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

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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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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.

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.


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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

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.

Correspondence to Michiyuki Matsuda.

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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.

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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.

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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.

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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

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