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.
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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).
All analysis code used in this study is available upon reasonable request.
Fan, L. Z. & Lin, M. Z. Optical control of biological processes by light‐switchable proteins. Wiley Interdiscip. Rev. Dev. Biol. 4, 545–554 (2015).
Spiltoir, J. I. & Tucker, C. L. Photodimerization systems for regulating protein–protein interactions with light. Curr. Opin. Struct. Biol. 57, 1–8 (2019).
Chen, D., Gibson, E. S. & Kennedy, M. J. A light-triggered protein secretion system. J. Cell Biol. 201, 631–640 (2013).
Lungu, O. I. et al. Designing photoswitchable peptides using the AsLOV2 domain. Chem. Biol. 19, 507–517 (2012).
Kennedy, M. J. et al. Rapid blue-light-mediated induction of protein interactions in living cells. Nat. Methods 7, 973–975 (2010).
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).
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).
Lee, S. et al. Reversible protein inactivation by optogenetic trapping in cells. Nat. Methods 11, 633–636 (2014).
Nguyen, M. K. et al. Optogenetic oligomerization of Rab GTPases regulates intracellular membrane trafficking. Nat. Chem. Biol. 12, 431 (2016).
Valon, L., Marín-Llauradó, A., Wyatt, T., Charras, G. & Trepat, X. Optogenetic control of cellular forces and mechanotransduction. Nat. Commun. 8, 14396 (2017).
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).
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).
Konermann, S. et al. Optical control of mammalian endogenous transcription and epigenetic states. Nature 500, 472–476 (2013).
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).
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).
Hughes, R. M. et al. Optogenetic apoptosis: light-triggered cell death. Angew. Chem. Int. Ed. Engl. 54, 12064–12068 (2015).
Katsura, Y. et al. An optogenetic system for interrogating the temporal dynamics of Akt. Sci. Rep. 5, 14589 (2015).
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).
Denk, W., Strickler, J. H. & Webb, W. W. Two-photon laser scanning fluorescence microscopy. Science 248, 73–76 (1990).
Prakash, R. et al. Two-photon optogenetic toolbox for fast inhibition, excitation and bistable modulation. Nat. Methods 9, 1171–1179 (2012).
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).
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).
Schindler, S. E. et al. Photo-activatable Cre recombinase regulates gene expression in vivo. Sci. Rep. 5, 13627 (2015).
Nishida, E. & Gotoh, Y. The MAP kinase cascade is essential for diverse signal transduction pathways. Trends Biochem. Sci. 18, 128–131 (1993).
Shaul, Y. D. & Seger, R. The MEK/ERK cascade: from signaling specificity to diverse functions. Biochim. Biophys. Acta 1773, 1213–1226 (2007).
Dhillon, A. S., Hagan, S., Rath, O. & Kolch, W. MAP kinase signalling pathways in cancer. Oncogene 26, 3279–3290 (2007).
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).
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).
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).
Hiratsuka, T. et al. Intercellular propagation of extracellular signal-regulated kinase activation revealed by in vivo imaging of mouse skin. eLife 4, e05178 (2015).
Muta, Y. et al. Composite regulation of ERK activity dynamics underlying tumour-specific traits in the intestine. Nat. Commun. 9, 2174 (2018).
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).
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).
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).
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).
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).
Yasuda, R. et al. Supersensitive Ras activation in dendrites and spines revealed by two-photon fluorescence lifetime imaging. Nat. Neurosci. 9, 283–291 (2006).
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).
Clegg, R. M. Fluorescence resonance energy transfer. Curr. Opin. Biotechnol. 6, 103–110 (1995).
Jares-Erijman, E. A. & Jovin, T. M. FRET imaging. Nat. Biotechnol. 21, 1387 (2003).
Wu, Y. I. et al. A genetically encoded photoactivatable Rac controls the motility of living cells. Nature 461, 104 (2009).
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).
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).
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).
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).
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).
Lo, C. A. et al. Quantification of protein levels in single living cells. Cell Rep. 13, 2634–2644 (2015).
Komatsu, N. et al. A platform of BRET-FRET hybrid biosensors for optogenetics, chemical screening, and in vivo imaging. Sci. Rep. 8, 8984 (2018).
Nishizuka, Y. Turnover of inositol phospholipids and signal transduction. Science 225, 1365–1370 (1984).
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).
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).
Homans, R. J. et al. Two photon spectroscopy and microscopy of the fluorescent flavoprotein, iLOV. Phys. Chem. Chem. Phys. 20, 16949–16955 (2018).
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).
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).
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).
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).
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).
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).
Tomosugi, W. et al. An ultramarine fluorescent protein with increased photostability and pH insensitivity. Nat. Methods 6, 351–353 (2009).
Niwa, H., Yamamura, K. & Miyazaki, J. Efficient selection for high-expression transfectants with a novel eukaryotic vector. Gene 108, 193–199 (1991).
Bindels, D. S. et al. mScarlet: a bright monomeric red fluorescent protein for cellular imaging. Nat. Methods 14, 53–56 (2017).
Komatsu, N. et al. Development of an optimized backbone of FRET biosensors for kinases and GTPases. Mol. Biol. Cell 22, 4647–4656 (2011).
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).
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).
Tarutani, M., Cai, T., Dajee, M. & Khavari, P. A. Inducible activation of Ras and Raf in adult epidermis. Cancer Res. 63, 319–323 (2003).
Taslimi, A. et al. Optimized second-generation CRY2-CIB dimerizers and photoactivatable Cre recombinase. Nat. Chem. Biol. 12, 425–430 (2016).
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).
Sali, A. & Blundell, T. L. Comparative protein modelling by satisfaction of spatial restraints. J. Mol. Biol. 234, 779–815 (1993).
Sato, T. et al. Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature 459, 262–265 (2009).
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).
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.).
The authors declare no competing interests.
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Integrated supplementary information
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.
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).
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.
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..
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 Figs. 1–6, Supplementary Tables 1 and 2, and Supplementary Notes 1 and 2.
Single-cell ERK activation in the 3D MDCK cyst expressing 2paRAF and EKAREV-nls.
Rotation images of the 3D MDCK cyst after single-cell ERK activation.
Induction of the ERK activation wave in the mouse auricular epidermis.
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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) doi:10.1038/s41592-019-0541-5