TY - JOUR
T1 - Direct multiplex imaging and optogenetics of Rho GTPases enabled by near-infrared FRET article
AU - Shcherbakova, Daria M.
AU - Cox Cammer, Natasha
AU - Huisman, Tsipora M.
AU - Verkhusha, Vladislav V.
AU - Hodgson, Louis
N1 - Funding Information:
We thank M. Brenowitz (Albert Einstein College of Medicine) for help with analytical ultracentrifugation, M. Baloban (Albert Einstein College of Medicine) for help with engineering miRFP720, and O. Oliinyk (University of Helsinki) for advice on kinase biosensors. We thank S. Donnelly (Albert Einstein College of Medicine) for critical reading of the manuscript. This work was supported by grants GM122567, NS099573, and NS103573 to V.V.V., and CA205262 to L.H. from the US National Institutes of Health and ERC-2013-ADG-340233 from the EU FP7 program to V.V.V. We thank K. Aoki (Kyoto University), K. Hahn (University of North Carolina at Chapel Hill) and J. van Buul (University of Amsterdam) for providing reagents.
Publisher Copyright:
© 2018 The Author(s).
PY - 2018/6/1
Y1 - 2018/6/1
N2 - Direct visualization and light control of several cellular processes is a challenge, owing to the spectral overlap of available genetically encoded probes. Here we report the most red-shifted monomeric near-infrared (NIR) fluorescent protein, miRFP720, and the fully NIR Förster resonance energy transfer (FRET) pair miRFP670-miRFP720, which together enabled design of biosensors compatible with CFP-YFP imaging and blue-green optogenetic tools. We developed a NIR biosensor for Rac1 GTPase and demonstrated its use in multiplexed imaging and light control of Rho GTPase signaling pathways. Specifically, we combined the Rac1 biosensor with CFP-YFP FRET biosensors for RhoA and for Rac1-GDI binding, and concurrently used the LOV-TRAP tool for upstream Rac1 activation. We directly observed and quantified antagonism between RhoA and Rac1 dependent on the RhoA-downstream effector ROCK; showed that Rac1 activity and GDI binding closely depend on the spatiotemporal coordination between these two molecules; and simultaneously observed Rac1 activity during optogenetic manipulation of Rac1.
AB - Direct visualization and light control of several cellular processes is a challenge, owing to the spectral overlap of available genetically encoded probes. Here we report the most red-shifted monomeric near-infrared (NIR) fluorescent protein, miRFP720, and the fully NIR Förster resonance energy transfer (FRET) pair miRFP670-miRFP720, which together enabled design of biosensors compatible with CFP-YFP imaging and blue-green optogenetic tools. We developed a NIR biosensor for Rac1 GTPase and demonstrated its use in multiplexed imaging and light control of Rho GTPase signaling pathways. Specifically, we combined the Rac1 biosensor with CFP-YFP FRET biosensors for RhoA and for Rac1-GDI binding, and concurrently used the LOV-TRAP tool for upstream Rac1 activation. We directly observed and quantified antagonism between RhoA and Rac1 dependent on the RhoA-downstream effector ROCK; showed that Rac1 activity and GDI binding closely depend on the spatiotemporal coordination between these two molecules; and simultaneously observed Rac1 activity during optogenetic manipulation of Rac1.
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U2 - 10.1038/s41589-018-0044-1
DO - 10.1038/s41589-018-0044-1
M3 - Article
C2 - 29686359
AN - SCOPUS:85045851039
VL - 14
SP - 591
EP - 600
JO - Nature Chemical Biology
JF - Nature Chemical Biology
SN - 1552-4450
IS - 6
ER -