0.08 sec per time interval, NA, 1.03 sec per time interval, 0.12 sec per time interval, 322.12 sec per time interval, 0.06 sec per time interval, 120.12 sec per time interval, 366.47 sec per time interval, 120.13 sec per time interval, 333.24 sec per time interval, 61.61 sec per time interval, 180.12 sec per time interval, 0.11 sec per time interval, 1 sec per time interval, 63.40 sec per time interval, 180.13 sec per time interval
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Related paper(s)
Tomoaki Kinjo, Kenta Terai, Shoichiro Horita, Norimichi Nomura, Kenta Sumiyama, Kaori Togashi, So Iwata, Michiyuki Matsuda (2019) FRET-assisted photoactivation of flavoproteins for in vivo two-photon optogenetics., Nature methods, Volume 16, Number 10, pp. 1029-1036
Published in 2019 Oct
(Electronic publication in Sept. 9, 2019, midnight )
(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 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.
Michiyuki Matsuda, Kenta Terai
, Graduate School of Biostudies, Kyoto University, Graduate School of Biostudies, Kyoto University
, Research Center for Dynamic Living Systems, Research Center for Dynamic Living Systems
, Laboratory of Bioimaging and Cell Signaling, Laboratory of Bioimaging and Cell Signaling