| RRC ID |
57649
|
| Author |
Kinjo T, Terai K, Horita S, Nomura N, Sumiyama K, Togashi K, Iwata S, Matsuda M.
|
| Title |
FRET-assisted photoactivation of flavoproteins for in vivo two-photon optogenetics.
|
| Journal |
Nat Methods
|
| 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 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.
|
| Volume |
16(10)
|
| Pages |
1029-1036
|
| Published |
2019-10-1
|
| DOI |
10.1038/s41592-019-0541-5
|
| PII |
10.1038/s41592-019-0541-5
|
| PMID |
31501546
|
| MeSH |
Animals
Enzyme Activation
Extracellular Signal-Regulated MAP Kinases / metabolism
Flavoproteins / metabolism*
Fluorescence Resonance Energy Transfer / methods*
Mice
Optogenetics*
Photons*
|
| IF |
30.822
|
| Times Cited |
5
|
| Resource |
| DNA material |
pCMV-VSV-G-RSV-Rev (RDB04393) |
| Human and Animal Cells |
MDCK(RCB0995) |
