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Optogenetic control of endogenous Ca2+ channels in vivo

Nature Biotechnology volume 33, pages 1092–1096 (2015)Cite this article

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Abstract

Calcium (Ca2+) signals that are precisely modulated in space and time mediate a myriad of cellular processes, including contraction, excitation, growth, differentiation and apoptosis1. However, study of Ca2+ responses has been hampered by technological limitations of existing Ca2+-modulating tools. Here we present OptoSTIM1, an optogenetic tool for manipulating intracellular Ca2+ levels through activation of Ca2+-selective endogenous Ca2+ release−activated Ca2+ (CRAC) channels. Using OptoSTIM1, which combines a plant photoreceptor2,3 and the CRAC channel regulator STIM1 (ref. 4), we quantitatively and qualitatively controlled intracellular Ca2+ levels in various biological systems, including zebrafish embryos and human embryonic stem cells. We demonstrate that activating OptoSTIM1 in the CA1 hippocampal region of mice selectively reinforced contextual memory formation. The broad utility of OptoSTIM1 will expand our mechanistic understanding of numerous Ca2+-associated processes and facilitate screening for drug candidates that antagonize Ca2+ signals.

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Figure 1: Light-induced Ca2+ influx and activation of Ca2+-responsive signaling pathways.
Figure 2: OptoSTIM1-activated Ca2+ increase in zebrafish embryos and human embryonic stem cells.
Figure 3: Reinforcement of context-specific memory induced by OptoSTIM1 activation in the CA1 hippocampus of mice.

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Acknowledgements

We thank C.L. Tucker (University of Colorado) for cDNA encoding CRY2PHR-mCherry, T. Inoue (Johns Hopkins University) for cDNA encoding Tom20-FRB, P. Gardner (Stanford University) for cDNA encoding NFATc1-GFP, D. Lee for fruitful discussions and N. Kim for advice on data analysis. This work was supported by the Institute for Basic Science (IBS-R001-G1), the NRF Stem Cell Program (2011-0019509) funded by MSIP, KAIST Institute for the BioCentury, and the National Leading Research Laboratory Program by the Ministry of Science, ICT and Future Planning (2011-0028772 to Daesoo K.), Republic of Korea.

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Author notes

  1. Taeyoon Kyung and Sangkyu Lee: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Biological Sciences, Korea Advanced Institute of Science and Technology (KAIST), Daejeon, Republic of Korea

    Taeyoon Kyung, Jung Eun Kim, Hyerim Park, Dongkyu Kim, Anna Shin, Sungsoo Kim, Jihoon Kim, Na Yeon Kim, Doyeon Woo, Yong-Mahn Han, Daesoo Kim & Won Do Heo

  2. Center for Cognition and Sociality, Institute for Basic Science (IBS), Daejeon, Republic of Korea

    Sangkyu Lee, Taesup Cho, Jinhee Baek, Hee-Sup Shin & Won Do Heo

  3. Department of Biology, Chungnam National University, Daejeon, Republic of Korea

    Yun-Mi Jeong & Cheol-Hee Kim

  4. Department of Bio and Brain Engineering, KAIST, Daejeon, Republic of Korea

    Jinhee Baek & Hee-Sup Shin

  5. KAIST Institute for the BioCentury, KAIST, Daejeon, Republic of Korea

    Sujin Chae & Won Do Heo

  6. Cancer Metastasis Control Center, KAIST Institute for the BioCentury, KAIST, Daejeon, Republic of Korea

    Won Do Heo

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  1. Taeyoon Kyung
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Contributions

W.D.H., T.K. and S.L. conceived the idea and directed the work. T.K., S.L., J.E.K., T.C., Y.-M.J., J.B., A.S., C.-H.K., H.-S.S., Y.-M.H., Daesoo K. and W.D.H. designed experiments; T.K., S.L., J.E.K., T.C., H.P., Y.-M.J., Dongkyu K., S.K., J.B., A.S., J.K., N.Y.K., D.W. and S.C. performed experiments, and T.K., S.L., Daesoo K. and W.D.H. wrote the manuscript.

Corresponding authors

Correspondence to Yong-Mahn Han, Daesoo Kim or Won Do Heo.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–20 (PDF 6312 kb)

Dynamic movement of OptoSTIM1 along microtubules upon blue-light illumination.

Fluorescent images of HeLa cells expressing OptoSTIM1 were captured at 3.3-s intervals through EGFP channel. Numbers indicate minutes:seconds. (AVI 1088 kb)

Induction of Ca2+ influx by OptoSTIM1 activation.

Fluorescent images of HeLa cells co-expressing R-GECO1 and OptoSTIM1 were captured at 20-s intervals through mCherry and EGFP channels. The movie only represents R-GECO1 signals. Numbers indicate minutes:seconds. (AVI 14467 kb)

Transient or sustained control of Ca2+ influx by pulsatile illumination.

Fluorescent images of HeLa cells co-expressing R-GECO1 and OptoSTIM1 were stimulated by blue light at 10-min (three times) and subsequently at 3-min (ten times) intervals for inducing transient or sustained Ca2+ influx, respectively. The video only represents R-GECO1 signals. Numbers indicate minutes:seconds. (AVI 4386 kb)

Reversible NFATc1 translocation by transient activation of OptoSTIM1.

Fluorescent images of HeLa cells co-expressing NFATc1-mCherry and OptoSTIM1 were captured at 1-min intervals through mCherry channel. Cells were illuminated by blue light for 3 s. The video only represents NFATc1 signals. Numbers indicate minutes:seconds. (AVI 3939 kb)

Reversible disassembly of cortical F-actin by transient activation of OptoSTIM1.

Fluorescent images of HeLa cells co-expressing iRFP670-Lifeact and OptoSTIM1 were captured at 1-min intervals through Alexa-647 and EGFP channels. The video only represents Lifeact signals. Numbers indicate minutes:seconds. (AVI 9204 kb)

Spatiotemporal control of Ca2+ influx in multiple cells upon blue-light illumination.

Fluorescent images of three HeLa cells co-expressing R-GECO1 and OptoSTIM1 were captured at 20-s intervals through mCherry channel. Three cells were sequentially illuminated by blue light (1.5 s) at different time points indicated by arrows. The video only represents R-GECO1 signals. Numbers indicate minutes:seconds. (AVI 3067 kb)

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Kyung, T., Lee, S., Kim, J. et al. Optogenetic control of endogenous Ca2+ channels in vivo. Nat Biotechnol 33, 1092–1096 (2015). https://doi.org/10.1038/nbt.3350

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