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Single-molecule imaging of EGFR signalling on the surface of living cells

Nature Cell Biology volume 2, pages 168–172 (2000)Cite this article

Abstract

The early events in signal transduction from the epidermal growth factor (EGF) receptor (EGFR) are dimerization and autophosphorylation of the receptor, induced by binding of EGF. Here we observe these events in living cells by visualizing single molecules of fluorescent-dye-labelled EGF in the plasma membrane of A431 carcinoma cells. Single-molecule tracking reveals that the predominant mechanism of dimerization involves the formation of a cell-surface complex of one EGF molecule and an EGFR dimer, followed by the direct arrest of a second EGF molecule, indicating that the EGFR dimers were probably preformed before the binding of the second EGF molecule. Single-molecule fluorescence-resonance energy transfer shows that EGF–EGFR complexes indeed form dimers at the molecular level. Use of a monoclonal antibody specific to the phosphorylated (activated) EGFR reveals that the EGFR becomes phosphorylated after dimerization.

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Figure 1: TIR microscopy for visualization of single molecules on the cell surface.
Figure 2: Visualization of Cy3–EGF on the plasma membrane of living A431 cells.
Figure 3: Distribution of the fluorescence intensity of Cy3–EGF and Cy3–EGFR1 1 on the surface of living cells.
Figure 4: Intracellular Ca2+ response induced by Cy3–EGF and the change in intensity distribution of Cy3–EGF spots.
Figure 5: Dimerization of EGF–EGFR complexes.
Figure 6: Single-molecule FRET between Cy3–EGF and Cy5–EGF.
Figure 7: Phosphorylation of EGF–EGFR complexes.

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Acknowledgements

We thank T. Wazawa at the Single-Molecular Processes Project for the computer program with which to measure fluorescence intensity; M. Murata for streptolysin O; and P. Conibear and F. Brozovich for critical reading of the manuscript.

Correspondence and requests for materials should be addressed to T.Y.

Supplementary information is available on Nature Cell Biology’s World-Wide Web site (http://cellbio.nature.com) or as paper copy from the London editorial office of Nature Cell Biology.

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Authors and Affiliations

  1. Department of Physiology and Biosignalling, Graduate School of Medicine, Osaka University, 2-2 Yamadaoka, Suita, 565-0871, Japan

    Yasushi Sako & Shigeru Minoghchi

  2. Single Molecule Processes Project, ICORP, JST, 2-4-14 Senba-Higashi, Mino, 562-0035, Japan

    Toshio Yanagida

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  1. Yasushi Sako
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Supplementary information

Figure 1

Effects of illumination mode on single-molecule imaging. (PDF 100 kb)

Figure 2 Single-molecule FRET from Cy3–EGF to Cy5–EGF.

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Sako, Y., Minoghchi, S. & Yanagida, T. Single-molecule imaging of EGFR signalling on the surface of living cells. Nat Cell Biol 2, 168–172 (2000). https://doi.org/10.1038/35004044

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