.org/10.1038/s41467-022-32994-7

Wang, L, Wu, C, Peng, W, Zhou, Z, Zeng, J, Li, X, Yang, Y, Yu, S, Zou, Y, Huang, M, Liu, C, Chen, Y, Li, Y, Ti, P, Liu, W, Gao, Y, Zheng, W, Zhong, H, Gao, S, Lu, Z, Ren, PG, Ng, HL, He, J, Chen, S, Xu, M, Li, Y & Chu, J 2022, 'A high-performance genetically encoded fluorescent indicator for in vivo cAMP imaging', Nature communications, vol. 13, no. 1, 5363. https://doi.org/10.1038/s41467-022-32994-7

@article{6de2d6c506ae4edf9c7779ce48f3abcb,

title = "A high-performance genetically encoded fluorescent indicator for in vivo cAMP imaging",

abstract = "cAMP is a key second messenger that regulates diverse cellular functions including neural plasticity. However, the spatiotemporal dynamics of intracellular cAMP in intact organisms are largely unknown due to low sensitivity and/or brightness of current genetically encoded fluorescent cAMP indicators. Here, we report the development of the new circularly permuted GFP (cpGFP)-based cAMP indicator G-Flamp1, which exhibits a large fluorescence increase (a maximum ΔF/F0 of 1100% in HEK293T cells), decent brightness, appropriate affinity (a Kd of 2.17 μM) and fast response kinetics (an association and dissociation half-time of 0.20 and 0.087 s, respectively). Furthermore, the crystal structure of the cAMP-bound G-Flamp1 reveals one linker connecting the cAMP-binding domain to cpGFP adopts a distorted β-strand conformation that may serve as a fluorescence modulation switch. We demonstrate that G-Flamp1 enables sensitive monitoring of endogenous cAMP signals in brain regions that are implicated in learning and motor control in living organisms such as fruit flies and mice.",

author = "Liang Wang and Chunling Wu and Wanling Peng and Ziliang Zhou and Jianzhi Zeng and Xuelin Li and Yini Yang and Shuguang Yu and Ye Zou and Mian Huang and Chang Liu and Yefei Chen and Yi Li and Panpan Ti and Wenfeng Liu and Yufeng Gao and Wei Zheng and Haining Zhong and Shangbang Gao and Zhonghua Lu and Ren, {Pei Gen} and Ng, {Ho Leung} and Jie He and Shoudeng Chen and Min Xu and Yulong Li and Jun Chu",

note = "Publisher Copyright: {\textcopyright} 2022, The Author(s).",

year = "2022",

month = dec,

doi = "10.1038/s41467-022-32994-7",

language = "English (US)",

volume = "13",

journal = "Nature communications",

issn = "2041-1723",

publisher = "Nature Publishing Group",

number = "1",

}

TY - JOUR

T1 - A high-performance genetically encoded fluorescent indicator for in vivo cAMP imaging

AU - Wang, Liang

AU - Wu, Chunling

AU - Peng, Wanling

AU - Zhou, Ziliang

AU - Zeng, Jianzhi

AU - Li, Xuelin

AU - Yang, Yini

AU - Yu, Shuguang

AU - Zou, Ye

AU - Huang, Mian

AU - Liu, Chang

AU - Chen, Yefei

AU - Li, Yi

AU - Ti, Panpan

AU - Liu, Wenfeng

AU - Gao, Yufeng

AU - Zheng, Wei

AU - Zhong, Haining

AU - Gao, Shangbang

AU - Lu, Zhonghua

AU - Ren, Pei Gen

AU - Ng, Ho Leung

AU - He, Jie

AU - Chen, Shoudeng

AU - Xu, Min

AU - Li, Yulong

AU - Chu, Jun

PY - 2022/12

Y1 - 2022/12

N2 - cAMP is a key second messenger that regulates diverse cellular functions including neural plasticity. However, the spatiotemporal dynamics of intracellular cAMP in intact organisms are largely unknown due to low sensitivity and/or brightness of current genetically encoded fluorescent cAMP indicators. Here, we report the development of the new circularly permuted GFP (cpGFP)-based cAMP indicator G-Flamp1, which exhibits a large fluorescence increase (a maximum ΔF/F0 of 1100% in HEK293T cells), decent brightness, appropriate affinity (a Kd of 2.17 μM) and fast response kinetics (an association and dissociation half-time of 0.20 and 0.087 s, respectively). Furthermore, the crystal structure of the cAMP-bound G-Flamp1 reveals one linker connecting the cAMP-binding domain to cpGFP adopts a distorted β-strand conformation that may serve as a fluorescence modulation switch. We demonstrate that G-Flamp1 enables sensitive monitoring of endogenous cAMP signals in brain regions that are implicated in learning and motor control in living organisms such as fruit flies and mice.

AB - cAMP is a key second messenger that regulates diverse cellular functions including neural plasticity. However, the spatiotemporal dynamics of intracellular cAMP in intact organisms are largely unknown due to low sensitivity and/or brightness of current genetically encoded fluorescent cAMP indicators. Here, we report the development of the new circularly permuted GFP (cpGFP)-based cAMP indicator G-Flamp1, which exhibits a large fluorescence increase (a maximum ΔF/F0 of 1100% in HEK293T cells), decent brightness, appropriate affinity (a Kd of 2.17 μM) and fast response kinetics (an association and dissociation half-time of 0.20 and 0.087 s, respectively). Furthermore, the crystal structure of the cAMP-bound G-Flamp1 reveals one linker connecting the cAMP-binding domain to cpGFP adopts a distorted β-strand conformation that may serve as a fluorescence modulation switch. We demonstrate that G-Flamp1 enables sensitive monitoring of endogenous cAMP signals in brain regions that are implicated in learning and motor control in living organisms such as fruit flies and mice.

UR - http://www.scopus.com/inward/record.url?scp=85137718549&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=85137718549&partnerID=8YFLogxK

U2 - 10.1038/s41467-022-32994-7

DO - 10.1038/s41467-022-32994-7

M3 - Article

C2 - 36097007

AN - SCOPUS:85137718549

SN - 2041-1723

VL - 13

JO - Nature communications

JF - Nature communications

IS - 1

M1 - 5363

ER -

A high-performance genetically encoded fluorescent indicator for in vivo cAMP imaging

Abstract

ASJC Scopus subject areas

Access to Document

Other files and links

Fingerprint

Cite this