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
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 2.17μM) 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.
附件下载：
A high-performance genetically encoded fluorescent indicator for in vivo cAMP imaging, Nat Commun, 12 Sep 2022
Nature Communications, 12 September, 2022, DOI：https://doi.org/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 Lu, Pei-Gen Ren, Ho Leung Ng, Jie He, Shoudeng Chen, Min Xu, Yulong Li & Jun Chu
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 2.17μM) 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.
文章链接：https://www.nature.com/articles/s41467-022-32994-7