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Direct observation of individual endogenous protein complexes in situ by proximity ligation

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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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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Correspondence to Ulf 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 complexes in situ by proximity ligation. Nat Methods 3, 995–1000 (2006). https://doi.org/10.1038/nmeth947

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