Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Optimized second-generation CRY2–CIB dimerizers and photoactivatable Cre recombinase

Abstract

Arabidopsis thaliana cryptochrome 2 (AtCRY2), a light-sensitive photosensory protein, was previously adapted for use in controlling protein–protein interactions through light-dependent binding to a partner protein, CIB1. While the existing CRY2–CIB dimerization system has been used extensively for optogenetic applications, some limitations exist. Here, we set out to optimize function of the CRY2–CIB system by identifying versions of CRY2–CIB that are smaller, show reduced dark interaction, and maintain longer or shorter signaling states in response to a pulse of light. We describe minimal functional CRY2 and CIB1 domains maintaining light-dependent interaction and new signaling mutations affecting AtCRY2 photocycle kinetics. The latter work implicates an α13–α14 turn motif within plant CRYs whose perturbation alters signaling-state lifetime. Using a long-lived L348F photocycle mutant, we engineered a second-generation photoactivatable Cre recombinase, PA-Cre2.0, that shows five-fold improved dynamic range, allowing robust recombination following exposure to a single, brief pulse of light.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Figure 1: Truncations of CRY2–CIB1 modules.
Figure 2: Identification of CRY2 photocycle mutants.
Figure 3: Sequence alignment and modeling of photocycle mutants.
Figure 4: Functional analysis of CRY2 L348F.

References

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

    CAS  PubMed  Google Scholar 

  2. Levskaya, A., Weiner, O.D., Lim, W.A. & Voigt, C.A. Spatiotemporal control of cell signalling using a light-switchable protein interaction. Nature 461, 997–1001 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  3. Yazawa, M., Sadaghiani, A.M., Hsueh, B. & Dolmetsch, R.E. Induction of protein-protein interactions in live cells using light. Nat. Biotechnol. 27, 941–945 (2009).

    CAS  PubMed  Google Scholar 

  4. Kennedy, M.J. et al. Rapid blue-light-mediated induction of protein interactions in living cells. Nat. Methods 7, 973–975 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  5. Strickland, D. et al. TULIPs: tunable, light-controlled interacting protein tags for cell biology. Nat. Methods 9, 379–384 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  6. Chen, D., Gibson, E.S. & Kennedy, M.J. A light-triggered protein secretion system. J. Cell Biol. 201, 631–640 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  7. Crefcoeur, R.P., Yin, R., Ulm, R. & Halazonetis, T.D. Ultraviolet-B-mediated induction of protein-protein interactions in mammalian cells. Nat. Commun. 4, 1779 (2013).

    PubMed  Google Scholar 

  8. Müller, K. et al. Multi-chromatic control of mammalian gene expression and signaling. Nucleic Acids Res. 41, e124 (2013).

    PubMed  PubMed Central  Google Scholar 

  9. Guntas, G. et al. Engineering an improved light-induced dimer (iLID) for controlling the localization and activity of signaling proteins. Proc. Natl. Acad. Sci. USA 112, 112–117 (2015).

    CAS  PubMed  Google Scholar 

  10. Kawano, F., Suzuki, H., Furuya, A. & Sato, M. Engineered pairs of distinct photoswitches for optogenetic control of cellular proteins. Nat. Commun. 6, 6256 (2015).

    CAS  PubMed  Google Scholar 

  11. Hughes, R.M., Bolger, S., Tapadia, H. & Tucker, C.L. Light-mediated control of DNA transcription in yeast. Methods 58, 385–391 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Konermann, S. et al. Optical control of mammalian endogenous transcription and epigenetic states. Nature 500, 472–476 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Polstein, L.R. & Gersbach, C.A. A light-inducible CRISPR-Cas9 system for control of endogenous gene activation. Nat. Chem. Biol. 11, 198–200 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Nihongaki, Y., Yamamoto, S., Kawano, F., Suzuki, H. & Sato, M. CRISPR-Cas9-based photoactivatable transcription system. Chem. Biol. 22, 169–174 (2015).

    CAS  PubMed  Google Scholar 

  15. Boulina, M., Samarajeewa, H., Baker, J.D., Kim, M.D. & Chiba, A. Live imaging of multicolor-labeled cells in Drosophila. Development 140, 1605–1613 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Idevall-Hagren, O., Dickson, E.J., Hille, B., Toomre, D.K. & De Camilli, P. Optogenetic control of phosphoinositide metabolism. Proc. Natl. Acad. Sci. USA 109, E2316–E2323 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  17. Giordano, F. et al. PI(4,5)P(2)-dependent and Ca(2+)-regulated ER-PM interactions mediated by the extended synaptotagmins. Cell 153, 1494–1509 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Kakumoto, T. & Nakata, T. Optogenetic control of PIP3: PIP3 is sufficient to induce the actin-based active part of growth cones and is regulated via endocytosis. PLoS One 8, e70861 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  19. Aoki, K. et al. Stochastic ERK activation induced by noise and cell-to-cell propagation regulates cell density-dependent proliferation. Mol. Cell 52, 529–540 (2013).

    CAS  PubMed  Google Scholar 

  20. O'Neill, P.R. & Gautam, N. Subcellular optogenetic inhibition of G proteins generates signaling gradients and cell migration. Mol. Biol. Cell 25, 2305–2314 (2014).

    PubMed  PubMed Central  Google Scholar 

  21. Maiuri, P. et al. Actin flows mediate a universal coupling between cell speed and cell persistence. Cell 161, 374–386 (2015).

    CAS  PubMed  Google Scholar 

  22. Duan, L. et al. Optogenetic control of molecular motors and organelle distributions in cells. Chem. Biol. 22, 671–682 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. Bugaj, L.J., Choksi, A.T., Mesuda, C.K., Kane, R.S. & Schaffer, D.V. Optogenetic protein clustering and signaling activation in mammalian cells. Nat. Methods 10, 249–252 (2013).

    CAS  PubMed  Google Scholar 

  24. Lee, S. et al. Reversible protein inactivation by optogenetic trapping in cells. Nat. Methods 11, 633–636 (2014).

    CAS  PubMed  Google Scholar 

  25. Taslimi, A. et al. An optimized optogenetic clustering tool for probing protein interaction and function. Nat. Commun. 5, 4925 (2014).

    CAS  PubMed  Google Scholar 

  26. Pathak, G.P., Strickland, D., Vrana, J.D. & Tucker, C.L. Benchmarking of optical dimerizer systems. ACS Synth. Biol. 3, 832–838 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  27. Liu, Y., Li, X., Li, K., Liu, H. & Lin, C. Multiple bHLH proteins form heterodimers to mediate CRY2-dependent regulation of flowering-time in Arabidopsis. PLoS Genet. 9, e1003861 (2013).

    PubMed  PubMed Central  Google Scholar 

  28. Liu, H. et al. Photoexcited CRY2 interacts with CIB1 to regulate transcription and floral initiation in Arabidopsis. Science 322, 1535–1539 (2008).

    CAS  PubMed  Google Scholar 

  29. Brautigam, C.A. et al. Structure of the photolyase-like domain of cryptochrome 1 from Arabidopsis thaliana. Proc. Natl. Acad. Sci. USA 101, 12142–12147 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. Müller, P. et al. ATP binding turns plant cryptochrome into an efficient natural photoswitch. Sci. Rep. 4, 5175 (2014).

    PubMed  PubMed Central  Google Scholar 

  31. Yang, H.Q. et al. The C termini of Arabidopsis cryptochromes mediate a constitutive light response. Cell 103, 815–827 (2000).

    CAS  PubMed  Google Scholar 

  32. Zeugner, A. et al. Light-induced electron transfer in Arabidopsis cryptochrome-1 correlates with in vivo function. J. Biol. Chem. 280, 19437–19440 (2005).

    CAS  PubMed  Google Scholar 

  33. Zoltowski, B.D. et al. Structure of full-length Drosophila cryptochrome. Nature 480, 396–399 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. Partch, C.L. & Sancar, A. Photochemistry and photobiology of cryptochrome blue-light photopigments: the search for a photocycle. Photochem. Photobiol. 81, 1291–1304 (2005).

    CAS  PubMed  Google Scholar 

  35. Engelhard, C. et al. Cellular metabolites enhance the light sensitivity of Arabidopsis cryptochrome through alternate electron transfer pathways. Plant Cell 26, 4519–4531 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  36. El-Esawi, M. et al. Cellular metabolites modulate in vivo signaling of Arabidopsis cryptochrome-1. Plant Signal. Behav. 10, e1063758 (2015).

    PubMed  PubMed Central  Google Scholar 

  37. Gao, J. et al. Trp triad-dependent rapid photoreduction is not required for the function of Arabidopsis CRY1. Proc. Natl. Acad. Sci. USA 112, 9135–9140 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  38. Zoltowski, B.D. Resolving cryptic aspects of cryptochrome signaling. Proc. Natl. Acad. Sci. USA 112, 8811–8812 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  39. Vaidya, A.T. et al. Flavin reduction activates Drosophila cryptochrome. Proc. Natl. Acad. Sci. USA 110, 20455–20460 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  40. Müller, P. & Bouly, J.-P. Searching for the mechanism of signalling by plant photoreceptor cryptochrome. FEBS Lett. 589, 189–192 (2015).

    PubMed  Google Scholar 

  41. Stanewsky, R. et al. The cryb mutation identifies cryptochrome as a circadian photoreceptor in Drosophila. Cell 95, 681–692 (1998).

    CAS  PubMed  Google Scholar 

  42. Oztürk, N., Song, S.-H., Selby, C.P. & Sancar, A. Animal type 1 cryptochromes. Analysis of the redox state of the flavin cofactor by site-directed mutagenesis. J. Biol. Chem. 283, 3256–3263 (2008).

    PubMed  Google Scholar 

  43. Schindler, S.E. et al. Photo-activatable Cre recombinase regulates gene expression in vivo. Sci. Rep. 5, 13627 (2015).

    PubMed  PubMed Central  Google Scholar 

  44. Jullien, N., Sampieri, F., Enjalbert, A. & Herman, J.P. Regulation of Cre recombinase by ligand-induced complementation of inactive fragments. Nucleic Acids Res. 31, e131 (2003).

    PubMed  PubMed Central  Google Scholar 

  45. Li, X. et al. Arabidopsis cryptochrome 2 (CRY2) functions by the photoactivation mechanism distinct from the tryptophan (trp) triad-dependent photoreduction. Proc. Natl. Acad. Sci. USA 108, 20844–20849 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  46. Thöing, C., Oldemeyer, S. & Kottke, T. Microsecond Deprotonation of Aspartic Acid and Response of the α/β Subdomain Precede C-Terminal Signaling in the Blue Light Sensor Plant Cryptochrome. J. Am. Chem. Soc. 137, 5990–5999 (2015).

    PubMed  Google Scholar 

  47. Solov'yov, I.A., Domratcheva, T., Moughal Shahi, A.R. & Schulten, K. Decrypting cryptochrome: revealing the molecular identity of the photoactivation reaction. J. Am. Chem. Soc. 134, 18046–18052 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  48. Herbel, V. et al. Lifetimes of Arabidopsis cryptochrome signaling states in vivo. Plant J. 74, 583–592 (2013).

    CAS  PubMed  Google Scholar 

  49. Bouly, J.P. et al. Cryptochrome blue light photoreceptors are activated through interconversion of flavin redox states. J. Biol. Chem. 282, 9383–9391 (2007).

    CAS  PubMed  Google Scholar 

  50. Banerjee, R. et al. The signaling state of Arabidopsis cryptochrome 2 contains flavin semiquinone. J. Biol. Chem. 282, 14916–14922 (2007).

    CAS  PubMed  Google Scholar 

  51. Matsuda, T. & Cepko, C.L. Controlled expression of transgenes introduced by in vivo electroporation. Proc. Natl. Acad. Sci. USA 104, 1027–1032 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  52. Cadwell, R.C. & Joyce, G.F. Randomization of genes by PCR mutagenesis. PCR Methods Appl. 2, 28–33 (1992).

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank Dr. Constance Cepko for the pCALVL-dsRed Cre reporter (13769), obtained through Addgene, Dr. Matthew Kennedy for critical reading of the manuscript, and Jessica Spiltoir and Qi Liu for experimental assistance. This work was supported by grants from the National Institutes of Health (GM100225) and the McKnight Endowment Fund for Neuroscience (Technological Innovations in Neuroscience Award) to C.L.T.

Author information

Authors and Affiliations

Authors

Contributions

A.T., B.Z., J.G.M., G.P.P., R.M.H., and C.L.T. carried out experiments. B.Z. carried out protein structural characterization and in vitro studies with OtCPF1. A.T., B.Z., and C.L.T. analyzed data and wrote the manuscript. C.L.T. conceived the project and edited the manuscript.

Corresponding author

Correspondence to Chandra L Tucker.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Results, Supplementary Figures 1–7. (PDF 2393 kb)

Supplementary Table 1

Sequences of constructs used in studies (XLS 247 kb)

Recruitment and dissociation of CRY2PHR-mCh with CIB81-pmEGFP (MOV 63 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Taslimi, A., Zoltowski, B., Miranda, J. et al. Optimized second-generation CRY2–CIB dimerizers and photoactivatable Cre recombinase. Nat Chem Biol 12, 425–430 (2016). https://doi.org/10.1038/nchembio.2063

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nchembio.2063

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing