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.

  • Article
  • Published:

Micropatterning for quantitative analysis of protein-protein interactions in living cells

Nature Methods volume 5, pages 1053–1060 (2008)Cite this article

Abstract

We present a method to identify and characterize interactions between a fluorophore-labeled protein ('prey') and a membrane protein ('bait') in live mammalian cells. Cells are plated on micropatterned surfaces functionalized with antibodies to the bait extracellular domain. Bait-prey interactions are assayed through the redistribution of the fluorescent prey. We used the method to characterize the interaction between human CD4, the major co-receptor in T-cell activation, and human Lck, the protein tyrosine kinase essential for early T-cell signaling. We measured equilibrium associations by quantifying Lck redistribution to CD4 micropatterns and studied interaction dynamics by photobleaching experiments and single-molecule imaging. In addition to the known zinc clasp structure, the Lck membrane anchor in particular had a major impact on the Lck-CD4 interaction, mediating direct binding and further stabilizing the interaction of other Lck domains. In total, membrane anchorage increased the interaction lifetime by two orders of magnitude.

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

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

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

Figure 1: Micropatterning of the bait CD4.
Figure 2: Specific interaction between CD4 and Lck-CFP-YFP.
Figure 3: CD4 interaction with Lck mutants.
Figure 4: Lck interaction with palmitoylation-deficient mutant CD4-C396S-C399S.
Figure 5: Interaction kinetics between CD4 and Lck.
Figure 6: Mobility and brightness of single Lck-CFP-YFP molecules.
Figure 7: Lck micropatterning on low-stringency microbiochips.

Similar content being viewed by others

References

  1. Papin, J.A., Hunter, T., Palsson, B.O. & Subramaniam, S. Reconstruction of cellular signalling networks and analysis of their properties. Nat. Rev. Mol. Cell Biol. 6, 99–111 (2005).

    Article  CAS  Google Scholar 

  2. Barrios-Rodiles, M. et al. High-throughput mapping of a dynamic signaling network in mammalian cells. Science 307, 1621–1625 (2005).

    Article  CAS  Google Scholar 

  3. Puig, O. et al. The tandem affinity purification (TAP) method: a general procedure of protein complex purification. Methods 24, 218–229 (2001).

    Article  CAS  Google Scholar 

  4. Fields, S. & Song, O. A novel genetic system to detect protein-protein interactions. Nature 340, 245–246 (1989).

    Article  CAS  Google Scholar 

  5. Broder, Y.C., Katz, S. & Aronheim, A. The ras recruitment system, a novel approach to the study of protein-protein interactions. Curr. Biol. 8, 1121–1124 (1998).

    Article  CAS  Google Scholar 

  6. Stagljar, I., Korostensky, C., Johnsson, N. & te Heesen, S. A genetic system based on split-ubiquitin for the analysis of interactions between membrane proteins in vivo. Proc. Natl. Acad. Sci. USA 95, 5187–5192 (1998).

    Article  CAS  Google Scholar 

  7. Wilson, C.G., Magliery, T.J. & Regan, L. Detecting protein-protein interactions with GFP-fragment reassembly. Nat. Methods 1, 255–262 (2004).

    Article  CAS  Google Scholar 

  8. Stagljar, I. & Fields, S. Analysis of membrane protein interactions using yeast-based technologies. Trends Biochem. Sci. 27, 559–563 (2002).

    Article  CAS  Google Scholar 

  9. Maurel, D. et al. Cell-surface protein-protein interaction analysis with time-resolved FRET and snap-tag technologies: application to GPCR oligomerization. Nat. Methods 5, 561–567 (2008).

    Article  CAS  Google Scholar 

  10. Valentin, G. et al. Photoconversion of YFP into a CFP-like species during acceptor photobleaching FRET experiments. Nat. Methods 2, 801 (2005).

    Article  CAS  Google Scholar 

  11. Harder, T., Scheiffele, P., Verkade, P. & Simons, K. Lipid domain structure of the plasma membrane revealed by patching of membrane components. J. Cell Biol. 141, 929–942 (1998).

    Article  CAS  Google Scholar 

  12. Digby, G.J., Lober, R.M., Sethi, P.R. & Lambert, N.A. Some G protein heterotrimers physically dissociate in living cells. Proc. Natl. Acad. Sci. USA 103, 17789–17794 (2006).

    Article  CAS  Google Scholar 

  13. Drbal, K. et al. Single-molecule microscopy reveals heterogeneous dynamics of lipid raft components upon TCR engagement. Int. Immunol. 19, 675–684 (2007).

    Article  CAS  Google Scholar 

  14. Suzuki, K.G. et al. GPI-anchored receptor clusters transiently recruit Lyn and G{alpha} for temporary cluster immobilization and Lyn activation: single-molecule tracking study 1. J. Cell Biol. 177, 717–730 (2007).

    Article  CAS  Google Scholar 

  15. Orth, R.N. et al. Mast cell activation on patterned lipid bilayers of subcellular dimensions. Langmuir 19, 1599–1605 (2003).

    Article  CAS  Google Scholar 

  16. Wu, M., Holowka, D., Craighead, H.G. & Baird, B. Visualization of plasma membrane compartmentalization with patterned lipid bilayers. Proc. Natl. Acad. Sci. USA 101, 13798–13803 (2004).

    Article  CAS  Google Scholar 

  17. Mossman, K.D., Campi, G., Groves, J.T. & Dustin, M.L. Altered TCR signaling from geometrically repatterned immunological synapses. Science 310, 1191–1193 (2005).

    Article  CAS  Google Scholar 

  18. Cavalcanti-Adam, E.A. et al. Lateral spacing of integrin ligands influences cell spreading and focal adhesion assembly. Eur. J. Cell Biol. 85, 219–224 (2006).

    Article  CAS  Google Scholar 

  19. Shaw, A.S. et al. Short related sequences in the cytoplasmic domains of CD4 and CD8 mediate binding to the amino-terminal domain of the p56lck tyrosine protein kinase. Mol. Cell. Biol. 10, 1853–1862 (1990).

    Article  CAS  Google Scholar 

  20. Turner, J.M. et al. Interaction of the unique N-terminal region of tyrosine kinase p56lck with cytoplasmic domains of CD4 and CD8 is mediated by cysteine motifs. Cell 60, 755–765 (1990).

    Article  CAS  Google Scholar 

  21. Li, Q.J. et al. CD4 enhances T cell sensitivity to antigen by coordinating Lck accumulation at the immunological synapse. Nat. Immunol. 5, 791–799 (2004).

    Article  CAS  Google Scholar 

  22. Filipp, D. et al. Enrichment of lck in lipid rafts regulates colocalized fyn activation and the initiation of proximal signals through TCR alpha beta. J. Immunol. 172, 4266–4274 (2004).

    Article  CAS  Google Scholar 

  23. Kim, P.W. et al. A zinc clasp structure tethers Lck to T cell coreceptors CD4 and CD8. Science 301, 1725–1728 (2003).

    Article  CAS  Google Scholar 

  24. Balamuth, F., Brogdon, J.L. & Bottomly, K. CD4 raft association and signaling regulate molecular clustering at the immunological synapse site. J. Immunol. 172, 5887–5892 (2004).

    Article  CAS  Google Scholar 

  25. Fragoso, R. et al. Lipid raft distribution of CD4 depends on its palmitoylation and association with Lck, and evidence for CD4-induced lipid raft aggregation as an additional mechanism to enhance CD3 signaling. J. Immunol. 170, 913–921 (2003).

    Article  CAS  Google Scholar 

  26. Stefanova, I. et al. GPI-anchored cell-surface molecules complexed to protein tyrosine kinases. Science 254, 1016–1019 (1991).

    Article  CAS  Google Scholar 

  27. Kabouridis, P.S., Magee, A.I. & Ley, S.C. S-acylation of LCK protein tyrosine kinase is essential for its signalling function in T lymphocytes. EMBO J. 16, 4983–4998 (1997).

    Article  CAS  Google Scholar 

  28. Zacharias, D.A., Violin, J.D., Newton, A.C. & Tsien, R.Y. Partitioning of lipid-modified monomeric GFPs into membrane microdomains of live cells. Science 296, 913–916 (2002).

    Article  CAS  Google Scholar 

  29. Moertelmaier, M. et al. Thinning out clusters while conserving stoichiometry of labeling. Appl. Phys. Lett. 87, 263903 (2005).

    Article  Google Scholar 

  30. Ulbrich, M.H. & Isacoff, E.Y. Subunit counting in membrane-bound proteins. Nat. Methods 4, 319–321 (2007).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work was supported by the Austrian Science Fund (project Y250-B10), the Competence Center for Biomolecular Therapeutics Research-Vienna and the GEN-AU project of the Austrian Federal Ministry for Science and Research. We thank V. Horejsi (Czech Academy of Sciences) for providing monoclonal antibodies, G. Nolan (Stanford University) for providing retroviral expression vector pBMN-Z, J. Lippincott-Schwartz (US National Institutes of Health) for providing plasmid pJB20 encoding GFP-GPI and J. Huppa and M.M. Davis (Stanford University) for providing biotinylated MHC class II I-Ek protein.

Author information

Authors and Affiliations

  1. Biophysics Institute, Johannes Kepler University Linz, Altenbergerstr. 69, Linz, A-4040, Austria

    Michaela Schwarzenbacher, Martin Kaltenbrunner, Mario Brameshuber, Clemens Hesch, Julian Weghuber & Gerhard J Schütz

  2. Department of Molecular Immunology, Center for Physiology, Pathophysiology and Immunology, Medical University of Vienna, Lazarettgasse 19, Vienna, A-1090, Austria

    Wolfgang Paster & Hannes Stockinger

  3. Department of Knowledge-based Mathematical Systems, Johannes Kepler University Linz, Altenbergerstr. 69, Linz, A-4040, Austria

    Bettina Heise

  4. Center for Biomedical Nanotechnology, Upper Austrian Research GmbH, Scharitzerstr. 6-8, Linz, A-4020, Austria

    Alois Sonnleitner

Authors
  1. Michaela Schwarzenbacher
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Martin Kaltenbrunner
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Mario Brameshuber
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Clemens Hesch
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Wolfgang Paster
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Julian Weghuber
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Bettina Heise
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Alois Sonnleitner
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Hannes Stockinger
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Gerhard J Schütz
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

M.S., A.S., H.S. and G.J.S. designed the research; M.S., M.K. and M.B. conducted experiments; W.P. and J.W. generated constructs; C.H. developed instrumentation; B.H. developed new analytical tools; M.S., M.K., M.B. and G.J.S. analyzed data; and M.S., H.S. and G.J.S. wrote the paper.

Corresponding authors

Correspondence to Hannes Stockinger or Gerhard J Schütz.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–8, Supplementary Discussion, Supplementary Methods (PDF 1086 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Schwarzenbacher, M., Kaltenbrunner, M., Brameshuber, M. et al. Micropatterning for quantitative analysis of protein-protein interactions in living cells. Nat Methods 5, 1053–1060 (2008). https://doi.org/10.1038/nmeth.1268

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nmeth.1268

This article is cited by

Search

Advanced 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