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Quantum dot ligands provide new insights into erbB/HER receptor–mediated signal transduction

Nature Biotechnology volume 22, pages 198–203 (2004)Cite this article

Abstract

The erbB/HER family of transmembrane receptor tyrosine kinases (RTKs) mediate cellular responses to epidermal growth factor (EGF) and related ligands. We have imaged the early stages of RTK-dependent signaling in living cells using: (i) stable expression of erbB1/2/3 fused with visible fluorescent proteins (VFPs), (ii) fluorescent quantum dots (QDs) bearing epidermal growth factor (EGF-QD) and (iii) continuous confocal laser scanning microscopy and flow cytometry. Here we demonstrate that EGF-QDs are highly specific and potent in the binding and activation of the EGF receptor (erbB1), being rapidly internalized into endosomes that exhibit active trafficking and extensive fusion. EGF-QDs bound to erbB1 expressed on filopodia revealed a previously unreported mechanism of retrograde transport to the cell body. When erbB2-monomeric yellow fluorescent protein (mYFP) or erbB3-monomeric Citrine (mCitrine) were coexpressed with erbB1, the rates and extent of endocytosis of EGF-QD and the RTK-VFP demonstrated that erbB2 but not erbB3 heterodimerizes with erbB1 after EGF stimulation, thereby modulating EGF-induced signaling. QD-ligands will find widespread use in basic research and biotechnological developments.

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Figure 1: QDs bind and activate EGFR, and are endocytosed into living cells.
Figure 2: Live cell activation by EGF-QDs.
Figure 3: Retrograde transport of EGF-QDs on filopodia.
Figure 4: Dynamics of endosomal fusion.
Figure 5: Quantitative analysis of real-time EGF-QD binding and internalization.
Figure 6: Quantitative analysis of EGF-QD–induced erbB1/2/3 internalization.

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Acknowledgements

The project was supported by funding from the Max Planck Gesellschaft, D.S.L., P.N., R.H by the European Union FP5 Project, J.N.P. by a DFG grant to D.J.A.-J., and E.A.J.-E. by CONICET.

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Authors and Affiliations

  1. Department of Molecular Biology, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, Göttingen, D-37077, Germany

    Diane S Lidke, Peter Nagy, Rainer Heintzmann, Donna J Arndt-Jovin, Janine N Post & Thomas M Jovin

  2. Departamento de Física, Laboratorio de Electrónica Cuántica, Facultad de Ciencias Exactas y Naturales, Intendente Güiraldes 2160, Pabellón I, Ciudad Universitaria, Universidad de Buenos Aires, Buenos Aires, C1428EHA, Argentina

    Hernan E Grecco

  3. Departamento de Química Orgánica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Pabellón II 3er piso, Ciudad Universitaria, Buenos Aires, C1428EHA, Argentina

    Elizabeth A Jares-Erijman

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Supplementary information

Supplementary Fig. 1 (PDF 27 kb)

41587_2004_BFnbt929_MOESM2_ESM.mov

Supplementary Movie 1. EGF-QD binding and internalization (Fig2a). Time series of CHO cells expressing erbB1-eGFP (green) with addition of 200 pM 6:1 EGF-QD (red). EGF-QD binding and internalization of the erbB1-eGFP-EGF-QD complex is observed. Note that the cell in the upper left corner undergoes cell division during EGF-QD-erbB1-eGFP endocytosis. 4 s/frame, playback 10 frames/s. Resolution: 0.14 -μm x 0.14 μm. (MOV 3952 kb)

41587_2004_BFnbt929_MOESM3_ESM.mov

Supplementary Movie 2. Z-stack projection of EGF-QD activation and internalization (Fig. 2b). Time series of CHO cells expressing erbB1-eGFP (green) with addition of 250 pM 30:1 EGF-QD (red). Ruffling of the cell surface in response to the EGF-QD binding is seen, followed by extensive internalization. Maximum projection of 12 slices to capture whole cell. 1 min/frame, playback 10 frames/s. _x,y,z: 0.14 μm x 0.14 μm x 0.62 μm. (MOV 556 kb)

41587_2004_BFnbt929_MOESM4_ESM.mov

Supplementary Movie 3. Selective internalization of EGF-QD-erbB1 on erbB3-mCitrine expressing cells (Fig. 3a). Time series of an A431 cell expressing erbB3-mCitrine (green) with addition of 200 pM 6:1 EGF-QD (red). EGF-QDs are seen to bind to the filopodia and travel to the cell body, while the erbB3-mCitrine remains on the cell surface. Maximum projection of 4 slices. 4.5 s/frame, playback 10 frames/s. _x,y,z: 0.1 μm x 0.1 μm x 0.5 μm. (MOV 1076 kb)

41587_2004_BFnbt929_MOESM5_ESM.mov

Supplementary Movie 4. ErbB1-eGFP-EGF-QD endosome fusion (Fig. 4). Time series of a CHO cell expressing erbB1-eGFP (green). Series begins 30 min after addition of 200 pM 6:1 EGF-QD (red). Brownian motion, directed movement and fusion of the erbB1-eGFP-EGF-QD endosomes is observed. Maximum projection of 3 slices. 3.6 s/frame, playback 5 frames/s. _x,y,z: 0.14 μm x 0.14 μm x 0.5 μm. (MOV 585 kb)

Supplementary Methods (PDF 32 kb)

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Lidke, D., Nagy, P., Heintzmann, R. et al. Quantum dot ligands provide new insights into erbB/HER receptor–mediated signal transduction. Nat Biotechnol 22, 198–203 (2004). https://doi.org/10.1038/nbt929

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