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Spatial control of membrane receptor function using ligand nanocalipers

Abstract

The spatial organization of membrane-bound ligands is thought to regulate receptor-mediated signaling. However, direct regulation of receptor function by nanoscale distribution of ligands has not yet been demonstrated, to our knowledge. We developed rationally designed DNA origami nanostructures modified with ligands at well-defined positions. Using these 'nanocalipers' to present ephrin ligands, we showed that the nanoscale spacing of ephrin-A5 directs the levels of EphA2 receptor activation in human breast cancer cells. Furthermore, we found that the nanoscale distribution of ephrin-A5 regulates the invasive properties of breast cancer cells. Our ligand nanocaliper approach has the potential to provide insight into the roles of ligand nanoscale spatial distribution in membrane receptor–mediated signaling.

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Figure 1: The nanocaliper principle and display of ephrin ligands.
Figure 2: Ephrin-A5 conjugate and ephrin-A5–nanocaliper binding abilities.
Figure 3: The spatial distribution of ephrin-A5 ligands directs the phosphorylation levels of the EphA2 receptor.
Figure 4: Ephrin-A5 nanocalipers modulate EphA2 mediated responses in human breast cancer cells.

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Acknowledgements

We thank U. Lendahl and his lab (Karolinska Institutet) for reagents and discussions, J. Avila-Cariño for help with the FACS experiments, O. Shupliakov for help with TEM, G. Bernardinelli for help with rendering ephrin-A5 and Y.-X. Zhao for help with initial experiments. This work was funded through grants from the Swedish Research Council to B.H. (repatriation grant 2010-6296 and project grant 2010-5060) and by the Strategic Research Program in Stem Cell Research and Regenerative Medicine at Karolinska Institutet (StratRegen), Sweden (A.I.T.). B.H. received startup funding from Carl Bennet AB, Karolinska Institutet and the Swedish Governmental Agency for Innovation Systems (Vinnova). V.L. and A.S. were supported by KID doctoral fellowships from the Karolinska Institutet, Sweden.

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Authors

Contributions

B.H. and A.I.T. conceived of and designed the study. V.L., A.S. and E.P. performed most of the experimental work. F.F. and E.B. contributed to nanostructure and conjugate production and nanocaliper characterization. A.A.-A., A.H. and A.B. contributed to cell culture and the setting up and validating of the PLA assay. A.B. performed immunoprecipitation and immunoblotting experiments. V.L., A.S., B.H. and A.I.T. wrote the manuscript. All authors contributed to manuscript proofing and discussion.

Corresponding authors

Correspondence to Björn Högberg or Ana I Teixeira.

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The authors declare no competing financial interests.

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Supplementary Figures 1–16 and Supplementary Table 1 (PDF 9518 kb)

Supplementary Data

Raw FACS source data for Figure 2b. (ZIP 738 kb)

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Shaw, A., Lundin, V., Petrova, E. et al. Spatial control of membrane receptor function using ligand nanocalipers. Nat Methods 11, 841–846 (2014). https://doi.org/10.1038/nmeth.3025

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