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Spatiotemporal control of cell signalling using a light-switchable protein interaction

Nature volume 461, pages 997–1001 (2009)Cite this article

Abstract

Genetically encodable optical reporters, such as green fluorescent protein, have revolutionized the observation and measurement of cellular states. However, the inverse challenge of using light to control precisely cellular behaviour has only recently begun to be addressed; semi-synthetic chromophore-tethered receptors1 and naturally occurring channel rhodopsins have been used to perturb directly neuronal networks2,3. The difficulty of engineering light-sensitive proteins remains a significant impediment to the optical control of most cell-biological processes. Here we demonstrate the use of a new genetically encoded light-control system based on an optimized, reversible protein–protein interaction from the phytochrome signalling network of Arabidopsis thaliana. Because protein–protein interactions are one of the most general currencies of cellular information, this system can, in principle, be generically used to control diverse functions. Here we show that this system can be used to translocate target proteins precisely and reversibly to the membrane with micrometre spatial resolution and at the second timescale. We show that light-gated translocation of the upstream activators of Rho-family GTPases, which control the actin cytoskeleton, can be used to precisely reshape and direct the cell morphology of mammalian cells. The light-gated protein–protein interaction that has been optimized here should be useful for the design of diverse light-programmable reagents, potentially enabling a new generation of perturbative, quantitative experiments in cell biology.

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Figure 1: The phytochrome–PIF interaction can be used to reversibly translocate proteins to the plasma membrane in a light-controlled fashion.
Figure 2: Confocal microscopy demonstrating the second-scale kinetics and photostability of the Phy–PIF photoswitchable membrane recruitment system.
Figure 3: Recruitment to the plasma membrane can be controlled spatially by simultaneously irradiating cells with patterned red and infrared light.
Figure 4: Rho-family G-protein signalling can be controlled by the light-activated translocation system.

References

  1. Szobota, S. et al. Remote control of neuronal activity with a light-gated glutamate receptor. Neuron 54, 535–545 (2007)

    Article  CAS  Google Scholar 

  2. Boyden, E. S., Zhang, F., Bamberg, E., Nagel, G. & Deisseroth, K. Millisecond-timescale, genetically targeted optical control of neural activity. Nature Neurosci. 8, 1263–1268 (2005)

    Article  CAS  Google Scholar 

  3. Han, X. & Boyden, E. S. Multiple-color optical activation, silencing, and desynchronization of neural activity, with single-spike temporal resolution. PLoS One 2, e299 (2007)

    Article  ADS  Google Scholar 

  4. Mettetal, J. T., Muzzey, D., Gómez-Uribe, C. & van Oudenaarden, A. The frequency dependence of osmo-adaptation in Saccharomyces cerevisiae . Science 319, 482–484 (2008)

    Article  ADS  CAS  Google Scholar 

  5. Bennett, M. R. et al. Metabolic gene regulation in a dynamically changing environment. Nature 454, 1119–1122 (2008)

    Article  ADS  CAS  Google Scholar 

  6. Ghosh, M. et al. Cofilin promotes actin polymerization and defines the direction of cell motility. Science 304, 743–746 (2004)

    Article  ADS  CAS  Google Scholar 

  7. Gorostiza, P. & Isacoff, E. Y. Optical switches for remote and noninvasive control of cell signaling. Science 322, 395–399 (2008)

    Article  ADS  MathSciNet  CAS  Google Scholar 

  8. Levskaya, A. et al. Synthetic biology: engineering Escherichia coli to see light. Nature 438, 441–442 (2005)

    Article  ADS  CAS  Google Scholar 

  9. Lee, J. et al. Surface sites for engineering allosteric control in proteins. Science 322, 438–442 (2008)

    Article  ADS  CAS  Google Scholar 

  10. Shimizu-Sato, S., Huq, E., Tepperman, J. M. & Quail, P. H. A light-switchable gene promoter system. Nature Biotechnol. 20, 1041–1044 (2002)

    Article  CAS  Google Scholar 

  11. Quail, P. H. Phytochrome photosensory signalling networks. Nature Rev. Mol. Cell Biol. 3, 85–93 (2002)

    Article  CAS  Google Scholar 

  12. Ni, M., Tepperman, J. M. & Quail, P. H. Binding of phytochrome B to its nuclear signalling partner PIF3 is reversibly induced by light. Nature 400, 781–784 (1999)

    Article  ADS  CAS  Google Scholar 

  13. Khanna, R. et al. A novel molecular recognition motif necessary for targeting photoactivated phytochrome signaling to specific basic helix-loop-helix transcription factors. Plant Cell 16, 3033–3044 (2004)

    Article  CAS  Google Scholar 

  14. Tyszkiewicz, A. B. & Muir, T. W. Activation of protein splicing with light in yeast. Nature Methods 5, 303–305 (2008)

    Article  CAS  Google Scholar 

  15. Leung, D. W., Otomo, C., Chory, J. & Rosen, M. K. Genetically encoded photoswitching of actin assembly through the Cdc42-WASP-Arp2/3 complex pathway. Proc. Natl Acad. Sci. USA 105, 12797–12802 (2008)

    Article  ADS  CAS  Google Scholar 

  16. Su, Y. & Lagarias, J. C. Light-independent phytochrome signaling mediated by dominant GAF domain tyrosine mutants of Arabidopsis phytochromes in transgenic plants. Plant Cell 19, 2124–2139 (2007)

    Article  CAS  Google Scholar 

  17. Heo, W. D. et al. PI(3,4,5)P3 and PI(4,5)P2 lipids target proteins with polybasic clusters to the plasma membrane. Science 314, 1458–1461 (2006)

    Article  ADS  CAS  Google Scholar 

  18. Al-Sady, B., Ni, W., Kircher, S., Schäfer, E. & Quail, P. H. Photoactivated phytochrome induces rapid PIF3 phosphorylation prior to proteasome-mediated degradation. Mol. Cell 23, 439–446 (2006)

    Article  CAS  Google Scholar 

  19. Genoud, T. et al. FHY1 mediates nuclear import of the light-activated phytochrome A photoreceptor. PLoS Genet. 4, e1000143 (2008)

    Article  Google Scholar 

  20. Chen, M., Tao, Y., Lim, J., Shaw, A. & Chory, J. Regulation of phytochrome B nuclear localization through light-dependent unmasking of nuclear-localization signals. Curr. Biol. 15, 637–642 (2005)

    Article  CAS  Google Scholar 

  21. Inoue, T., Heo, W. D., Grimley, J. S., Wandless, T. J. & Meyer, T. An inducible translocation strategy to rapidly activate and inhibit small GTPase signaling pathways. Nature Methods 2, 415–418 (2005)

    Article  CAS  Google Scholar 

  22. Wang, S. et al. All optical interface for parallel, remote, and spatiotemporal control of neuronal activity. Nano Lett. 7, 3859–3863 (2007)

    Article  ADS  CAS  Google Scholar 

  23. Castellano, F. et al. Inducible recruitment of Cdc42 or WASP to a cell-surface receptor triggers actin polymerization and filopodium formation. Curr. Biol. 9, 351–361 (1999)

    Article  CAS  Google Scholar 

  24. Suh, B., Inoue, T., Meyer, T. & Hille, B. Rapid chemically induced changes of PtdIns(4,5)P2 gate KCNQ ion channels. Science 314, 1454–1457 (2006)

    Article  ADS  CAS  Google Scholar 

  25. Inoue, T. & Meyer, T. Synthetic activation of endogenous PI3K and Rac identifies an AND-gate switch for cell polarization and migration. PLoS One 3, e3068 (2008)

    Article  ADS  Google Scholar 

  26. Gambetta, G. A. & Lagarias, J. C. Genetic engineering of phytochrome biosynthesis in bacteria. Proc. Natl Acad. Sci. USA 98, 10566–10571 (2001)

    Article  ADS  CAS  Google Scholar 

Download references

Acknowledgements

We thank B. El-Sady, J. Tepperman, K. Thorn, G. Kapp and members of the Voigt, Weiner and Lim laboratories for assistance and discussion. We thank Molecular Devices and Photonics Instruments for the loan and customization of a Mosaic spatial light modulator. Data for this study were acquired at the Nikon Imaging Center at UCSF. This work was supported by a NSFGR fellowship (A.L.); NIH R01 GM084040 and Searles Scholar Award.(O.D.W.); Packard Fellowship, the Howard Hughes Medical Institute, and NIH grants GM55040, GM62583 and EY016546 (NIH Roadmap Nanomedicine Development Centers) (W.A.L.); Pew Fellowship, the Office of Naval Research, Packard Fellowship, NIH EY016546, NIH AI067699, NSF BES-0547637, UC-Discovery and the SynBERC NSF ERC (C.A.V.).

Author Contributions Concept was conceived by A.L., W.A.L. and C.A.V.; experiments were executed by A.L.; spatiotemporal microscopy methods were developed by A.L. and O.D.W.; all authors were involved in interpretation of results and preparation of the manuscript.

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Authors and Affiliations

  1. The Cell Propulsion Lab, UCSF/UCB NIH Nanomedicine Development Center,,

    Anselm Levskaya, Orion D. Weiner, Wendell A. Lim & Christopher A. Voigt

  2. Graduate Program in Biophysics,,

    Anselm Levskaya

  3. Department of Pharmaceutical Chemistry,,

    Anselm Levskaya & Christopher A. Voigt

  4. Cardiovascular Research Institute,,

    Orion D. Weiner

  5. Howard Hughes Medical Institute and Department of Cellular and Molecular Pharmacology, University of California, San Francisco, California 94158-2517, USA,

    Wendell A. Lim

Authors
  1. Anselm Levskaya
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  2. Orion D. Weiner
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  3. Wendell A. Lim
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  4. Christopher A. Voigt
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Corresponding author

Correspondence to Wendell A. Lim.

Additional information

Plasmids will be available from Addgene (http://www.addgene.org).

Supplementary information

Supplementary Information

This file contains Supplementary Methods, Supplementary Table S1, Supplementary Data, Supplementary Movie Legends, Supplementary References and Supplementary Figures S1-S5 with Legends. (PDF 826 kb)

Supplementary Movie 1

This movie shows YFP membrane recruitment - see file s1 for full Legend. (MOV 246 kb)

Supplementary Movie 2

This movie shows YFP membrane dissociation - see file s1 for full Legend. (MOV 149 kb)

Supplementary Movie 3

This movie shows oscillating YFP translocation - see file s1 for full Legend. (MOV 1986 kb)

Supplementary Movie 4

This movie shows YFP point recruitment - see file s1 for full Legend. (MOV 185 kb)

Supplementary Movie 5

This movie shows patterned recruitment of YFP - see file s1 for full Legend. (MOV 583 kb)

Supplementary Movie 6

This movie shows titrated recruitment of YFP - see file s1 for full Legend. (MOV 755 kb)

Supplementary Movie 7

This movie shows cell extrusion by Tiam(Rac GEF) recruitment - see file s1 for full Legend. (MOV 764 kb)

Supplementary Movie 8

This movie shows dynamic lamellipodia by Tiam(Rac GEF - see file s1 for full Legend. (MOV 7019 kb)

Supplementary Movie 9

This movie shows periodic contractions by Tim(Rho GEF) recruitment - see file s1 for full Legend. (MOV 481 kb)

Supplementary Movie 10

This movie shows PIF-YFP only recruitment control - see file s1 for full Legend. (MOV 806 kb)

Supplementary Movie 11

This movie shows monitoring membrane-bound Cdc42-GTP dynamics via recruited mCherry-WASP-GBD in TIRF - see file s1 for full Legend. (MOV 4621 kb)

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Levskaya, A., Weiner, O., Lim, W. et al. Spatiotemporal control of cell signalling using a light-switchable protein interaction. Nature 461, 997–1001 (2009). https://doi.org/10.1038/nature08446

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