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Detecting the conformation of individual proteins in live cells

Abstract

We combined single-molecule fluorescence resonance energy transfer (smFRET) with single-particle tracking in live cells to detect the in vivo conformation of individual proteins. We site-specifically labeled recombinant SNARE proteins with a FRET donor and acceptor before microinjecting them into cultured cells. Individual proteins rapidly incorporated into folded complexes at the cell membrane, demonstrating the potential of this method to reveal dynamic interactions within cells.

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Figure 1: smFRET detection in live cells.
Figure 2: smFRET studies of SNARE proteins in BS-C-1 cells.

References

  1. Joo, C., Balci, H., Ishitsuka, Y., Buranachai, C. & Ha, T. Annu. Rev. Biochem. 77, 51–76 (2008).

    CAS  Article  Google Scholar 

  2. Inomata, K. et al. Nature 458, 106–109 (2009).

    CAS  Article  Google Scholar 

  3. Wieser, S. & Schutz, G.J. Methods 46, 131–140 (2008).

    CAS  Article  Google Scholar 

  4. Sako, Y., Minoghchi, S. & Yanagida, T. Nat. Cell Biol. 2, 168–172 (2000).

    CAS  Article  Google Scholar 

  5. Murakoshi, H. et al. Proc. Natl. Acad. Sci. USA 101, 7317–7322 (2004).

    CAS  Article  Google Scholar 

  6. Brustad, E.M., Lemke, E.A., Schultz, P.G. & Deniz, A.A. J. Am. Chem. Soc. 130, 17664–17665 (2008).

    CAS  Article  Google Scholar 

  7. Jahn, R. & Scheller, R.H. Nat. Rev. Mol. Cell Biol. 7, 631–643 (2006).

    CAS  Article  Google Scholar 

  8. An, S.J. & Almers, W. Science 306, 1042–1046 (2004).

    CAS  Article  Google Scholar 

  9. Xie, X.S., Yu, J. & Yang, W.Y. Science 312, 228–230 (2006).

    CAS  Article  Google Scholar 

  10. Weninger, K., Bowen, M.E., Chu, S. & Brunger, A.T. Proc. Natl. Acad. Sci. USA 100, 14800–14805 (2003).

    CAS  Article  Google Scholar 

  11. Loranger, S.S. & Linder, M.E. J. Biol. Chem. 277, 34303–34309 (2002).

    CAS  Article  Google Scholar 

  12. Bajohrs, M., Darios, F., Peak-Chew, S.Y. & Davletov, B. Biochem. J. 392, 283–289 (2005).

    CAS  Article  Google Scholar 

  13. Bar-On, D. et al. FEBS Lett. 582, 3563–3568 (2008).

    CAS  Article  Google Scholar 

  14. Lang, T., Margittai, M., Holzler, H. & Jahn, R. J. Cell Biol. 158, 751–760 (2002).

    CAS  Article  Google Scholar 

  15. Yildiz, A. et al. Science 300, 2061–2065 (2003).

    CAS  Article  Google Scholar 

  16. Bowen, M.E., Weninger, K., Brunger, A.T. & Chu, S. Biophys. J. 87, 3569–3584 (2004).

    CAS  Article  Google Scholar 

  17. Bowen, M.E., Weninger, K., Ernst, J., Chu, S. & Brunger, A.T. Biophys. J. 89, 690–702 (2005).

    CAS  Article  Google Scholar 

  18. Weninger, K., Bowen, M.E., Choi, U.B., Chu, S. & Brunger, A.T. Structure 16, 308–320 (2008).

    CAS  Article  Google Scholar 

  19. Steegmaier, M. et al. J. Biol. Chem. 273, 34171–34179 (1998).

    CAS  Article  Google Scholar 

  20. Su, Q., Mochida, S., Tian, J.H., Mehta, R. & Sheng, Z.H. Proc. Natl. Acad. Sci. USA 98, 14038–14043 (2001).

    CAS  Article  Google Scholar 

  21. Li, Y., Augustine, G.J. & Weninger, K. Biophys. J. 93, 2178–2187 (2007).

    CAS  Article  Google Scholar 

  22. Boukobza, E., Sonnenfeld, A. & Haran, G. J. Phys. Chem. B 105, 12165–12170 (2001).

    CAS  Article  Google Scholar 

  23. Roy, R., Hohng, S. & Ha, T. Nat. Methods 5, 507–516 (2008).

    CAS  Article  Google Scholar 

  24. Stuurman, N. & Vale, R. in Live Cell Imaging: A laboratory manual (eds. Goldman, R.D. & Spector, D.L.) 585–602 (Cold Springs Harbor Laboratory Press, Woodbury, 2005).

  25. Cordes, T., Vogelsang, J. & Tinnefeld, P. J. Am. Chem. Soc. 131, 5018–5019 (2009).

    CAS  Article  Google Scholar 

  26. Sutton, R.B., Fasshauer, D., Jahn, R. & Brunger, A.T. Nature 395, 347–353 (1998).

    CAS  Article  Google Scholar 

  27. Brunger, A.T. Q. Rev. Biophys. 38, 1–47 (2005).

    CAS  Article  Google Scholar 

  28. Rasnik, I., McKinney, S.A. & Ha, T. Nat. Methods 3, 891–893 (2006).

    CAS  Article  Google Scholar 

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Acknowledgements

We thank B. Choi and T. Anderson for assistance with protein preparation, G. Ribeill and E. Cady for software development, G. Feng and Z. Lu for assistance with PC-12 cells, Z.-H. Sheng (US National Institutes of Health) for the gift of a SNAP-29 expression vector, and M. Bowen, J. Ernst and G. Augustine for useful discussions. K.R.W. is supported in part by a Career Award at the Scientific Interface from the Burroughs Wellcome Fund. This work was funded by the US National Institutes of Health (GM076039).

Author information

Authors and Affiliations

Authors

Contributions

J.J.S. performed all measurements and data analysis. J.J.S. and K.R.W. contributed substantially to all other aspects.

Corresponding author

Correspondence to Keith R Weninger.

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Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–6, Supplementary Table 1 and Supplementary Note (PDF 814 kb)

Supplementary Video 1

Microinjection of Cy3-Cy5 labeled SNAP-25–CC into a BS-C-1 cell under 'near'-total internal reflection (Online Methods) with green illumination. The left half of the screen is the acceptor channel while the right half is the donor channel. Recorded at 10 frames s−1. (MOV 2604 kb)

Supplementary Video 2

Microinjection of Cy3-Cy5 labeled SNAP-25–CC into a PC-12 cell under full total internal reflection with lower protein concentration injected than in Supplementary Video 1. Recorded at 10 frames s−1. (MOV 2883 kb)

Supplementary Video 3

Microinjection of Alexa Fluor 555-Alexa Fluor 647 labeled G43D mutant of SNAP-25–CC into a BS-C-1 cell with the same conditions as in Supplementary Video 1. Recorded at 10 frames s−1. (MOV 2420 kb)

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Sakon, J., Weninger, K. Detecting the conformation of individual proteins in live cells. Nat Methods 7, 203–205 (2010). https://doi.org/10.1038/nmeth.1421

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