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.

TULIPs: tunable, light-controlled interacting protein tags for cell biology

Abstract

Naturally photoswitchable proteins offer a means of directly manipulating the formation of protein complexes that drive a diversity of cellular processes. We developed tunable light-inducible dimerization tags (TULIPs) based on a synthetic interaction between the LOV2 domain of Avena sativa phototropin 1 (AsLOV2) and an engineered PDZ domain (ePDZ). TULIPs can recruit proteins to diverse structures in living yeast and mammalian cells, either globally or with precise spatial control using a steerable laser. The equilibrium binding and kinetic parameters of the interaction are tunable by mutation, making TULIPs readily adaptable to signaling pathways with varying sensitivities and response times. We demonstrate the utility of TULIPs by conferring light sensitivity to functionally distinct components of the yeast mating pathway and by directing the site of cell polarization.

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

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Design and characterization of TULIPs.
Figure 2: Mutational and chemical control of binding.
Figure 3: Optical control of MAPK activation and polarity establishment in yeast.

Similar content being viewed by others

References

  1. Hartman, N.C. & Groves, J.T. Signaling clusters in the cell membrane. Curr. Opin. Cell Biol. 23, 370–376 (2011).

    Article  CAS  Google Scholar 

  2. Toettcher, J.E., Voigt, C.A., Weiner, O.D. & Lim, W.A. The promise of optogenetics in cell biology: interrogating molecular circuits in space and time. Nat. Methods 8, 35–38 (2011).

    Article  CAS  Google Scholar 

  3. Wu, Y.I. et al. A genetically encoded photoactivatable Rac controls the motility of living cells. Nature 461, 104–108 (2009).

    Article  CAS  Google Scholar 

  4. Yoo, S.K. et al. Differential regulation of protrusion and polarity by PI3K during neutrophil motility in live zebrafish. Dev. Cell 18, 226–236 (2010).

    Article  CAS  Google Scholar 

  5. Wang, X., He, L., Wu, Y.I., Hahn, K.M. & Montell, D.J. Light-mediated activation reveals a key role for Rac in collective guidance of cell movement in vivo. Nat. Cell Biol. 12, 591–597 (2010).

    Article  CAS  Google Scholar 

  6. 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).

    Article  CAS  Google Scholar 

  7. 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).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  9. Christie, J.M. et al. Arabidopsis NPH1: a flavoprotein with the properties of a photoreceptor for phototropism. Science 282, 1698–1701 (1998).

    Article  CAS  Google Scholar 

  10. Halavaty, A.S. & Moffat, K. N- and C-terminal flanking regions modulate light-induced signal transduction in the LOV2 domain of the blue light sensor phototropin 1 from Avena sativa. Biochemistry 46, 14001–14009 (2007).

    Article  CAS  Google Scholar 

  11. Harper, S.M., Neil, L.C. & Gardner, K.H. Structural basis of a phototropin light switch. Science 301, 1541–1544 (2003).

    Article  CAS  Google Scholar 

  12. Yao, X., Rosen, M.K. & Gardner, K.H. Estimation of the available free energy in a LOV2-J alpha photoswitch. Nat. Chem. Biol. 4, 491–497 (2008).

    Article  CAS  Google Scholar 

  13. Harper, S.M., Christie, J.M. & Gardner, K.H. Disruption of the LOV-Jalpha helix interaction activates phototropin kinase activity. Biochemistry 43, 16184–16192 (2004).

    Article  CAS  Google Scholar 

  14. Strickland, D., Moffat, K. & Sosnick, T.R. Light-activated DNA binding in a designed allosteric protein. Proc. Natl. Acad. Sci. USA 105, 10709–10714 (2008).

    Article  CAS  Google Scholar 

  15. Christie, J.M. et al. Steric interactions stabilize the signaling state of the LOV2 domain of phototropin 1. Biochemistry 46, 9310–9319 (2007).

    Article  CAS  Google Scholar 

  16. Zoltowski, B.D., Vaccaro, B. & Crane, B.R. Mechanism-based tuning of a LOV domain photoreceptor. Nat. Chem. Biol. 5, 827–834 (2009).

    Article  CAS  Google Scholar 

  17. Strickland, D. et al. Rationally improving LOV domain-based photoswitches. Nat. Methods 7, 623–626 (2010).

    Article  CAS  Google Scholar 

  18. Alexandre, M.T., Arents, J.C., van Grondelle, R., Hellingwerf, K.J. & Kennis, J.T. A base-catalyzed mechanism for dark state recovery in the Avena sativa phototropin-1 LOV2 domain. Biochemistry 46, 3129–3137 (2007).

    Article  CAS  Google Scholar 

  19. Möglich, A. & Moffat, K. Engineered photoreceptors as novel optogenetic tools. Photochem. Photobiol. Sci. 9, 1286–1300 (2010).

    Article  Google Scholar 

  20. Huang, J., Koide, A., Makabe, K. & Koide, S. Design of protein function leaps by directed domain interface evolution. Proc. Natl. Acad. Sci. USA 105, 6578–6583 (2008).

    Article  CAS  Google Scholar 

  21. Huang, J., Makabe, K., Biancalana, M., Koide, A. & Koide, S. Structural basis for exquisite specificity of affinity clamps, synthetic binding proteins generated through directed domain-interface evolution. J. Mol. Biol. 392, 1221–1231 (2009).

    Article  CAS  Google Scholar 

  22. Huh, W.K. et al. Global analysis of protein localization in budding yeast. Nature 425, 686–691 (2003).

    Article  CAS  Google Scholar 

  23. 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. Nat. Methods 2, 415–418 (2005).

    Article  CAS  Google Scholar 

  24. Pryciak, P.M. Designing new cellular signaling pathways. Chem. Biol. 16, 249–254 (2009).

    Article  CAS  Google Scholar 

  25. Pryciak, P.M. & Huntress, F.A. Membrane recruitment of the kinase cascade scaffold protein Ste5 by the Gβγ complex underlies activation of the yeast pheromone response pathway. Genes Dev. 12, 2684–2697 (1998).

    Article  CAS  Google Scholar 

  26. Winters, M.J., Lamson, R.E., Nakanishi, H., Neiman, A.M. & Pryciak, P.M. A membrane binding domain in the ste5 scaffold synergizes with Gβγ binding to control localization and signaling in pheromone response. Mol. Cell 20, 21–32 (2005).

    Article  CAS  Google Scholar 

  27. Park, H.O. & Bi, E. Central roles of small GTPases in the development of cell polarity in yeast and beyond. Microbiol. Mol. Biol. Rev. 71, 48–96 (2007).

    Article  CAS  Google Scholar 

  28. Strack, R.L. et al. A noncytotoxic DsRed variant for whole-cell labeling. Nat. Methods 5, 955–957 (2008).

    Article  CAS  Google Scholar 

  29. Shimada, Y., Wiget, P., Gulli, M.P., Bi, E. & Peter, M. The nucleotide exchange factor Cdc24p may be regulated by auto-inhibition. EMBO J. 23, 1051–1062 (2004).

    Article  CAS  Google Scholar 

  30. Dueber, J.E., Yeh, B.J., Chak, K. & Lim, W.A. Reprogramming control of an allosteric signaling switch through modular recombination. Science 301, 1904–1908 (2003).

    Article  CAS  Google Scholar 

  31. Jansen, G., Wu, C., Schade, B., Thomas, D.Y. & Whiteway, M. Drag&Drop cloning in yeast. Gene 344, 43–51 (2005).

    Article  CAS  Google Scholar 

  32. Gietz, R.D. & Sugino, A. New yeast-Escherichia coli shuttle vectors constructed with in vitro mutagenized yeast genes lacking six-base pair restriction sites. Gene 74, 527–534 (1988).

    Article  CAS  Google Scholar 

  33. Janke, C. et al. A versatile toolbox for PCR-based tagging of yeast genes: new fluorescent proteins, more markers and promoter substitution cassettes. Yeast 21, 947–962 (2004).

    Article  CAS  Google Scholar 

  34. Gietz, R.D. & Woods, R.A. Transformation of yeast by lithium acetate/single-stranded carrier DNA/polyethylene glycol method. Methods Enzymol. 350, 87–96 (2002).

    Article  CAS  Google Scholar 

  35. Picard, D. Posttranslational regulation of proteins by fusions to steroid-binding domains. Methods Enzymol. 327, 385–401 (2000).

    Article  CAS  Google Scholar 

  36. Storici, F. & Resnick, M.A. The delitto perfetto approach to in vivo site-directed mutagenesis and chromosome rearrangements with synthetic oligonucleotides in yeast. Methods Enzymol. 409, 329–345 (2006).

    Article  CAS  Google Scholar 

  37. Longtine, M.S. et al. Additional modules for versatile and economical PCR-based gene deletion and modification in Saccharomyces cerevisiae. Yeast 14, 953–961 (1998).

    Article  CAS  Google Scholar 

  38. Gibson, D.G. et al. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat. Methods 6, 343–345 (2009).

    Article  CAS  Google Scholar 

  39. Thorn, K. Spinning-disk confocal microscopy of yeast. Methods Enzymol. 470, 581–602 (2010).

    Article  Google Scholar 

  40. Thevenaz, P., Ruttimann, U.E. & Unser, M. A pyramid approach to subpixel registration based on intensity. IEEE Trans. Image Process. 7, 27–41 (1998).

    Article  CAS  Google Scholar 

  41. Bodvard, K. et al. Continuous light exposure causes cumulative stress that affects the localization oscillation dynamics of the transcription factor Msn2p. Biochim. Biophys. Acta 1813, 358–366 (2011).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank B. Glick (University of Chicago), S. Koide (University of Chicago) and F. Cross (Rockefeller University) for sharing plasmids, and members of the Glotzer, Weiss, Sosnick, Munro, Kovar and Glick laboratories for helpful discussions. This work was supported by research (GM088668, M.G. and T.R.S.) and training grants from the US National Institutes of Health, a grant from the Chicago Biomedical Consortium with support from the Searle Funds at the Chicago Community Trust (M.G., T.R.S. and E.L.W.), and by an American Cancer Society Postdoctoral Fellowship to D.S. (119248-PF-10-134-01-CCG).

Author information

Authors and Affiliations

Authors

Contributions

D.S. and M.G. conceived of the TULIPs strategy. D.S., Y.L., E.W., E.L.W. and M.G. designed experiments. D.S., Y.L., E.W. and C.M.H. performed experiments. D.S., Y.L., E.W., C.M.H., T.R.S., E.L.W. and M.G. analyzed data. J.Z., C.A. and T.R.S. provided new AsLOV2 mutations. D.S., E.L.W. and M.G. wrote the paper.

Corresponding author

Correspondence to Michael Glotzer.

Ethics declarations

Competing interests

A provisional patent application that includes portions of the research described in this manuscript has been filed by the University of Chicago Office of Technology and Intellectual Property.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–11, Supplementary Tables 1–5 and Supplementary Notes 1–2 (PDF 3795 kb)

Supplementary Video 1

Repeated photoexcitation, mitochondrial recruitment of ePDZ-mCherry and dark-state recovery in a HeLa cell expressing Tom70-GFP-LOVpep (K–6R, T–2S) and ePDZb1-mCherry. mCherry fluorescence images were taken every 5 s. (MOV 2253 kb)

Supplementary Video 2

Photoexcitation, PM recruitment of ePDZ-mCherry and dark-state recovery in cells expressing Mid2-GFP-LOVpep (T406A, T407A). Left, ePDZb-mCherry. Right, ePDZb1-mCherry. Photoexcitation occurs between the first and second frames. mCherry fluorescence images were taken every 5 s. (MOV 106 kb)

Supplementary Video 3

Spot photoexcitation, PM recruitment of ePDZb1-mCherry and dark-state recovery in cells expressing Mid2GFP-LOVpep (V416I,T406A,T407A) and ePDZb1-mCherry. Left, spot recruitment of ePDZb1-mCherry only. Right, spot recruitment of ePDZb1-mCherry followed by global recruitment and cytoplasmic depletion. mCherry fluorescence images 1–17 were taken every 2 s, 18–30 every 10 s and 31–42 every 60 s. (MOV 144 kb)

Supplementary Video 4

Light-directed polarized growth in budding yeast. Cells were exposed to mating pheromone to induce cell cycle arrest, then stimulated with spot photoexcitation to recruit Cdc24-ePDZb1 to PM-tethered LOVpep. Red, actin labeled using Abp1-mCherry. Blue, phase contrast image processed using an edge detection filter. Frames were taken every 1 s. Spot photoexcitation occurs at the white crosshairs, only in the frames where they are visible. (MOV 1143 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Strickland, D., Lin, Y., Wagner, E. et al. TULIPs: tunable, light-controlled interacting protein tags for cell biology. Nat Methods 9, 379–384 (2012). https://doi.org/10.1038/nmeth.1904

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nmeth.1904

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