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.

A naturally monomeric infrared fluorescent protein for protein labeling in vivo

Abstract

Infrared fluorescent proteins (IFPs) provide an additional color to GFP and its homologs in protein labeling. Drawing on structural analysis of the dimer interface, we identified a bacteriophytochrome in the sequence database that is monomeric in truncated form and engineered it into a naturally monomeric IFP (mIFP). We demonstrate that mIFP correctly labels proteins in live cells, Drosophila and zebrafish. It should be useful in molecular, cell and developmental biology.

Your institute does not have access to this article

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: Rational design of mIFP, a naturally monomeric infrared FP.
Figure 2: Expression of mIFP fusions in vivo.

Accession codes

Primary accessions

NCBI Reference Sequence

Referenced accessions

NCBI Reference Sequence

Protein Data Bank

References

  1. Day, R.N. & Davidson, M.W. Chem. Soc. Rev. 38, 2887–2921 (2009).

    CAS  Article  Google Scholar 

  2. Tsien, R.Y. Angew. Chem. Int. Ed. Engl. 48, 5612–5626 (2009).

    CAS  Article  Google Scholar 

  3. Shu, X. et al. Science 324, 804–807 (2009).

    Article  Google Scholar 

  4. Filonov, G.S. et al. Nat. Biotechnol. 29, 757–761 (2011).

    CAS  Article  Google Scholar 

  5. Auldridge, M.E., Satyshur, K.A., Anstrom, D.M. & Forest, K.T. J. Biol. Chem. 287, 7000–7009 (2012).

    CAS  Article  Google Scholar 

  6. Shcherbakova, D.M. & Verkhusha, V.V. Nat. Methods 10, 751–754 (2013).

    CAS  Article  Google Scholar 

  7. Piatkevich, K.D., Subach, F.V. & Verkhusha, V.V. Nat. Commun. 4, 2153–2162 (2013).

    Article  Google Scholar 

  8. Giraud, E. & Verméglio, A. Photosynth. Res. 97, 141–153 (2008).

    CAS  Article  Google Scholar 

  9. Rockwell, N.C., Su, Y.S. & Lagarias, J.C. Annu. Rev. Plant Biol. 57, 837–858 (2006).

    CAS  Article  Google Scholar 

  10. Karniol, B., Wagner, J.R., Walker, J.M. & Vierstra, R.D. Biochem. J. 392, 103–116 (2005).

    CAS  Article  Google Scholar 

  11. Yu, D. et al. Nat. Commun. 5, 3626–3632 (2014).

    CAS  Article  Google Scholar 

  12. Geiler-Samerotte, K.A. et al. Proc. Natl. Acad. Sci. USA 108, 680–685 (2011).

    CAS  Article  Google Scholar 

  13. Wagner, J.R., Brunzelle, J.S., Forest, K.T. & Vierstra, R.D. Nature 438, 325–331 (2005).

    CAS  Article  Google Scholar 

  14. Stemmer, W.P. Nature 370, 389–391 (1994).

    CAS  Article  Google Scholar 

  15. Szymczak, A.L. et al. Nat. Biotechnol. 22, 589–594 (2004).

    CAS  Article  Google Scholar 

  16. Cui, L. et al. Biochem. Biophys. Res. Commun. 377, 1156–1161 (2008).

    CAS  Article  Google Scholar 

  17. Han, C., Jan, L.Y. & Jan, Y.-N. Proc. Natl. Acad. Sci. USA 108, 9673–9678 (2011).

    CAS  Article  Google Scholar 

  18. Shaner, N.C. et al. Nat. Biotechnol. 22, 1567–1572 (2004).

    CAS  Article  Google Scholar 

  19. Shemiakina, I.I. et al. Nat. Commun. 3, 1204–1207 (2012).

    CAS  Article  Google Scholar 

  20. To, T.-L. et al. Proc. Natl. Acad. Sci. USA 112, 3338–3343 (2015).

    CAS  Article  Google Scholar 

Download references

Acknowledgements

This work was supported by the Program for Breakthrough Biomedical Research (to X.S.); US National Institute of Health (NIH) GM030637 (to T.B.K.), GM084040 and GM096164 (to O.D.W.); and the Howard Hughes Medical Institute (to Y.-N.J.). S.-Q.Z. was supported by NIH GM054616 (to W.F. DeGrado) and US National Science Foundation DMR-1120901. We thank N. Joh for assistance in gel filtration chromatography; A. Royant for size-exclusion chromatography; W.F. DeGrado for providing access to the analytical ultracentrifuge; L.D. Wilsbacher, A. Schepis and S.R. Coughlin for initial testing of mIFP in zebrafish; C.S. Craik for constructive comments; S. Woo at University of California, San Francisco (UCSF) for providing the pCS2+ vector; and S. Roy (UCSF) for providing the UAS-mCherry Drosophila line.

Author information

Authors and Affiliations

Authors

Contributions

X.S. conceived the project. D.Y. and X.S. designed mIFP and the H2B fusion. K.M. and X.S. planned the Drosophila embryo imaging. M.W.D. planned the fusion constructs. T.B.K. planned the histone H3.3 fusion construct and the transgenic Drosophila. A.R. and O.D.W. planned the imaging of zebrafish. M.P.K., Y.S. and Y.-N.J. planned the imaging of epithelia, muscle and neurons in Drosophila larvae and adults. D.Y., M.A.B., J.R.A., E.S.H., M.P.K., A.R., K.M., Y.S., S.L., Z.M. and S.-Q.Z. performed the experiments. D.Y. and X.S. wrote the manuscript. All the authors contributed to the final draft.

Corresponding author

Correspondence to Xiaokun Shu.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–21 and Supplementary Tables 1 and 2 (PDF 29544 kb)

Z-section confocal imaging of mIFP-α-tubulin in live cells.

HEK293 cells transiently transfected with the fusion construct were imaged with z-section (step size 1 μm). The entire field of view is shown. (AVI 1664 kb)

Time lapse imaging of mIFP-α-tubulin in live cells.

HEK293 cells transiently transfected with the fusion construct were imaged every 15 min. The entire field of view is shown. (AVI 2039 kb)

Time lapse imaging of mIFP-β-actin in live cells.

Ptk2 cells transiently transfected with the fusion construct were imaged every 3 min. (AVI 2222 kb)

Time lapse imaging of mIFP-EB3 in live cells.

HeLa cells transiently transfected with the fusion construct were imaged every 2 seconds. (AVI 438 kb)

Time lapse imaging of mIFP-H1 in live cells.

HeLa cells transiently transfected with the fusion construct were imaged every 2 min. (AVI 3536 kb)

Z-section two-color confocal imaging.

HEK293 cells transiently transfected with the mRuby-H2B (in yellow) and mIFP-α-tubulin (in red) were imaged with z-section (step size 1 μm). (AVI 125 kb)

Time lapse imaging of CD8-GFP in Drosophila.

Entire Drosophila embryo expressing UAS-CD8-GFP driven by elav-GAL4 was imaged every 10 min, which revealed the ventral nerve cord condensation. (AVI 502 kb)

Time lapse imaging of mIFP-H3.3 T2A HO1 and CD8-GFP in Drosophila.

Entire Drosophila embryo expressing UAS-mIFP-H3.3 T2A HO1 (in red) and UAS-CD8-GFP (in green) driven by elav-GAL4 was imaged every 10 min, which revealed the ventral nerve cord condensation. (AVI 531 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Yu, D., Baird, M., Allen, J. et al. A naturally monomeric infrared fluorescent protein for protein labeling in vivo. Nat Methods 12, 763–765 (2015). https://doi.org/10.1038/nmeth.3447

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nmeth.3447

Further reading

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