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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.

References

  1. Machacek, M. et al. Coordination of Rho GTPase activities during cell protrusion. Nature 461, 99–103 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Hodgson, L. et al. FRET binding antenna reports spatiotemporal dynamics of GDI–Cdc42 GTPase interactions. Nat. Chem. Biol. 12, 802–809 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Ouyang, M. et al. Simultaneous visualization of protumorigenic Src and MT1-MMP activities with fluorescence resonance energy transfer. Cancer Res. 70, 2204–2212 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Grant, D. M. et al. Multiplexed FRET to image multiple signaling events in live cells. Biophys. J. 95, L69–L71 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Demeautis, C. et al. Multiplexing PKA and ERK1&2 kinases FRET biosensors in living cells using single excitation wavelength dual colour FLIM. Sci. Rep. 7, 41026 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Shcherbakova, D. M., Hink, M. A., Joosen, L., Gadella, T. W. & Verkhusha, V. V. An orange fluorescent protein with a large Stokes shift for single-excitation multicolor FCCS and FRET imaging. J. Am. Chem. Soc. 134, 7913–7923 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Shcherbakova, D. M., Shemetov, A. A., Kaberniuk, A. A. & Verkhusha, V. V. Natural photoreceptors as a source of fluorescent proteins, biosensors, and optogenetic tools. Annu. Rev. Biochem. 84, 519–550 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Chernov, K. G., Redchuk, T. A., Omelina, E. S. & Verkhusha, V. V. Near-infrared fluorescent proteins, biosensors, and optogenetic tools engineered from phytochromes. Chem. Rev. 117, 6423–6446 (2017).

    Article  CAS  PubMed  Google Scholar 

  9. Shcherbakova, D. M. & Verkhusha, V. V. Near-infrared fluorescent proteins for multicolor in vivo imaging. Nat. Methods 10, 751–754 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Shcherbakova, D. M. et al. Bright monomeric near-infrared fluorescent proteins as tags and biosensors for multiscale imaging. Nat. Commun. 7, 12405 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Nobes, C. & Hall, A. Regulation and function of the Rho subfamily of small GTPases. Curr. Opin. Genet. Dev. 4, 77–81 (1994).

    Article  CAS  PubMed  Google Scholar 

  12. García-Mata, R. & Burridge, K. Catching a GEF by its tail. Trends Cell Biol. 17, 36–43 (2007).

    Article  CAS  PubMed  Google Scholar 

  13. Garcia-Mata, R., Boulter, E. & Burridge, K. The ‘invisible hand’: regulation of RHO GTPases by RHOGDIs. Nat. Rev. Mol. Cell Biol. 12, 493–504 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Pai, S. Y., Kim, C. & Williams, D. A. Rac GTPases in human diseases. Dis. Markers 29, 177–187 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Pertz, O., Hodgson, L., Klemke, R. L. & Hahn, K. M. Spatiotemporal dynamics of RhoA activity in migrating cells. Nature 440, 1069–1072 (2006).

    Article  CAS  PubMed  Google Scholar 

  16. Wang, H. et al. LOVTRAP: an optogenetic system for photoinduced protein dissociation. Nat. Methods 13, 755–758 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Bellini, D. & Papiz, M. Z. Dimerization properties of the RpBphP2 chromophore-binding domain crystallized by homologue-directed mutagenesis. Acta Crystallogr. D Biol. Crystallogr. 68, 1058–1066 (2012).

    Article  CAS  PubMed  Google Scholar 

  18. Müller, S. M., Galliardt, H., Schneider, J., Barisas, B. G. & Seidel, T. Quantification of Förster resonance energy transfer by monitoring sensitized emission in living plant cells. Front. Plant Sci. 4, 413 (2013).

    PubMed  PubMed Central  Google Scholar 

  19. Patterson, G. H. & Piston, D. W. Photobleaching in two-photon excitation microscopy. Biophys. J. 78, 2159–2162 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Moshfegh, Y., Bravo-Cordero, J. J., Miskolci, V., Condeelis, J. & Hodgson, L. A Trio–Rac1–Pak1 signalling axis drives invadopodia disassembly. Nat. Cell Biol. 16, 574–586 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Quilliam, L. A. et al. Identification of residues critical for Ras(17N) growth-inhibitory phenotype and for Ras interaction with guanine nucleotide exchange factors. Mol. Cell. Biol. 14, 1113–1121 (1994).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Wu, B. et al. Synonymous modification results in high-fidelity gene expression of repetitive protein and nucleotide sequences. Genes Dev. 29, 876–886 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Miskolci, V., Wu, B., Moshfegh, Y., Cox, D. & Hodgson, L. Optical tools to study the isoform-specific roles of small GTPases in immune cells. J. Immunol. 196, 3479–3493 (2016).

    Article  CAS  PubMed  Google Scholar 

  24. Martin, E., Ouellette, M. H. & Jenna, S. Rac1/RhoA antagonism defines cell-to-cell heterogeneity during epidermal morphogenesis in nematodes. J. Cell Biol. 215, 483–498 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Nakamura, F. FilGAP and its close relatives: a mediator of Rho-Rac antagonism that regulates cell morphology and migration. Biochem. J. 453, 17–25 (2013).

    Article  CAS  PubMed  Google Scholar 

  26. Shcherbakova, D. M., Baloban, M. & Verkhusha, V. V. Near-infrared fluorescent proteins engineered from bacterial phytochromes. Curr. Opin. Chem. Biol. 27, 52–63 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Hodgson, L., Nalbant, P., Shen, F. & Hahn, K. Imaging and photobleach correction of Mero-CBD, sensor of endogenous Cdc42 activation. Methods Enzymol. 406, 140–156 (2006).

    Article  CAS  PubMed  Google Scholar 

  28. Martin, K. et al. Spatio-temporal co-ordination of RhoA, Rac1 and Cdc42 activation during prototypical edge protrusion and retraction dynamics. Sci. Rep. 6, 21901 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. del Pozo, M. A., Price, L. S., Alderson, N. B., Ren, X. D. & Schwartz, M. A. Adhesion to the extracellular matrix regulates the coupling of the small GTPase Rac to its effector PAK. EMBO J. 19, 2008–2014 (2000).

    Article  PubMed  PubMed Central  Google Scholar 

  30. Bellanger, J. M. et al. The two guanine nucleotide exchange factor domains of Trio link the Rac1 and the RhoA pathways in vivo. Oncogene 16, 147–152 (1998).

    Article  CAS  PubMed  Google Scholar 

  31. Chen, Y., Saulnier, J. L., Yellen, G. & Sabatini, B. L. A PKA activity sensor for quantitative analysis of endogenous GPCR signaling via 2-photon FRET-FLIM imaging. Front. Pharmacol. 5, 56 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Fosbrink, M., Aye-Han, N. N., Cheong, R., Levchenko, A. & Zhang, J. Visualization of JNK activity dynamics with a genetically encoded fluorescent biosensor. Proc. Natl. Acad. Sci. USA 107, 5459–5464 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

  33. Komatsu, N. et al. Development of an optimized backbone of FRET biosensors for kinases and GTPases. Mol. Biol. Cell 22, 4647–4656 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Piston, D. W. & Kremers, G. J. Fluorescent protein FRET: the good, the bad and the ugly. Trends Biochem. Sci. 32, 407–414 (2007).

    Article  CAS  PubMed  Google Scholar 

  35. Piatkevich, K. D., Subach, F. V. & Verkhusha, V. V. Far-red light photoactivatable near-infrared fluorescent proteins engineered from a bacterial phytochrome. Nat. Commun. 4, 2153 (2013).

    Article  CAS  PubMed  Google Scholar 

  36. Filonov, G. S. et al. Bright and stable near-infrared fluorescent protein for in vivo imaging. Nat. Biotechnol. 29, 757–761 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Shu, X. et al. Mammalian expression of infrared fluorescent proteins engineered from a bacterial phytochrome. Science 324, 804–807 (2009).

    Article  PubMed  PubMed Central  Google Scholar 

  38. Sens, R. & Drexhage, K. H. Fluorescence quantum yield of oxazine and carbazine laser dyes. J. Lumin. 24, 709–712 (1981).

    Article  Google Scholar 

  39. Philo, J. S. Improved methods for fitting sedimentation coefficient distributions derived by time-derivative techniques. Anal. Biochem. 354, 238–246 (2006).

    Article  CAS  PubMed  Google Scholar 

  40. Stafford, W. F. III. Boundary analysis in sedimentation transport experiments: a procedure for obtaining sedimentation coefficient distributions using the time derivative of the concentration profile. Anal. Biochem. 203, 295–301 (1992).

    Article  CAS  PubMed  Google Scholar 

  41. Subach, O. M., Cranfill, P. J., Davidson, M. W. & Verkhusha, V. V. An enhanced monomeric blue fluorescent protein with the high chemical stability of the chromophore. PLoS One 6, e28674 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Subach, O. M. et al. Conversion of red fluorescent protein into a bright blue probe. Chem. Biol. 15, 1116–1124 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. van Rijssel, J., Hoogenboezem, M., Wester, L., Hordijk, P. L. & Van Buul, J. D. The N-terminal DH-PH domain of Trio induces cell spreading and migration by regulating lamellipodia dynamics in a Rac1-dependent fashion. PLoS One 7, e29912 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Brunet, J. P. et al. Rotavirus infection induces cytoskeleton disorganization in human intestinal epithelial cells: implication of an increase in intracellular calcium concentration. J. Virol. 74, 10801–10806 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Whitlow, M. et al. An improved linker for single-chain Fv with reduced aggregation and enhanced proteolytic stability. Protein Eng. 6, 989–995 (1993).

    Article  CAS  PubMed  Google Scholar 

  46. Hodgson, L., Pertz, O. & Hahn, K. M. Design and optimization of genetically encoded fluorescent biosensors: GTPase biosensors. Methods Cell Biol. 85, 63–81 (2008).

    Article  CAS  PubMed  Google Scholar 

  47. Benard, V. & Bokoch, G. M. Assay of Cdc42, Rac, and Rho GTPase activation by affinity methods. Methods Enzymol. 345, 349–359 (2002).

    Article  PubMed  Google Scholar 

  48. Spiering, D. & Hodgson, L. Multiplex imaging of Rho family GTPase activities in living cells. Methods Mol. Biol. 827, 215–234 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Spiering, D., Bravo-Cordero, J. J., Moshfegh, Y., Miskolci, V. & Hodgson, L. Quantitative ratiometric imaging of FRET-biosensors in living cells. Methods Cell Biol. 114, 593–609 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

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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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