TY - JOUR
T1 - Sensitive genetically encoded sensors for population and subcellular imaging of cAMP in vivo
AU - Massengill, Crystian I.
AU - Bayless-Edwards, Landon
AU - Ceballos, Cesar C.
AU - Cebul, Elizabeth R.
AU - Cahill, James
AU - Bharadwaj, Arpita
AU - Wilson, Evan
AU - Qin, Maozhen
AU - Whorton, Matthew R.
AU - Baconguis, Isabelle
AU - Ye, Bing
AU - Mao, Tianyi
AU - Zhong, Haining
N1 - Funding Information:
We thank Ryohei Yasuda (Max Planck Florida) for 2pFLIM acquisition software; Jin Zhang (University of California, San Diego) for ICUE3 cDNA; Kees Jalink (Netherlands Cancer Institute) for Epac-S cDNA; Laurinda Jaffe (University of Connecticut) and Viacheslav Nikolaev (University of Hamburg) for Epac1-camps and Epac2-camps300 cDNA; Lei Ma, Vivek Unni and Sydney Boutros (Oregon Health & Science University, OHSU) and Josh Melander (Stanford University) for training and advice on in vivo surgery; Kevin Wright (Vollum Institute) for immunoprecipitation reagents; Caitlynn De Preter for contributing to in vitro data collection; Makayla Freitas for assistance in spectrophotometer measurements; the OHSU Shared Resources Biophysics Core for use of the stopped-flow device; and Bart Jongbloets, Michael Muniak, Lei Ma, Yang Chen, and the entire Zhong lab and Mao lab at OHSU for helpful comments and discussions. Fly stocks from the Bloomington Drosophila Stock Center (NIH P40OD018537) were used. This work was supported by NIH BRAIN Initiative awards R01NS104944 (H.Z. and T.M.) and RF1MH120119 (H.Z. and T.M.), National Institute of Neurological Disorders and Stroke (NINDS) R01 grants R01NS127013 (H.Z.), R01NS081071 (T.M.), and R01NS104299 (B.Y.), and National Institute of General Medical Sciences (NIGMS) R01 grant R01GM138862 (I.B.). H187
Funding Information:
We thank Ryohei Yasuda (Max Planck Florida) for 2pFLIM acquisition software; Jin Zhang (University of California, San Diego) for ICUE3 cDNA; Kees Jalink (Netherlands Cancer Institute) for Epac-SH187cDNA; Laurinda Jaffe (University of Connecticut) and Viacheslav Nikolaev (University of Hamburg) for Epac1-camps and Epac2-camps300 cDNA; Lei Ma, Vivek Unni and Sydney Boutros (Oregon Health & Science University, OHSU) and Josh Melander (Stanford University) for training and advice on in vivo surgery; Kevin Wright (Vollum Institute) for immunoprecipitation reagents; Caitlynn De Preter for contributing to in vitro data collection; Makayla Freitas for assistance in spectrophotometer measurements; the OHSU Shared Resources Biophysics Core for use of the stopped-flow device; and Bart Jongbloets, Michael Muniak, Lei Ma, Yang Chen, and the entire Zhong lab and Mao lab at OHSU for helpful comments and discussions. Fly stocks from the Bloomington Drosophila Stock Center (NIH P40OD018537) were used. This work was supported by NIH BRAIN Initiative awards R01NS104944 (H.Z. and T.M.) and RF1MH120119 (H.Z. and T.M.), National Institute of Neurological Disorders and Stroke (NINDS) R01 grants R01NS127013 (H.Z.), R01NS081071 (T.M.), and R01NS104299 (B.Y.), and National Institute of General Medical Sciences (NIGMS) R01 grant R01GM138862 (I.B.).
Publisher Copyright:
© 2022, The Author(s), under exclusive licence to Springer Nature America, Inc.
PY - 2022/11
Y1 - 2022/11
N2 - Cyclic adenosine monophosphate (cAMP) signaling integrates information from diverse G-protein-coupled receptors, such as neuromodulator receptors, to regulate pivotal biological processes in a cellular-specific and subcellular-specific manner. However, in vivo cellular-resolution imaging of cAMP dynamics remains challenging. Here, we screen existing genetically encoded cAMP sensors and further develop the best performer to derive three improved variants, called cAMPFIREs. Compared with their parental sensor, these sensors exhibit up to 10-fold increased sensitivity to cAMP and a cytosolic distribution. cAMPFIREs are compatible with both ratiometric and fluorescence lifetime imaging and can detect cAMP dynamics elicited by norepinephrine at physiologically relevant, nanomolar concentrations. Imaging of cAMPFIREs in awake mice reveals tonic levels of cAMP in cortical neurons that are associated with wakefulness, modulated by opioids, and differentially regulated across subcellular compartments. Furthermore, enforced locomotion elicits neuron-specific, bidirectional cAMP dynamics. cAMPFIREs also function in Drosophila. Overall, cAMPFIREs may have broad applicability for studying intracellular signaling in vivo.
AB - Cyclic adenosine monophosphate (cAMP) signaling integrates information from diverse G-protein-coupled receptors, such as neuromodulator receptors, to regulate pivotal biological processes in a cellular-specific and subcellular-specific manner. However, in vivo cellular-resolution imaging of cAMP dynamics remains challenging. Here, we screen existing genetically encoded cAMP sensors and further develop the best performer to derive three improved variants, called cAMPFIREs. Compared with their parental sensor, these sensors exhibit up to 10-fold increased sensitivity to cAMP and a cytosolic distribution. cAMPFIREs are compatible with both ratiometric and fluorescence lifetime imaging and can detect cAMP dynamics elicited by norepinephrine at physiologically relevant, nanomolar concentrations. Imaging of cAMPFIREs in awake mice reveals tonic levels of cAMP in cortical neurons that are associated with wakefulness, modulated by opioids, and differentially regulated across subcellular compartments. Furthermore, enforced locomotion elicits neuron-specific, bidirectional cAMP dynamics. cAMPFIREs also function in Drosophila. Overall, cAMPFIREs may have broad applicability for studying intracellular signaling in vivo.
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UR - http://www.scopus.com/inward/citedby.url?scp=85140607367&partnerID=8YFLogxK
U2 - 10.1038/s41592-022-01646-5
DO - 10.1038/s41592-022-01646-5
M3 - Article
C2 - 36303019
AN - SCOPUS:85140607367
SN - 1548-7091
VL - 19
SP - 1461
EP - 1471
JO - Nature Methods
JF - Nature Methods
IS - 11
ER -