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Nature Methods
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Direct observation of individual endogenous protein complexesin situ by proximity ligation

Nature Methodsvolume 3, pages995–1000 (2006)Cite this article

Abstract

Cellular processes can only be understood as the dynamic interplay of molecules. There is a need for techniques to monitor interactions of endogenous proteins directly in individual cells and tissues to reveal the cellular and molecular architecture and its responses to perturbations. Here we report our adaptation of the recently developed proximity ligation method to examine the subcellular localization of protein-protein interactions at single-molecule resolution. Proximity probes—oligonucleotides attached to antibodies against the two target proteins—guided the formation of circular DNA strands when bound in close proximity. The DNA circles in turn served as templates for localized rolling-circle amplification (RCA), allowing individual interacting pairs of protein molecules to be visualized and counted in human cell lines and clinical specimens. We used this method to show specific regulation of protein-protein interactions between endogenous Myc and Max oncogenic transcription factors in response to interferon-γ (IFN-γ) signaling and low-molecular-weight inhibitors.

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Figure 1: Detection of endogenous c-Myc/Max heterodimers in cultured cells using P-LISA.
Figure 2: Detection of protein-protein interactions.
Figure 3: Visualization of c-Myc/Max heterodimerization in tissue sections.
Figure 4: Perturbation of Myc/Max heterodimerization.

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References

  1. Fredriksson, S. et al. Protein detection using proximity-dependent DNA ligation assays.Nat. Biotechnol.20, 473–477 (2002).

    Article CAS  Google Scholar 

  2. Gullberg, M. et al. Cytokine detection by antibody-based proximity ligation.Proc. Natl. Acad. Sci. USA101, 8420–8424 (2004).

    Article CAS  Google Scholar 

  3. Baner, J., Nilsson, M., Mendel-Hartvig, M. & Landegren, U. Signal amplification of padlock probes by rolling circle replication.Nucleic Acids Res.26, 5073–5078 (1998).

    Article CAS  Google Scholar 

  4. Grandori, C., Cowley, S.M., James, L.P. & Eisenman, R.N. The Myc/Max/Mad network and the transcriptional control of cell behavior.Annu. Rev. Cell Dev. Biol.16, 653–699 (2000).

    Article CAS  Google Scholar 

  5. Oster, S.K., Ho, C.S., Soucie, E.L. & Penn, L.Z. The myc oncogene: MarvelouslY Complex.Adv. Cancer Res.84, 81–154 (2002).

    Article CAS  Google Scholar 

  6. Patel, J.H., Loboda, A.P., Showe, M.K., Showe, L.C. & McMahon, S.B. Analysis of genomic targets reveals complex functions of MYC.Nat. Rev. Cancer4, 562–568 (2004).

    Article CAS  Google Scholar 

  7. Pelengaris, S., Khan, M. & Evan, G. c-MYC: more than just a matter of life and death.Nat. Rev. Cancer2, 764–776 (2002).

    Article CAS  Google Scholar 

  8. Hu, C.D., Chinenov, Y. & Kerppola, T.K. Visualization of interactions among bZIP and Rel family proteins in living cells using bimolecular fluorescence complementation.Mol. Cell9, 789–798 (2002).

    Article CAS  Google Scholar 

  9. Venetsanakos, E. et al. Induction of tubulogenesis in telomerase-immortalized human microvascular endothelial cells by glioblastoma cells.Exp. Cell Res.273, 21–33 (2002).

    Article CAS  Google Scholar 

  10. Grinberg, A.V., Hu, C.D. & Kerppola, T.K. Visualization of Myc/Max/Mad family dimers and the competition for dimerization in living cells.Mol. Cell. Biol.24, 4294–4308 (2004).

    Article CAS  Google Scholar 

  11. Winqvist, R., Saksela, K. & Alitalo, K. The myc proteins are not associated with chromatin in mitotic cells.EMBO J.3, 2947–2950 (1984).

    Article CAS  Google Scholar 

  12. Mateyak, M.K., Obaya, A.J., Adachi, S. & Sedivy, J.M. Phenotypes of c-Myc-deficient rat fibroblasts isolated by targeted homologous recombination.Cell Growth Differ.8, 1039–1048 (1997).

    CAS  Google Scholar 

  13. Alroy, I. & Yarden, Y. The ErbB signaling network in embryogenesis and oncogenesis: signal diversification through combinatorial ligand-receptor interactions.FEBS Lett.410, 83–86 (1997).

    Article CAS  Google Scholar 

  14. Bouchard, C., Marquardt, J., Bras, A., Medema, R.H. & Eilers, M. Myc-induced proliferation and transformation require Akt-mediated phosphorylation of FoxO proteins.EMBO J.23, 2830–2840 (2004).

    Article CAS  Google Scholar 

  15. Adhikary, S. & Eilers, M. Transcriptional regulation and transformation by Myc proteins.Nat. Rev. Mol. Cell Biol.6, 635–645 (2005).

    Article CAS  Google Scholar 

  16. Öberg, F., Larsson, L.G., Anton, R. & Nilsson, K. Interferon gamma abrogates the differentiation block in v-myc-expressing U-937 monoblasts.Proc. Natl. Acad. Sci. USA88, 5567–5571 (1991).

    Article  Google Scholar 

  17. Guzhova, I. et al. Interferon-gamma cooperates with retinoic acid and phorbol ester to induce differentiation and growth inhibition of human neuroblastoma cells.Int. J. Cancer94, 97–108 (2001).

    Article CAS  Google Scholar 

  18. Bahram, F., Wu, S., Öberg, F., Lüscher, B. & Larsson, L.G. Posttranslational regulation of Myc function in response to phorbol ester/interferon-gamma-induced differentiation of v-Myc-transformed U-937 monoblasts.Blood93, 3900–3912 (1999).

    CAS  Google Scholar 

  19. Eilers, M., Picard, D., Yamamoto, K.R. & Bishop, J.M. Chimaeras of myc oncoprotein and steroid receptors cause hormone-dependent transformation of cells.Nature340, 66–68 (1989).

    Article CAS  Google Scholar 

  20. Littlewood, T.D., Hancock, D.C., Danielian, P.S., Parker, M.G. & Evan, G.I. A modified oestrogen receptor ligand-binding domain as an improved switch for the regulation of heterologous proteins.Nucleic Acids Res.23, 1686–1690 (1995).

    Article CAS  Google Scholar 

  21. Solomon, D.L., Philipp, A., Land, H. & Eilers, M. Expression of cyclin D1 mRNA is not upregulated by Myc in rat fibroblasts.Oncogene11, 1893–1897 (1995).

    CAS  Google Scholar 

  22. Yin, X., Giap, C., Lazo, J.S. & Prochownik, E.V. Low molecular weight inhibitors of Myc-Max interaction and function.Oncogene22, 6151–6159 (2003).

    Article CAS  Google Scholar 

  23. Arabi, A. et al. c-Myc associates with ribosomal DNA in the nucleolus and activates RNA polymerase I transcription.Nat. Cell Biol.7, 303–310 (2005).

    Article CAS  Google Scholar 

  24. Beckstead, J.H. A simple technique for preservation of fixation-sensitive antigens in paraffin-embedded tissues.J. Histochem. Cytochem.42, 1127–1134 (1994).

    Article CAS  Google Scholar 

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Acknowledgements

We thank A.-C. Andersson for technical assistance; C. Wählby for image analysis; M. Taussig and S. Fredriksson for valuable comments on the manuscript; J.M. Sedivy and M. Eilers for providing cells; A.-C. Steffen and J. Carlsson for providing EGFR and Her-2 antibodies; and G.R. Adolf for providing IFN-γ. We obtained frozen human tissues from the Fresh Tissue Biobank at the Department of Clinical Pathology, Uppsala University Hospital (supported by the SWEGENE/Wallenberg Consortium North Biobank Program). This project was supported by the Wallenberg Foundation, the EU Integrated Project MolTools, the Research Councils of Sweden for natural science and for medicine (to U.L.); and the Swedish Cancer Foundation, the Swedish Children Cancer Foundation and the Human Frontiers Science Program (to L.-G.L.).

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Author notes

  1. Ola Söderberg, Mats Gullberg and Malin Jarvius: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Genetics and Pathology, Rudbeck Laboratory, University of Uppsala, Uppsala, SE-75185, Sweden

    Ola Söderberg, Mats Gullberg, Malin Jarvius, Karl-Johan Leuchowius, Jonas Jarvius, Kenneth Wester & Ulf Landegren

  2. Department of Plant Biology and Forest Genetics, Uppsala Genetic Center, Swedish University of Agricultural Sciences, Uppsala, SE-75007, Sweden

    Karin Ridderstråle, Per Hydbring, Fuad Bahram & Lars-Gunnar Larsson

Authors
  1. Ola Söderberg

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  2. Mats Gullberg

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  4. Karin Ridderstråle

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  5. Karl-Johan Leuchowius

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  6. Jonas Jarvius

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  7. Kenneth Wester

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  9. Fuad Bahram

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  10. Lars-Gunnar Larsson

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  11. Ulf Landegren

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Corresponding author

Correspondence toUlf Landegren.

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Competing interests

U.L., M.G. and J.J. are cofounders of the company Olink AB, which exploits this technology and holds the right to patents US6558928, US6878515, US7074564, US09/785657, US 10/496385, US 11/011438, and corresponding patents and patent applications in other geographies describing the technology. The inventions described in the patents have been jointly or separately invented by U.L. and Simon Fredriksson.

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Söderberg, O., Gullberg, M., Jarvius, M.et al. Direct observation of individual endogenous protein complexesin situ by proximity ligation.Nat Methods3, 995–1000 (2006). https://doi.org/10.1038/nmeth947

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