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Published September 15, 2014 | Supplemental Material + Accepted Version
Journal Article Open

Archaerhodopsin variants with enhanced voltage-sensitive fluorescence in mammalian and Caenorhabditis elegans neurons


Probing the neural circuit dynamics underlying behaviour would benefit greatly from improved genetically encoded voltage indicators. The proton pump Archaerhodopsin-3 (Arch), an optogenetic tool commonly used for neuronal inhibition, has been shown to emit voltage-sensitive fluorescence. Here we report two Arch variants with enhanced radiance (Archers) that in response to 655 nm light have 3–5 times increased fluorescence and 55–99 times reduced photocurrents compared with Arch WT. The most fluorescent variant, Archer1, has 25–40% fluorescence change in response to action potentials while using 9 times lower light intensity compared with other Arch-based voltage sensors. Archer1 is capable of wavelength-specific functionality as a voltage sensor under red light and as an inhibitory actuator under green light. As a proof-of-concept for the application of Arch-based sensors in vivo, we show fluorescence voltage sensing in behaving Caenorhabditis elegans. Archer1's characteristics contribute to the goal of all-optical detection and modulation of activity in neuronal networks in vivo.

Additional Information

© 2014, Nature Publishing Group. Received 21 June 2014. Accepted 04 August 2014. Published 15 September 2014. We thank the entire Gradinaru lab for helpful discussions. We also thank Prof. David Anderson for helpful discussions and suggestions; Dr Benjamin Judkewitz for valuable input on the optical setup for imaging; Bin Yang for assistance with optical setup and cultured neurons; Christopher Cronin and Ravi Nath for assistance with C. elegans experiments; and Dr John Bedbrook and Dr Jennifer Treweek for critical reading of the manuscript. This work was funded by the NIH/NINDS New Innovator (NIH IDP20D017782-01); startup funds from the President and Provost of California Institute of Technology and the Biology and Biological Engineering Division of California Institute of Technology; the Beckman Institute of Caltech; the Gordon and Betty Moore Foundation through Grant GBMF2809 to the Caltech Programmable Molecular Technology Initiative (to V.G.); by NIH 1R21MH103824-01 (to V.G. and F.H.A.); and by the Institute for Collaborative Biotechnologies through grant W911NF-09-0001 from the U.S. Army Research Office (to F.H.A.). The content of the information does not necessarily reflect the position or the policy of the Government, and no official endorsement should be inferred. V.G. is also supported by Human Frontiers in Science Program, the Mallinckrodt Foundation, the Pew Charitable Trust, the Michael J. Fox Foundation, the Kimmel Foundation, Caltech-GIST, NIH 1R01NS085910-01, NIH 1R01AG047664-01. P.W.S. is an investigator with the HHMI, which supported this research. N.C.F., C.N.B. and K.Y.C. acknowledge support from the Caltech Biology Division Training grant (NIH/NRSA 5T32GM07616). M.K.M.E. acknowledges support from the German Research Foundation (DFG) under programme EN 957/1-1. Nicholas C. Flytzanis & Claire N. Bedbrook: These authors contributed equally to this work. Author Contributions: N.C.F., M.K.M.E., F.H.A. and V.G. conceived the project. N.C.F., C.N.B., H.C., P.S.W. and V.G. designed the experiments. N.C.F., C.N.B., H.C., C.X. and K.Y.C. performed the experiments. N.C.F. and C.N.B. analysed all of the data. N.C.F., C.N.B. and V.G. wrote the manuscript with support from all authors. V.G. supervised all aspects of the work. The authors declare no competing financial interests.

Attached Files

Accepted Version - nihms619133.pdf

Supplemental Material - ncomms5894-s1.pdf

Supplemental Material - ncomms5894-s2.mov

Supplemental Material - ncomms5894-s3.mov


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