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Optimized labeling of membrane proteins for applications to super-resolution imaging in confined cellular environments using monomeric streptavidin

Nature Protocols volume 12, pages 748–763 (2017)Cite this article

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Abstract

Recent progress in super-resolution imaging (SRI) has created a strong need to improve protein labeling with probes of small size that minimize the target-to-label distance, increase labeling density, and efficiently penetrate thick biological tissues. This protocol describes a method for labeling genetically modified proteins incorporating a small biotin acceptor peptide with a 3-nm fluorescent probe, monomeric streptavidin. We show how to express, purify, and conjugate the probe to organic dyes with different fluorescent properties, and how to label selectively biotinylated membrane proteins for SRI techniques (point accumulation in nanoscale topography (PAINT), stimulated emission depletion (STED), stochastic optical reconstruction microscopy (STORM)). This method is complementary to the previously described anti-GFP-nanobody/SNAP-tag strategies, with the main advantage being that it requires only a short 15-amino-acid tag, and can thus be used with proteins resistant to fusion with large tags and for multicolor imaging. The protocol requires standard molecular biology/biochemistry equipment, making it easily accessible for laboratories with only basic skills in cell biology and biochemistry. The production/purification/conjugation steps take ∼5 d, and labeling takes a few minutes to an hour.

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Figure 1: Site-specific protein labeling using mSA.
Figure 2: mSA-labeled β3-integrin efficiently penetrates cell-matrix adhesions.
Figure 3: Assessing functionality of the fluorophore-conjugated mSA.
Figure 4: Analysis of representative fluorescent dye–mSA conjugates.
Figure 5: Cell-surface protein labeling with mSA in brain slices, cell line cultures, and primary neurons.

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References

  1. Stefan, W.H. et al. The 2015 super-resolution microscopy roadmap. J. Phys. D Appl. Phys. 48, 443001 (2015).

    Article  CAS  Google Scholar 

  2. Fernandez-Suarez, M. & Ting, A.Y. Fluorescent probes for super-resolution imaging in living cells. Nat. Rev. Mol. Cell Biol. 9, 929–943 (2008).

    Article  CAS  PubMed  Google Scholar 

  3. Ha, T. & Tinnefeld, P. Photophysics of fluorescent probes for single-molecule biophysics and super-resolution imaging. Annu. Rev. Phys. Chem. 63, 595–617 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Cranfill, P.J. et al. Quantitative assessment of fluorescent proteins. Nat. Methods 13, 557–562 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Vreja, I.C. et al. Super-resolution microscopy of clickable amino acids reveals the effects of fluorescent protein tagging on protein assemblies. ACS Nano 9, 11034–11041 (2015).

    Article  CAS  PubMed  Google Scholar 

  6. Stadler, C. et al. Immunofluorescence and fluorescent-protein tagging show high correlation for protein localization in mammalian cells. Nat. Methods 10, 315–323 (2013).

    Article  CAS  PubMed  Google Scholar 

  7. Landgraf, D., Okumus, B., Chien, P., Baker, T.A. & Paulsson, J. Segregation of molecules at cell division reveals native protein localization. Nat. Methods 9, 480–482 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Deschout, H. et al. Precisely and accurately localizing single emitters in fluorescence microscopy. Nat. Methods 11, 253–266 (2014).

    Article  CAS  PubMed  Google Scholar 

  9. Ries, J., Kaplan, C., Platonova, E., Eghlidi, H. & Ewers, H. A simple, versatile method for GFP-based super-resolution microscopy via nanobodies. Nat. Methods 9, 582–584 (2012).

    Article  CAS  PubMed  Google Scholar 

  10. Chamma, I. et al. Mapping the dynamics and nanoscale organization of synaptic adhesion proteins using monomeric streptavidin. Nat. Commun. 7, 10773 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Rothbauer, U. et al. Targeting and tracing antigens in live cells with fluorescent nanobodies. Nat. Methods 3, 887–889 (2006).

    Article  CAS  PubMed  Google Scholar 

  12. Keppler, A. et al. A general method for the covalent labeling of fusion proteins with small molecules in vivo. Nat. Biotechnol. 21, 86–89 (2003).

    Article  CAS  PubMed  Google Scholar 

  13. Los, G.V. et al. HaloTag: a novel protein labeling technology for cell imaging and protein analysis. ACS Chem. Biol. 3, 373–382 (2008).

    Article  CAS  PubMed  Google Scholar 

  14. Jespersen, L.K., Kuusinen, A., Orellana, A., Keinanen, K. & Engberg, J. Use of proteoliposomes to generate phage antibodies against native AMPA receptor. Eur. J. Biochem. 267, 1382–1389 (2000).

    Article  CAS  PubMed  Google Scholar 

  15. Mikhaylova, M. et al. Resolving bundled microtubules using anti-tubulin nanobodies. Nat. Commun. 6, 7933 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Nikic, I., Kang, J.H., Girona, G.E., Aramburu, I.V. & Lemke, E.A. Labeling proteins on live mammalian cells using click chemistry. Nat. Protoc. 10, 780–791 (2015).

    Article  CAS  PubMed  Google Scholar 

  17. Lim, K.H., Huang, H., Pralle, A. & Park, S. Stable, high-affinity streptavidin monomer for protein labeling and monovalent biotin detection. Biotechnol. Bioeng. 110, 57–67 (2013).

    Article  CAS  PubMed  Google Scholar 

  18. Demonte, D., Drake, E.J., Lim, K.H., Gulick, A.M. & Park, S. Structure-based engineering of streptavidin monomer with a reduced biotin dissociation rate. Proteins 81, 1621–1633 (2013).

    Article  CAS  PubMed  Google Scholar 

  19. Studier, F.W. Protein production by auto-induction in high density shaking cultures. Protein Expr. Purif. 41, 207–234 (2005).

    Article  CAS  PubMed  Google Scholar 

  20. Schatz, P.J. Use of peptide libraries to map the substrate specificity of a peptide-modifying enzyme: a 13 residue consensus peptide specifies biotinylation in Escherichia coli. Biotechnology 11, 1138–1143 (1993).

    CAS  PubMed  Google Scholar 

  21. Beckett, D., Kovaleva, E. & Schatz, P.J. A minimal peptide substrate in biotin holoenzyme synthetase-catalyzed biotinylation. Protein Sci. 8, 921–929 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Parrott, M.B. & Barry, M.A. Metabolic biotinylation of secreted and cell surface proteins from mammalian cells. Biochem. Biophys. Res. Commun. 281, 993–1000 (2001).

    Article  CAS  PubMed  Google Scholar 

  23. Saxton, M.J. & Jacobson, K. Single-particle tracking: applications to membrane dynamics. Annu. Rev. Biophys. Biomol. Struct. 26, 373–399 (1997).

    Article  CAS  PubMed  Google Scholar 

  24. Sharonov, A. & Hochstrasser, R.M. Wide-field subdiffraction imaging by accumulated binding of diffusing probes. Proc. Natl. Acad. Sci. USA 103, 18911–18916 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Giannone, G. et al. Dynamic superresolution imaging of endogenous proteins on living cells at ultra-high density. Biophys. J. 99, 1303–1310 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Klar, T.A., Jakobs, S., Dyba, M., Egner, A. & Hell, S.W. Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission. Proc. Natl. Acad. Sci. USA 97, 8206–8210 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Rust, M.J., Bates, M. & Zhuang, X. Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nat. Methods 3, 793–795 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  28. Endesfelder, U. et al. Chemically induced photoswitching of fluorescent probes--a general concept for super-resolution microscopy. Molecules 16, 3106–3118 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Lukinavicius, G. et al. Fluorogenic probes for multicolor imaging in living cells. J. Am. Chem. Soc. 138, 9365–9368 (2016).

    Article  CAS  PubMed  Google Scholar 

  30. Lavis, L.D. et al. Bright photoactivatable fluorophores for single-molecule imaging. bioRxiv 13, 985–988 (2016).

    Google Scholar 

  31. Howarth, M. et al. A monovalent streptavidin with a single femtomolar biotin binding site. Nat. Methods 3, 267–273 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Howarth, M. & Ting, A.Y. Imaging proteins in live mammalian cells with biotin ligase and monovalent streptavidin. Nat. Protoc. 3, 534–545 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Grunwald, C. et al. Quantum-yield-optimized fluorophores for site-specific labeling and super-resolution imaging. J. Am. Chem. Soc. 133, 8090–8093 (2011).

    Article  CAS  PubMed  Google Scholar 

  34. Wieneke, R., Raulf, A., Kollmannsperger, A., Heilemann, M. & Tampe, R. SLAP: small labeling pair for single-molecule super-resolution imaging. Angew. Chem. Int. Ed. Engl. 54, 10216–10219 (2015).

    Article  CAS  PubMed  Google Scholar 

  35. Yin, J., Lin, A.J., Golan, D.E. & Walsh, C.T. Site-specific protein labeling by Sfp phosphopantetheinyl transferase. Nat. Protoc. 1, 280–285 (2006).

    Article  CAS  PubMed  Google Scholar 

  36. Yagi, T., Terada, N., Baba, T. & Ohno, S. Localization of endogenous biotin-containing proteins in mouse Bergmann glial cells. Histochem. J. 34, 567–572 (2002).

    Article  CAS  PubMed  Google Scholar 

  37. Viens, A. et al. Use of protein biotinylation in vivo for immunoelectron microscopic localization of a specific protein isoform. J. Histochem. Cytochem. 56, 911–919 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Paetzel, M., Karla, A., Strynadka, N.C. & Dalbey, R.E. Signal peptidases. Chem. Rev. 102, 4549–4580 (2002).

    Article  CAS  PubMed  Google Scholar 

  39. Lim, K.H., Huang, H., Pralle, A. & Park, S. Engineered streptavidin monomer and dimer with improved stability and function. Biochemistry 50, 8682–8691 (2011).

    Article  CAS  PubMed  Google Scholar 

  40. Demonte, D., Dundas, C.M. & Park, S. Expression and purification of soluble monomeric streptavidin in Escherichia coli. Appl. Microbiol. Biotechnol. 98, 6285–6295 (2014).

    Article  CAS  PubMed  Google Scholar 

  41. Chamma, I., Levet, F., Sibarita, J.B., Sainlos, M. & Thoumine, O. Nanoscale organization of synaptic adhesion proteins revealed by single-molecule localization microscopy. Neurophotonics 3, 041810 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  42. Hughes, L.D., Rawle, R.J. & Boxer, S.G. Choose your label wisely: water-soluble fluorophores often interact with lipid bilayers. PLoS One 9, e87649 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Dempsey, G.T., Vaughan, J.C., Chen, K.H., Bates, M. & Zhuang, X. Evaluation of fluorophores for optimal performance in localization-based super-resolution imaging. Nat. Methods 8, 1027–1036 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. van de Linde, S. et al. Direct stochastic optical reconstruction microscopy with standard fluorescent probes. Nat. Protoc. 6, 991–1009 (2011).

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

We thank D. Perrais (Interdisciplinary Institute for Neuroscience, University of Bordeaux (IINS)), A. Ting (Stanford University), and S. Park (Buffalo University) for the generous gift of DNA plasmids; M. Letellier (IINS) for the slice cultures; Z. Karatas, B. Tessier, S. Antoine, and I. Gauthereau for molecular biology and biochemistry; and C. Poujol and the Bordeaux Imaging Center for providing access and support to confocal and STED setups. This work received funding from the Centre National de la Recherche Scientifique, Agence Nationale pour la Recherche (grants Synapse-2Dt and Nanodom (O.T.), Integractome and FastNano (O.R., G.G.), and SynAdh (O.T., M.S.), Conseil Régional Aquitaine, Fondation pour la Recherche Médicale, the National Infrastructure France BioImaging (grant ANR-10INBS-04-01), and the Labex BRAIN.

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Author notes

  1. Olivier Thoumine and Matthieu Sainlos: These authors contributed equally to this work.

Authors and Affiliations

  1. Interdisciplinary Institute for Neuroscience, UMR 5297, Centre National de la Recherche Scientifique, Bordeaux, France

    Ingrid Chamma, Olivier Rossier, Grégory Giannone, Olivier Thoumine & Matthieu Sainlos

  2. Interdisciplinary Institute for Neuroscience, University of Bordeaux, Bordeaux, France

    Ingrid Chamma, Olivier Rossier, Grégory Giannone, Olivier Thoumine & Matthieu Sainlos

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  1. Ingrid Chamma
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Contributions

I.C., M.S., and O.T. designed research and coordinated the research project. O.R. and G.G. provided their expertise and tools for the integrin model and contributed to related experiments. I.C. and M.S. performed experiments and analysis, and wrote the article. All authors discussed the results and commented on the manuscript.

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Correspondence to Matthieu Sainlos.

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Chamma, I., Rossier, O., Giannone, G. et al. Optimized labeling of membrane proteins for applications to super-resolution imaging in confined cellular environments using monomeric streptavidin. Nat Protoc 12, 748–763 (2017). https://doi.org/10.1038/nprot.2017.010

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