FRET-assisted photoactivation of flavoproteins for in vivo two-photon optogenetics
FRET-assisted photoactivation of flavoproteins for in vivo two-photon optogenetics
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DOI：
10.1038/s41592-019-0541-5
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发表时间：
2019-10-01
期刊：
影响因子：
48
通讯作者：
Matsuda, Michiyuki
中科院分区：
文献类型：
--
作者：
Kinjo, Tomoaki;Terai, Kenta;Matsuda, Michiyuki
光学二聚体已经被开发用来解开缠绕的信号通路，但它们在体内的应用有限，部分原因是它们在双光子(2P)激发下的激活效率低下。为了克服这个问题，我们开发了Forster共振能量转移(FRET)辅助光激活，或FRAPA。在2P激发下，mTagBFP2有效地吸收能量并将能量传递给CRY2的发色团。基于结构导向工程，开发了一种具有40%FRET效率的嵌合蛋白，命名为2P-Actiatable CRY2，或2paCRY2。2paCRY2用于构建RAF1激活系统，命名为2paRAF。在三维培养的表达2paRAF的细胞中，细胞外信号调节激酶(ERK)在单细胞分辨率下可被2P有效激活。在表达2paRAF的小鼠的表皮细胞中也完成了ERK的光激活。我们进一步开发了mTFP1融合的LOV结构域，该结构域对2P激发表现出有效的响应。总的来说，FRAPA将为体内信号通路的单细胞光学控制铺平道路。
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.