Abstract
The flagellate Euglena gracilis contains a photoactivated adenylyl cyclase (PAC), consisting of the flavoproteins PACα and PACβ. Here we report functional expression of PACs in Xenopus laevis oocytes, HEK293 cells and in Drosophila melanogaster, where neuronal expression yields light-induced changes in behavior. The activity of PACs is strongly and reversibly enhanced by blue light, providing a powerful tool for light-induced manipulation of cAMP in animal cells.
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Acknowledgements
We are indebted to J.W. Karpen (Oregon Health & Science University) for a generous gift of the CNGA2-C460W,E583M plasmid. S.S.-L. and G.N. thank E. Bamberg (Max-Planck-Institut für Biophysik) for generous support. D. Ollig, M. Dörr, T. Schiereis, A. Jaeckel, M. Glander and J. Wiegant provided excellent technical support. Members of the Drosophila Genomic Resource Center (Bloomington University) provided us with T. Murphey's GATEWAY collection of expression vectors. The work was supported by the Deutsche Forschungsgemeinschaft (to P.H. and G.N.) and the Max-Planck-Gesellschaft (to G.N.).
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Supplementary information
Supplementary Fig. 1
Detection of PACα and PACβ in oocytes.
Supplementary Fig. 2
Light-dependence of PAC activation.
Supplementary Fig. 3
Photostimulation of PACβ does not alter behavior in freely moving Drosophila.
Supplementary Table 1
ON- and OFF kinetics of light-induced behavioral changes for two consecutive irradiations of 1 min.
Supplementary Video 1
Change in grooming behavior of PACα expressing flies (elav-Gal4 / UAS-PACα) upon illumination with bright blue light. An individual fly is illuminated during grooming behavior (note the diode placed underneath the observation chamber turning on) resulting in nearly instant stop of activity and unusual leg shaking. After turning off blue light the animal returns to grooming activity. Note that the video was taped using a red filter that extinguishes blue light to protect the camera; blue light intensity was set to 55.7 microMol × (m−2 s−1).
Supplementary Video 2
Illumination with blue light does not alter grooming behavior in wild type Canton-S flies.
Supplementary Video 3
Illumination with amber light (590 ± 10 nm) does not alter grooming activity in Pac expressing flies (elav-Gal4 / UAS-PACα). Note that the video was taped using a blue filter that partly extinguishes amber light to protect the camera; light intensity was set to 55.7 microMol × (m−2 s−1).
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Schröder-Lang, S., Schwärzel, M., Seifert, R. et al. Fast manipulation of cellular cAMP level by light in vivo. Nat Methods 4, 39–42 (2007). https://doi.org/10.1038/nmeth975
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DOI: https://doi.org/10.1038/nmeth975
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