Journal
NATURE METHODS
Volume 16, Issue 10, Pages 1029-+Publisher
NATURE PUBLISHING GROUP
DOI: 10.1038/s41592-019-0541-5
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Funding
- Platform Project for Supporting Drug Discovery and Life Science Research (Basis for Supporting Innovative Drug Discovery and Life Science Research from AMED) [JP18am0101079]
- Japan Society for the Promotion of Science (JSPS) [KAKENHI17J07950, KAKENHI18K07066, AMED16gm0610010h004, KAKENHI15H02397, KAKENHI15H05949, KAKENHI16H06280, CRESTJPMJCR1654]
- Nakatani Foundation
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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 Forster 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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