Abstract
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.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 1-10 |
| Number of pages | 10 |
| Journal | Nature Chemical Biology |
| DOIs | |
| State | Accepted/In press - Apr 23 2018 |
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ASJC Scopus subject areas
- Molecular Biology
- Cell Biology
Cite this
Direct multiplex imaging and optogenetics of Rho GTPases enabled by near-infrared FRET. / Shcherbakova, Daria; Cox Cammer, Natasha; Huisman, Tsipora M.; Verkhusha, Vladislav; Hodgson, Louis.
In: Nature Chemical Biology, 23.04.2018, p. 1-10.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Direct multiplex imaging and optogenetics of Rho GTPases enabled by near-infrared FRET
AU - Shcherbakova, Daria
AU - Cox Cammer, Natasha
AU - Huisman, Tsipora M.
AU - Verkhusha, Vladislav
AU - Hodgson, Louis
PY - 2018/4/23
Y1 - 2018/4/23
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
SP - 1
EP - 10
JO - Nature Chemical Biology
JF - Nature Chemical Biology
SN - 1552-4450
ER -