Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

FRET-assisted photoactivation of flavoproteins for in vivo two-photon optogenetics

Nature Methods volume 16, pages 1029–1036 (2019)Cite this article

Subjects

Abstract

Optical dimerizers have been developed to untangle signaling pathways, but they are of limited use in vivo, partly due to their inefficient activation under two-photon (2P) excitation. To overcome this problem, we developed Förster resonance energy transfer (FRET)-assisted photoactivation, or FRAPA. On 2P excitation, mTagBFP2 efficiently absorbs and transfers the energy to the chromophore of CRY2. Based on structure-guided engineering, a chimeric protein with 40% FRET efficiency was developed and named 2P-activatable CRY2, or 2paCRY2. 2paCRY2 was employed to develop a RAF1 activation system named 2paRAF. In three-dimensionally cultured cells expressing 2paRAF, extracellular signal-regulated kinase (ERK) was efficiently activated by 2P excitation at single-cell resolution. Photoactivation of ERK was also accomplished in the epidermal cells of 2paRAF-expressing mice. We further developed an mTFP1-fused LOV domain that exhibits efficient response to 2P excitation. Collectively, FRAPA will pave the way to single-cell optical control of signaling pathways in vivo.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Development of 2paCRY2.
Fig. 2: 2P activation of 2paCRY2.
Fig. 3: ERK activation by 2paRAF.
Fig. 4: Single-cell control of ERK activity in the 3D MDCK cyst and organoids.
Fig. 5: Induction of ERK activation waves in the mouse auricular epidermis.
Fig. 6: 2P activation of 2paLINX.

Similar content being viewed by others

Data availability

The data that support the findings of this study are available within the article and its Supplementary Information or from the corresponding author upon reasonable request. Some plasmids encoding 2paCRY2, 2paRAF and 2paLINX are available through AddGene (IDs: 129651 to 129655). The 2paRAF mouse strain is available through the Center for Animal Resources and Development, Kumamoto University (ID: 2853).

Code availability

All analysis code used in this study is available upon reasonable request.

References

  1. Fan, L. Z. & Lin, M. Z. Optical control of biological processes by light‐switchable proteins. Wiley Interdiscip. Rev. Dev. Biol. 4, 545–554 (2015).

    Article  CAS  Google Scholar 

  2. Spiltoir, J. I. & Tucker, C. L. Photodimerization systems for regulating protein–protein interactions with light. Curr. Opin. Struct. Biol. 57, 1–8 (2019).

    Article  CAS  Google Scholar 

  3. Chen, D., Gibson, E. S. & Kennedy, M. J. A light-triggered protein secretion system. J. Cell Biol. 201, 631–640 (2013).

    Article  CAS  Google Scholar 

  4. Lungu, O. I. et al. Designing photoswitchable peptides using the AsLOV2 domain. Chem. Biol. 19, 507–517 (2012).

    Article  CAS  Google Scholar 

  5. Kennedy, M. J. et al. Rapid blue-light-mediated induction of protein interactions in living cells. Nat. Methods 7, 973–975 (2010).

    Article  CAS  Google Scholar 

  6. Zhou, X. X., Fan, L. Z., Li, P., Shen, K. & Lin, M. Z. Optical control of cell signaling by single-chain photoswitchable kinases. Science 355, 836–842 (2017).

    Article  Google Scholar 

  7. Levskaya, A., Weiner, O. D., Lim, W. A. & Voigt, C. A. Spatiotemporal control of cell signalling using a light-switchable protein interaction. Nature 461, 997 (2009).

    Article  CAS  Google Scholar 

  8. Lee, S. et al. Reversible protein inactivation by optogenetic trapping in cells. Nat. Methods 11, 633–636 (2014).

    Article  CAS  Google Scholar 

  9. Nguyen, M. K. et al. Optogenetic oligomerization of Rab GTPases regulates intracellular membrane trafficking. Nat. Chem. Biol. 12, 431 (2016).

    Article  CAS  Google Scholar 

  10. Valon, L., Marín-Llauradó, A., Wyatt, T., Charras, G. & Trepat, X. Optogenetic control of cellular forces and mechanotransduction. Nat. Commun. 8, 14396 (2017).

    Article  CAS  Google Scholar 

  11. Idevall-Hagren, O., Dickson, E. J., Hille, B., Toomre, D. K. & De Camilli, P. Optogenetic control of phosphoinositide metabolism. Proc. Natl Acad. Sci. USA 109, E2316–E2323 (2012).

    Article  CAS  Google Scholar 

  12. Zhou, X. X. et al. A single-chain photoswitchable CRISPR-Cas9 architecture for light-inducible gene editing and transcription. ACS Chem. Biol. 13, 443–448 (2018).

    Article  CAS  Google Scholar 

  13. Konermann, S. et al. Optical control of mammalian endogenous transcription and epigenetic states. Nature 500, 472–476 (2013).

    Article  CAS  Google Scholar 

  14. Polstein, L. R. & Gersbach, C. A. A light-inducible CRISPR-Cas9 system for control of endogenous gene activation. Nat. Chem. Biol. 11, 198–200 (2015).

    Article  CAS  Google Scholar 

  15. Hallett, R. A., Zimmerman, S. P., Yumerefendi, H., Bear, J. E. & Kuhlman, B. Correlating in vitro and in vivo activities of light-inducible dimers: a cellular optogenetics guide. ACS Synth. Biol. 5, 53–64 (2016).

    Article  CAS  Google Scholar 

  16. Hughes, R. M. et al. Optogenetic apoptosis: light-triggered cell death. Angew. Chem. Int. Ed. Engl. 54, 12064–12068 (2015).

    Article  CAS  Google Scholar 

  17. Katsura, Y. et al. An optogenetic system for interrogating the temporal dynamics of Akt. Sci. Rep. 5, 14589 (2015).

    Article  CAS  Google Scholar 

  18. O’Banion, C. P. et al. Design and profiling of a subcellular targeted optogenetic cAMP-dependent protein kinase. Cell Chem. Biol. 25, 100–109.e108 (2018).

    Article  Google Scholar 

  19. Denk, W., Strickler, J. H. & Webb, W. W. Two-photon laser scanning fluorescence microscopy. Science 248, 73–76 (1990).

    Article  CAS  Google Scholar 

  20. Prakash, R. et al. Two-photon optogenetic toolbox for fast inhibition, excitation and bistable modulation. Nat. Methods 9, 1171–1179 (2012).

    Article  CAS  Google Scholar 

  21. Andrasfalvy, B. K., Zemelman, B. V., Tang, J. & Vaziri, A. Two-photon single-cell optogenetic control of neuronal activity by sculpted light. Proc. Natl Acad. Sci. USA 107, 11981–11986 (2010).

    Article  CAS  Google Scholar 

  22. Guglielmi, G., Barry, J. D., Huber, W. & De Renzis, S. An optogenetic method to modulate cell contractility during tissue morphogenesis. Dev. Cell 35, 646–660 (2015).

    Article  CAS  Google Scholar 

  23. Schindler, S. E. et al. Photo-activatable Cre recombinase regulates gene expression in vivo. Sci. Rep. 5, 13627 (2015).

    Article  Google Scholar 

  24. Nishida, E. & Gotoh, Y. The MAP kinase cascade is essential for diverse signal transduction pathways. Trends Biochem. Sci. 18, 128–131 (1993).

    Article  CAS  Google Scholar 

  25. Shaul, Y. D. & Seger, R. The MEK/ERK cascade: from signaling specificity to diverse functions. Biochim. Biophys. Acta 1773, 1213–1226 (2007).

    Article  CAS  Google Scholar 

  26. Dhillon, A. S., Hagan, S., Rath, O. & Kolch, W. MAP kinase signalling pathways in cancer. Oncogene 26, 3279–3290 (2007).

    Article  CAS  Google Scholar 

  27. Albeck, J. G., Mills, G. B. & Brugge, J. S. Frequency-modulated pulses of ERK activity transmit quantitative proliferation signals. Mol. Cell 49, 249–261 (2013).

    Article  CAS  Google Scholar 

  28. Aoki, K. et al. Stochastic ERK activation induced by noise and cell-to-cell propagation regulates cell density-dependent proliferation. Mol. Cell 52, 529–540 (2013).

    Article  CAS  Google Scholar 

  29. Toettcher, J. E., Weiner, O. D. & Lim, W. A. Using optogenetics to interrogate the dynamic control of signal transmission by the Ras/Erk module. Cell 155, 1422–1434 (2013).

    Article  CAS  Google Scholar 

  30. Hiratsuka, T. et al. Intercellular propagation of extracellular signal-regulated kinase activation revealed by in vivo imaging of mouse skin. eLife 4, e05178 (2015).

    Article  Google Scholar 

  31. Muta, Y. et al. Composite regulation of ERK activity dynamics underlying tumour-specific traits in the intestine. Nat. Commun. 9, 2174 (2018).

    Article  Google Scholar 

  32. Ogura, Y., Wen, F. L., Sami, M. M., Shibata, T. & Hayashi, S. A switch-like activation relay of EGFR-ERK signaling regulates a wave of cellular contractility for epithelial invagination. Dev. Cell 46, 162–172.e165 (2018).

    Article  CAS  Google Scholar 

  33. Hancock, J. F., Paterson, H. & Marshall, C. J. A polybasic domain or palmitoylation is required in addition to the CAAX motif to localize p21ras to the plasma membrane. Cell 63, 133–139 (1990).

    Article  CAS  Google Scholar 

  34. Drobizhev, M. et al. Strong two-photon absorption in new asymmetrically substituted porphyrins: interference between charge-transfer and intermediate-resonance pathways. J. Phys. Chem. B 110, 9802–9814 (2006).

    Article  CAS  Google Scholar 

  35. Rebane, A. et al. New all-optical method for measuring molecular permanent dipole moment difference using two-photon absorption spectroscopy. J. Lumin. 130, 1619–1623 (2010).

    Article  CAS  Google Scholar 

  36. Subach, O. M., Cranfill, P. J., Davidson, M. W. & Verkhusha, V. V. An enhanced monomeric blue fluorescent protein with the high chemical stability of the chromophore. PLoS ONE 6, e28674 (2011).

    Article  CAS  Google Scholar 

  37. Yasuda, R. et al. Supersensitive Ras activation in dendrites and spines revealed by two-photon fluorescence lifetime imaging. Nat. Neurosci. 9, 283–291 (2006).

    Article  CAS  Google Scholar 

  38. Gratton, E., Breusegem, S., Sutin, J., Ruan, Q. & Barry, N. Fluorescence lifetime imaging for the two-photon microscope: time-domain and frequency-domain methods. J. Biomed. Opt. 8, 381–390 (2003).

    Article  Google Scholar 

  39. Clegg, R. M. Fluorescence resonance energy transfer. Curr. Opin. Biotechnol. 6, 103–110 (1995).

    Article  CAS  Google Scholar 

  40. Jares-Erijman, E. A. & Jovin, T. M. FRET imaging. Nat. Biotechnol. 21, 1387 (2003).

    Article  CAS  Google Scholar 

  41. Wu, Y. I. et al. A genetically encoded photoactivatable Rac controls the motility of living cells. Nature 461, 104 (2009).

    Article  CAS  Google Scholar 

  42. Subach, O. M. et al. Structural characterization of acylimine-containing blue and red chromophores in mTagBFP and TagRFP fluorescent proteins. Chem. Biol. 17, 333–341 (2010).

    Article  CAS  Google Scholar 

  43. Brautigam, C. A. et al. Structure of the photolyase-like domain of cryptochrome 1 from Arabidopsis thaliana. Proc. Natl Acad. Sci. USA 101, 12142–12147 (2004).

    Article  CAS  Google Scholar 

  44. Xu, D., Jaroszewski, L., Li, Z. & Godzik, A. AIDA: ab initio domain assembly for automated multi-domain protein structure prediction and domain–domain interaction prediction. Bioinformatics 31, 2098–2105 (2015).

    Article  CAS  Google Scholar 

  45. Zhang, K. et al. Light-mediated kinetic control reveals the temporal effect of the Raf/MEK/ERK pathway in PC12 cell neurite outgrowth. PLoS ONE 9, e92917 (2014).

    Article  Google Scholar 

  46. Akagi, T., Sasai, K. & Hanafusa, H. Refractory nature of normal human diploid fibroblasts with respect to oncogene-mediated transformation. Proc. Natl Acad. Sci. USA 100, 13567–13572 (2003).

    Article  CAS  Google Scholar 

  47. Lo, C. A. et al. Quantification of protein levels in single living cells. Cell Rep. 13, 2634–2644 (2015).

    Article  CAS  Google Scholar 

  48. Komatsu, N. et al. A platform of BRET-FRET hybrid biosensors for optogenetics, chemical screening, and in vivo imaging. Sci. Rep. 8, 8984 (2018).

    Article  Google Scholar 

  49. Nishizuka, Y. Turnover of inositol phospholipids and signal transduction. Science 225, 1365–1370 (1984).

    Article  CAS  Google Scholar 

  50. Lerner, A. M., Yumerefendi, H., Goudy, O. J., Strahl, B. D. & Kuhlman, B. Engineering improved photoswitches for the control of nucleocytoplasmic distribution. ACS Synth. Biol. 7, 2898–2907 (2018).

    Article  CAS  Google Scholar 

  51. Drobizhev, M., Makarov, N. S., Tillo, S. E., Hughes, T. E. & Rebane, A. Two-photon absorption properties of fluorescent proteins. Nat. Methods 8, 393–399 (2011).

    Article  CAS  Google Scholar 

  52. Homans, R. J. et al. Two photon spectroscopy and microscopy of the fluorescent flavoprotein, iLOV. Phys. Chem. Chem. Phys. 20, 16949–16955 (2018).

    Article  CAS  Google Scholar 

  53. Hayek, A. et al. Cell‐permeant cytoplasmic blue fluorophores optimized for in vivo two‐photon microscopy with low‐power excitation. Microsc. Res. Tech. 70, 880–885 (2007).

    Article  CAS  Google Scholar 

  54. Cornejo, J., Beale, S. I., Terry, M. J. & Lagarias, J. C. Phytochrome assembly. The structure and biological activity of 2(R),3(E)-phytochromobilin derived from phycobiliproteins. J. Biol. Chem. 267, 14790–14798 (1992).

    CAS  PubMed  Google Scholar 

  55. Conlon, P., Gelin-Licht, R., Ganesan, A., Zhang, J. & Levchenko, A. Single-cell dynamics and variability of MAPK activity in a yeast differentiation pathway. Proc. Natl Acad. Sci. USA 113, E5896–E5905 (2016).

    Article  CAS  Google Scholar 

  56. Stanley, P. L., Steiner, S., Havens, M. & Tramposch, K. M. Mouse skin inflammation induced by multiple topical applications of 12-O-tetradecanoylphorbol-13-acetate. Ski. Pharmacol. Physiol. 4, 262–271 (1991).

    Article  CAS  Google Scholar 

  57. Lee, S. H. et al. Suppression of 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced skin inflammation in mice by transduced Tat-Annexin protein. BMB Rep. 45, 354–359 (2012).

    Article  CAS  Google Scholar 

  58. Ai, H. W., Shaner, N. C., Cheng, Z., Tsien, R. Y. & Campbell, R. E. Exploration of new chromophore structures leads to the identification of improved blue fluorescent proteins. Biochemistry 46, 5904–5910 (2007).

    Article  CAS  Google Scholar 

  59. Tomosugi, W. et al. An ultramarine fluorescent protein with increased photostability and pH insensitivity. Nat. Methods 6, 351–353 (2009).

    Article  CAS  Google Scholar 

  60. Niwa, H., Yamamura, K. & Miyazaki, J. Efficient selection for high-expression transfectants with a novel eukaryotic vector. Gene 108, 193–199 (1991).

    Article  CAS  Google Scholar 

  61. Bindels, D. S. et al. mScarlet: a bright monomeric red fluorescent protein for cellular imaging. Nat. Methods 14, 53–56 (2017).

    Article  CAS  Google Scholar 

  62. Komatsu, N. et al. Development of an optimized backbone of FRET biosensors for kinases and GTPases. Mol. Biol. Cell 22, 4647–4656 (2011).

    Article  CAS  Google Scholar 

  63. Urasaki, A., Morvan, G. & Kawakami, K. Functional dissection of the Tol2 transposable element identified the minimal cis-sequence and a highly repetitive sequence in the subterminal region essential for transposition. Genetics 174, 639–649 (2006).

    Article  CAS  Google Scholar 

  64. Sumiyama, K., Kawakami, K. & Yagita, K. A simple and highly efficient transgenesis method in mice with the Tol2 transposon system and cytoplasmic microinjection. Genomics 95, 306–311 (2010).

    Article  CAS  Google Scholar 

  65. Tarutani, M., Cai, T., Dajee, M. & Khavari, P. A. Inducible activation of Ras and Raf in adult epidermis. Cancer Res. 63, 319–323 (2003).

    CAS  PubMed  Google Scholar 

  66. Taslimi, A. et al. Optimized second-generation CRY2-CIB dimerizers and photoactivatable Cre recombinase. Nat. Chem. Biol. 12, 425–430 (2016).

    Article  CAS  Google Scholar 

  67. Lillo, M. P., Beechem, J. M., Szpikowska, B. K., Sherman, M. A. & Mas, M. T. Design and characterization of a multisite fluorescence energy-transfer system for protein folding studies: a steady-state and time-resolved study of yeast phosphoglycerate kinase. Biochemistry 36, 11261–11272 (1997).

    Article  CAS  Google Scholar 

  68. Sali, A. & Blundell, T. L. Comparative protein modelling by satisfaction of spatial restraints. J. Mol. Biol. 234, 779–815 (1993).

    Article  CAS  Google Scholar 

  69. Sato, T. et al. Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature 459, 262–265 (2009).

    Article  CAS  Google Scholar 

  70. Sano, T., Kobayashi, T., Ogawa, O. & Matsuda, M. Gliding basal cell migration of the urothelium during wound healing. Am. J. Pathol. 188, 2564–2573 (2018).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank J. Miyazaki, T. Nagai (Osaka University), A. Hotta (Kyoto University), K. Kawakami (National Institute of Genetics), H. Miyoshi (Keio University) and C. Tucker (University of Colorado, Denver) for the plasmids. We also thank Y. Suzuki, J. Kawamata (Yamaguchi University) and T. Furuta (Toho University) for helpful discussion. We are grateful to the members of the Matsuda Laboratory for their helpful input; K. Hirano, K. Takakura, A. Kawagishi and Y. Takeshita for their technical assistance and the Medical Research Support Center of Kyoto University for in vivo imaging. This research was partially supported by the Platform Project for Supporting Drug Discovery and Life Science Research (Basis for Supporting Innovative Drug Discovery and Life Science Research from AMED under Grant No. JP18am0101079 (to S.I.)). This work was also supported by Japan Society for the Promotion of Science (JSPS) grant nos. KAKENHI17J07950 (to T.K.), KAKENHI18K07066, AMED16gm0610010h004 (to K.Terai), KAKENHI15H02397, KAKENHI15H05949 ‘Resonance Bio’, KAKENHI16H06280 ‘ABiS’, CRESTJPMJCR1654 and the Nakatani Foundation (to M.M.).

Author information

Authors and Affiliations

  1. Department of Pathology and Biology of Diseases, Graduate School of Medicine, Kyoto University, Kyoto, Japan

    Tomoaki Kinjo & Michiyuki Matsuda

  2. Department of Diagnostic Imaging and Nuclear Medicine, Graduate School of Medicine, Kyoto University, Kyoto, Japan

    Tomoaki Kinjo & Kaori Togashi

  3. Research Center for Dynamic Living Systems, Graduate School of Biostudies, Kyoto University, Kyoto, Japan

    Kenta Terai & Michiyuki Matsuda

  4. Department of Cell Biology, Graduate School of Medicine, Kyoto University, Kyoto, Japan

    Shoichiro Horita, Norimichi Nomura & So Iwata

  5. Laboratory for Mouse Genetic Engineering, RIKEN Center for Biosystems Dynamics Research, Osaka, Japan

    Kenta Sumiyama

  6. RIKEN SPring-8 Center, Sayo, Hyogo, Japan

    So Iwata

Authors
  1. Tomoaki Kinjo
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Kenta Terai
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Shoichiro Horita
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Norimichi Nomura
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Kenta Sumiyama
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Kaori Togashi
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. So Iwata
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Michiyuki Matsuda
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

T.K., K. Terai and M.M. conceptualized the study. T.K., S.H., K. Terai, N.N., K.S., S.I. and M.M. designed the methodology. T.K., K. Terai and M.M. performed validation. T.K., K. Terai, S.H., N.N., S.I. and M.M. conducted the formal analysis. The investigation was conducted by T.K., K. Terai and M.M. The data curation was done by T.K., K. Terai and M.M. Resources were collected by T.K., K. Terai and M.M. The original draft was written by T.K. Writing, review and editing was done by K. Terai, N.N., K.S., K. Togashi, S.I. and M.M. K. Terai and M.M. supervised the work. Project administration was done by K. Terai and M.M. T.K., K. Terai, S.I. and M.M. were responsible for funding acquisition.

Corresponding author

Correspondence to Michiyuki Matsuda.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Peer review information: Rita Strack was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Integrated supplementary information

Supplementary Figure 1 Structure model of CRY2PHR with FAD.

The model is visualized with PyMOL. The structure model of CRY2PHR is shown as gray-colored cartoon. The mTagBFP2 insertion loop for 2paCRY2 (a.a. 161–170) is shown in blue. Other representative loops for insertion are shown in orange. Residues of FAD were shown as sticks representation with carbon, oxygen, phosphorus, and nitrogen atoms; Green: Carbon, Red: Oxygen, Blue: Nitrogen, Orange: Phosphorus.

Supplementary Figure 2 Inter-chromophore distances predicted by the structure model.

a–c, Structure models of representative mTagBFP2-CRY2PHR chimeric proteins: mTagBFP2-QFT-CRY2PHR, mTagBFP (a.a. 1–220)-CRY2PHR (a.a. 2–498, K2Y) and 2paCRY2. The chromophores of mTagBFP2 (Leu63-Tyr64-Gly65) are shown in blue. FADs are shown in cyan. d, Shown here are inter-chromophore distance measured in each structure model and the FRET efficiency quantified by FLIM (Fig. 1).

Supplementary Figure 3 Kinetics and repeatability of 2paCRY2 activation.

HeLa cells expressing CIBN-EGFP-CAAX and 2paCRY2-mScarlet-H were kept under a dark condition for at least 1 h. Then, the cells were exposed to the 2P activation (810 nm, 5.0/7.5 mW, 0.207 μm/pixel, 2 µs/pixel, 128 × 128 pixels, 150 scans), which took approximately 30 sec. The plasma membrane translocation of 2paCRY2-mScarlet-H was monitored at 1040 nm and quantified by the intensity ratio of the cytosol versus nucleus. The gray and black lines in squared boxes represent the raw and the fitted data, respectively. In this experiment, the nuclear mScarlet-H intensity was used as the reference, because the kinetics of the plasma membrane translocation of 2paCRY2-mScarlet-H was markedly faster than that of the nucleocytoplasmic shuttling. Representative data among n = 3 independent experiments.

Supplementary Figure 4 2P activation spectra of CRY2PHR and 2paCRY2.

HeLa cells expressing CIBN-EGFP-CAAX and CRY2PHR- or 2paCRY2-mScarlet-H were kept under a dark condition for at least 1 h. Then, the cells were exposed to the 2P activation at the indicated wavelength (5 mW, 0.207 μm/pixel, 2 µs/pixel, 512 × 512 pixels, every 5 sec, 30 scans) or 1P activation (480 nm, 5 mW/cm2, for 1 sec after 2P activation). Membrane versus cytosol ratio (mem/cyto ratio) of mScarlet-H intensity was quantified at each wavelength. The increase in mem/cyto ratio after 2P activation was normalized by that after 1P activation. n = 6 cells; Representative data among n = 2 independent experiments. Blue and cyan dots represent mean; error bars, s.d..

Supplementary Figure 5 Optical control of 2paCRY2 localization at subcellular resolution.

A HeLa cell expressing CIBN-EGFP-CAAX and 2paCRY2-mScarlet-H was kept under a dark condition for at least 1 h. Then, the red squared region including plasma membrane was exposed to the 2P activation (810 nm, 7.5 mW, 0.103 μm/pixel, 2 µs/pixel, 30 × 30 pixels, 50 scans). The mScarlet-H intensities along the yellow broken lines are shown in the lower panels. Representative data among n = 3 independent experiments.

Supplementary Figure 6 Quantification of the ERK activity (FRET/CFP) in HeLa cells expressing 2paRAF variants with P2A and full-length RAF1, IRES and full-length RAF1, or P2A and the RAF1 catalytic domain (a.a. 307-648) before and after the 2P activation.

HeLa cells stably expressing EKAREV-nls, an ERK FRET biosensor, were transfected with the plasmids encoding 2paRAF connected with full-length RAF1 or the RAF1 catalytic domain (a.a. 307–648) via P2A or IRES. Cells were photoactivated and imaged by 2P excitation (840 nm, 10mW, 0.828 μm/pixel, 2 µs/pixel, 20 scans). The ERK activity was plotted against mScarlet-I intensity to examine the correlation between the expression level of the plasmid and ERK activity. The ERK activity (FRET/CFP) in each cell before (black dots) and after (magenta dots) 2P activation (840 nm, 10 mW, 0.828 μm/pixel, 2 µs/pixel, 20 scans) was quantified and plotted as a function of mScarlet-I intensity; n = 21 cells; Representative data among n = 2 independent experiments. a.u., arbitrary units.

Supplementary information

Supplementary Information

Supplementary Figs. 1–6, Supplementary Tables 1 and 2, and Supplementary Notes 1 and 2.

Reporting Summary

Supplementary Video 1

Single-cell ERK activation in the 3D MDCK cyst expressing 2paRAF and EKAREV-nls.

Supplementary Video 2

Rotation images of the 3D MDCK cyst after single-cell ERK activation.

Supplementary Video 3

Induction of the ERK activation wave in the mouse auricular epidermis.

Source data

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Kinjo, T., Terai, K., Horita, S. et al. FRET-assisted photoactivation of flavoproteins for in vivo two-photon optogenetics. Nat Methods 16, 1029–1036 (2019). https://doi.org/10.1038/s41592-019-0541-5

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41592-019-0541-5

This article is cited by

Search

Advanced search

Quick links

Nature Briefing: Translational Research

Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology, drug discovery and pharma.

Get what matters in translational research, free to your inbox weekly. Sign up for Nature Briefing: Translational Research