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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.

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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.

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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.

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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
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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.

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

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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

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