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A photoactivatable marker protein for pulse-chase imaging with superresolution

Nature Methods volume 7, pages 627–630 (2010)Cite this article

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Abstract

IrisFP is a photoactivatable fluorescent protein that combines irreversible photoconversion from a green- to a red-emitting form with reversible photoswitching between a fluorescent and a nonfluorescent state in both forms. Here we introduce a monomeric variant, mIrisFP, and demonstrate how its multiple photoactivation modes can be used for pulse-chase experiments combined with subdiffraction-resolution imaging in living cells by using dual-color photoactivation localization microscopy (PALM).

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Figure 1: mIrisFP for combined pulse-chase experiments and superresolution imaging.
Figure 2: PALM imaging of paxillin-mIrisFP dynamics in a live HeLa cell.
Figure 3: Focal adhesion dynamics in a live HeLa cell expressing paxillin-mIrisFP.

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References

  1. Adam, V. et al. Proc. Natl. Acad. Sci. USA 105, 18343–18348 (2008).

    Article  CAS  Google Scholar 

  2. Betzig, E. et al. Science 313, 1642–1645 (2006).

    Article  CAS  Google Scholar 

  3. Hess, S.T., Girirajan, T.P. & Mason, M.D Biophys. J. 91, 4258–4272 (2006).

    Article  CAS  Google Scholar 

  4. Bates, M., Huang, B., Dempsey, G.T. & Zhuang, X . Science 317, 1749–1753 (2007).

    Article  CAS  Google Scholar 

  5. Hell, S.W Nat. Methods 6, 24–32 (2009).

    Article  CAS  Google Scholar 

  6. Hedde, P.N., Fuchs, J., Oswald, F., Wiedenmann, J. & Nienhaus, G.U. Nat. Methods 6, 689–690 (2009).

    Article  CAS  Google Scholar 

  7. Lippincott-Schwartz, J. & Patterson, G.H. Trends Cell Biol. 19, 555–565 (2009).

    Article  CAS  Google Scholar 

  8. Shaner, N.C., Patterson, G.H. & Davidson, M.W. J. Cell Sci. 120, 4247–4260 (2007).

    Article  CAS  Google Scholar 

  9. Wiedenmann, J. & Nienhaus, G.U. Expert Rev. Proteomics 3, 361–374 (2006).

    Article  CAS  Google Scholar 

  10. Wiedenmann, J. et al. Proc. Natl. Acad. Sci. USA 101, 15905–15910 (2004).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  12. Riedl, J. et al. Nat. Methods 5, 605–607 (2008).

    Article  CAS  Google Scholar 

  13. Choi, C.K. et al. Nat. Cell Biol. 10, 1039–1050 (2008).

    Article  CAS  Google Scholar 

  14. Webb, D.J., Parsons, J.T. & Horwitz, A.F. Nat. Cell Biol. 4, E97–E100 (2002).

    Article  CAS  Google Scholar 

  15. Shroff, H., Galbraith, C.G., Galbraith, J.A. & Betzig, E. Nat. Methods 5, 417–423 (2008).

    Article  CAS  Google Scholar 

  16. Wiedenmann, J., Oswald, F. & Nienhaus, G.U. IUBMB Life 61, 1029–1042 (2009).

    Article  CAS  Google Scholar 

  17. Karasawa, S., Araki, T., Yamamoto-Hino, M. & Miyawaki, A. J. Biol. Chem. 278, 34167–34171 (2003).

    Article  CAS  Google Scholar 

  18. Campbell, R.E. et al. Proc. Natl. Acad. Sci. USA 99, 7877–7882 (2002).

    Article  CAS  Google Scholar 

  19. Oswald, F. et al. EMBO J. 21, 5417–5426 (2002).

    Article  CAS  Google Scholar 

  20. Kredel, S. et al. Chem. Biol. 15, 224–233 (2008).

    Article  CAS  Google Scholar 

  21. Kredel, S. et al. PLoS One 4, e4391 (2009).

    Article  Google Scholar 

  22. Oswald, F., Liptay, S., Adler, G. & Schmid, R.M. Mol. Cell. Biol. 18, 2077–2088 (1998).

    Article  CAS  Google Scholar 

  23. Wahl, C., Liptay, S., Adler, G. & Schmid, R.M. J. Clin. Invest. 101, 1163–1174 (1998).

    Article  CAS  Google Scholar 

  24. Azoitei, N., Wirth, T. & Baumann, B. J. Neurochem. 93, 1487–1501 (2005).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank F. Schmitt for his help with generating the A69V variant of EosFP. The expression vector pmRuby–α-actinin was provided by M.W. Davidson (Florida State University). This work was supported by the Deutsche Forschungsgemeinschaft and the State of Baden-Württemberg through the Center for Functional Nanostructures, by Deutsche Forschungsgemeinschaft grant NI 291/9, Landesstiftung Baden-Württemberg and Fonds der Chemischen Industrie.

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Author notes

  1. Jochen Fuchs, Susan Böhme and Franz Oswald: These authors contributed equally to this work.

Authors and Affiliations

  1. Institute of Applied Physics and Center for Functional Nanostructures, Karlsruhe Institute of Technology, Karlsruhe, Germany

    Jochen Fuchs, Susan Böhme, Per Niklas Hedde & G Ulrich Nienhaus

  2. Department of Internal Medicine I, University Medical Center Ulm, Ulm, Germany

    Franz Oswald

  3. Institute of Biophysics, University of Ulm, Ulm, Germany

    Maike Krause

  4. National Oceanography Centre, University of Southampton, Southampton, UK

    Jörg Wiedenmann

  5. Department of Physics, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA

    G Ulrich Nienhaus

Authors
  1. Jochen Fuchs
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  2. Susan Böhme
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  3. Franz Oswald
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  4. Per Niklas Hedde
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  5. Maike Krause
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  6. Jörg Wiedenmann
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  7. G Ulrich Nienhaus
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Contributions

G.U.N. supervised the project in conception and discussion. S.B. and M.K. generated mIrisFP. S.B. carried out biophysical characterization of mIrisFP. F.O. produced fusion constructs, transfected HeLa cells and performed widefield microscopy. J.W. supplied materials. J.F. and P.N.H. collected and analyzed PALM images. S.B., J.F., F.O. and G.U.N. wrote the manuscript.

Corresponding author

Correspondence to G Ulrich Nienhaus.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–15 and Supplementary Tables 1–3 (PDF 2585 kb)

Supplementary Video 1

Paxillin-mIrisFP migration in a live HeLa cell. A time sequence of PALM images shows protein migration after pulse labeling. (MOV 971 kb)

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Fuchs, J., Böhme, S., Oswald, F. et al. A photoactivatable marker protein for pulse-chase imaging with superresolution. Nat Methods 7, 627–630 (2010). https://doi.org/10.1038/nmeth.1477

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