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Direct multiplex imaging and optogenetics of Rho GTPases enabled by near-infrared FRET

Nature Chemical Biology volume 14, pages 591–600 (2018)Cite this article

A Correction to this article was published on 04 May 2018

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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.

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Fig. 1: Characterization of the engineered monomeric miRFP720.
Fig. 2: NIR Rac1 biosensor for live-cell imaging.
Fig. 3: Morphodynamic analysis of RhoA–Rac1 antagonism.
Fig. 4: Morphodynamic analysis of Rac1 activity and Rac1–GDI binding.
Fig. 5: Concurrent measurement of Rac1 activity during LOV-TRAP optogenetics.
Fig. 6: NIR-FRET pair of fluorescent proteins in kinase-substrate biosensors.

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  • 04 May 2018

    In the version of this article originally published, the values for time shown on the x axis of Figure 5c were incorrect. The error has been corrected in all versions of the paper.

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Acknowledgements

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.

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Authors and Affiliations

  1. Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, NY, USA

    Daria M. Shcherbakova, Natasha Cox Cammer, Tsipora M. Huisman, Vladislav V. Verkhusha & Louis Hodgson

  2. Gruss-Lipper Biophotonics Center, Albert Einstein College of Medicine, Bronx, NY, USA

    Daria M. Shcherbakova, Vladislav V. Verkhusha & Louis Hodgson

  3. Department of Biochemistry and Developmental Biology, Faculty of Medicine, University of Helsinki, Helsinki, Finland

    Vladislav V. Verkhusha

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Contributions

D.M.S. and V.V.V. developed the miRFP720 and characterized it in vitro, in cells and as a FRET acceptor. L.H., N.C.C. and T.M.H. engineered the NIR Rac1 biosensor, characterized it in cells and performed live-cell imaging. L.H., D.M.S. and  V.V.V. designed the project, planned the experiments and discussed the data; V.V.V., D.M.S. and L.H. wrote the manuscript. All authors reviewed the manuscript.

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Correspondence to Vladislav V. Verkhusha or Louis Hodgson.

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Supplementary information

Supplementary Text and Figures

Supplementary Tables 1–3 and Supplementary Figures 1–16

Reporting Summary

Supplementary Video 1: A representative live cell movie of a control MEF cell with CFP-YFP FRET RhoA and NIR Rac1 biosensors imaged concurrently

Differential interference contrast, RhoA activity, Rac1 activity, and binary overlay of high RhoA/Rac1 activities are shown (yellow: top 2.5% of Rac1 activity; blue: top 2.5% of RhoA activity; white: colocalization). White bar = 20 µm. Frame rate: 7 frames per second, timelapse imaging rate: 10 s intervals. Example cell taken from data set of 16 cells from 6 independent experiments.

Supplementary Video 2: A representative segment of a leading edge protrusion used in morphodynamic analysis from control MEF cell with CFP-YFP FRET RhoA and NIR Rac1 biosensors imaged concurrently

RhoA activity, Rac1 activity, and binary overlay of high RhoA/Rac1 activities are shown (yellow: top 6% of Rac1 activity; blue: top 6% of RhoA activity; white: colocalization). White bar = 10 µm. Frame rate: 7 frames per second, timelapse imaging rate: 10 s intervals. Example cell taken from data set of 16 cells, from 6 independent experiments.

Supplementary Video 3: A representative segment of a leading edge protrusion used in morphodynamic analysis from control MEF cell with CFP-YFP FRET RhoA and NIR Rac1 biosensors imaged concurrently

RhoA activity, Rac1 activity, and binary overlay of high RhoA/Rac1 activities are shown (yellow: top 5.5% of Rac1 activity; blue: top 5.5% of RhoA activity; white: colocalization). White bar = 10 µm. Frame rate: 7 frames per second, timelapse imaging rate: 10 s intervals. Example cell taken from data set of 16 cells, from 6 independent experiments.

Supplementary Video 4: A representative segment of a leading edge protrusion used in morphodynamic analysis from MEF cell treated with ROCK-inhibitor, with CFP-YFP FRET RhoA and NIR Rac1 biosensors imaged concurrently

RhoA activity, Rac1 activity, and binary overlay of high RhoA/Rac1 activities are shown (yellow: top 8% of Rac1 activity; blue: top 8% of RhoA activity; white: colocalization). White bar = 10 µm. Frame rate: 7 frames per second, timelapse imaging rate: 10 s intervals. Example cell taken from data set of 18 cells, from 3 independent experiments.

Supplementary Video 5: A representative segment of a leading edge protrusion used in morphodynamic analysis from MEF cell treated with ROCK-inhibitor, with CFP-YFP FRET RhoA and NIR Rac1 biosensors imaged concurrently

RhoA activity, Rac1 activity, and binary overlay of high RhoA/Rac1 activities are shown (yellow: top 3% of Rac1 activity; blue: top 3% of RhoA activity; white: colocalization). White bar = 10 µm. Frame rate: 7 frames per second, timelapse imaging rate: 10 s intervals. Example cell taken from data set of 18 cells, from 3 independent experiments.

Supplementary Video 6: A representative segment of a leading edge protrusion used in morphodynamic analysis from MEF cell with CFP-YFP FRET Rac1-GDI binding biosensor and NIR Rac1 biosensor imaged concurrently

Rac1-GDI binding, Rac1 activity, and binary overlay of high Rac1-GDI binding/Rac1 activity are shown (yellow: top 5% of Rac1 activity; blue: top 5% of Rac1-GDI binding; white: colocalization). White bar = 10 µm. Frame rate: 7 frames per second, timelapse imaging rate: 10 s intervals. Example cell taken from data set of 10 cells from 3 independent experiments.

Supplementary Video 7: A representative live cell movie of LOV-TRAP optogenetics of Rac1 activity and concurrent measurement of Rac1 activity using the NIR Rac1 biosensor in a MEF cell

457 nm light was used to illuminate the whole field of view during indicated time points. White bar = 20 µm. Frame rate: 7 frames per second, timelapse imaging rate: 10 s intervals. Example cell taken from data set of 17 independent photoactivation experiments.

Supplementary Video 8: A representative live cell movie of a HeLa cell with AKAR biosensor

White bar = 20 µm. Frame rate: 7 frames per second, timelapse imaging rate: 2 min intervals. * indicates addition of 1 mM dibutyryl cAMP. Example cell taken from data set of 3 independent stimulation experiments.

Supplementary Video 9: A representative live cell movie of a HeLa cell with JNKAR biosensor

White bar = 20 µm. Frame rate: 7 frames per second, timelapse imaging rate: 2 min intervals. * indicates addition of 1 µg/mL anisomycin. Example cell taken from data set of 3 independent stimulation experiments.

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Shcherbakova, D.M., Cox Cammer, N., Huisman, T.M. et al. Direct multiplex imaging and optogenetics of Rho GTPases enabled by near-infrared FRET. Nat Chem Biol 14, 591–600 (2018). https://doi.org/10.1038/s41589-018-0044-1

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