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Imaging the lateral diffusion of membrane molecules with quantum dots

Nature Protocols volume 1, pages 2628–2634 (2006)Cite this article

Abstract

This protocol describes a sensitive approach to tracking the motion of membrane molecules such as lipids and proteins with molecular resolution in live cells. This technique makes use of fluorescent semiconductor nanocrystals, quantum dots (QDs), as a probe to detect membrane molecules of interest. The photostability and brightness of QDs allow them to be tracked at a single particle level for longer periods than previous fluorophores, such as fluorescent proteins and organic dyes. QDs are bound to the extracellular part of the object to be followed, and their movements can be recorded with a fluorescence microscope equipped with a spectral lamp and a sensitive cooled charge-coupled device camera. The experimental procedure described for neurons below takes about 45 min. This technique is applicable to various cultured cells.

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Figure 1: Labeling molecules on the cell surface with QDs.
Figure 2
Figure 3: Moist chamber and medium exchange procedure.
Figure 4: QD-trajectory analysis.
Figure 5: Example of QD labeling combined with other dyes.

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References

  1. Saxton, M.J. & Jacobson, K. Single-particle tracking: applications to membrane dynamics. Annu. Rev. Biophys. Biomol. Struct. 26, 373–399 (1997).

    Article  CAS  Google Scholar 

  2. Dahan, M. From analog to digital: exploring cell dynamics with single quantum dots. Histochem. Cell. Biol. 125, 451–456 (2006).

    Article  CAS  Google Scholar 

  3. Geerts, H. et al. Nanovid tracking: a new automatic method for the study of mobility in living cells based on colloidal gold and video microscopy. Biophys. J. 52, 775–782 (1987).

    Article  CAS  Google Scholar 

  4. Schnapp, B.J., Gelles, J. & Sheetz, M.P. Nanometer-scale measurements using video light microscopy. Cell. Motil. Cytoskeleton 10, 47–53 (1988).

    Article  CAS  Google Scholar 

  5. Kusumi, A., Sako, Y. & Yamamoto, M. Confined lateral diffusion of membrane receptors as studied by single particle tracking (nanovid microscopy). Effects of calcium-induced differentiation in cultured epithelial cells. Biophys. J. 65, 2021–2040 (1993).

    Article  CAS  Google Scholar 

  6. Meier, J., Vannier, C., Serge, A., Triller, A. & Choquet, D. Fast and reversible trapping of surface glycine receptors by gephyrin. Nat. Neurosci. 4, 253–260 (2001).

    Article  CAS  Google Scholar 

  7. Borgdorff, A.J. & Choquet, D. Regulation of AMPA receptor lateral movements. Nature 417, 649–653 (2002).

    Article  CAS  Google Scholar 

  8. Iino, R., Koyama, I. & Kusumi, A. Single molecule imaging of green fluorescent proteins in living cells: E-cadherin forms oligomers on the free cell surface. Biophys. J. 80, 2667–2677 (2001).

    Article  CAS  Google Scholar 

  9. Schmidt, T., Schutz, G.J., Baumgartner, W., Gruber, H.J. & Schindler, H. Imaging of single molecule diffusion. Proc. Natl. Acad. Sci. USA 93, 2926–2929 (1996).

    Article  CAS  Google Scholar 

  10. Tardin, C., Cognet, L., Bats, C., Lounis, B. & Choquet, D. Direct imaging of lateral movements of AMPA receptors inside synapses. EMBO J. 22, 4656–4665 (2003).

    Article  CAS  Google Scholar 

  11. Schutz, G.J., Sonnleitner, M., Hinterdorfer, P. & Schindler, H. Single molecule microscopy of biomembranes (review). Mol. Membr. Biol. 17, 17–29 (2000).

    Article  CAS  Google Scholar 

  12. Alivisatos, P. The use of nanocrystals in biological detection. Nat. Biotechnol. 22, 47–52 (2004).

    Article  CAS  Google Scholar 

  13. Jaiswal, J.K. & Simon, S.M. Potentials and pitfalls of fluorescent quantum dots for biological imaging. Trends Cell Biol. 14, 497–504 (2004).

    Article  CAS  Google Scholar 

  14. Michalet, X. et al. Quantum dots for live cells, in vivo imaging, and diagnostics. Science 307, 538–544 (2005).

    Article  CAS  Google Scholar 

  15. Giepmans, B.N., Adams, S.R., Ellisman, M.H. & Tsien, R.Y. The fluorescent toolbox for assessing protein location and function. Science 312, 217–224 (2006).

    Article  CAS  Google Scholar 

  16. Wu, X. et al. Immunofluorescent labeling of cancer marker Her2 and other cellular targets with semiconductor quantum dots. Nat. Biotechnol. 21, 41–46 (2003).

    Article  CAS  Google Scholar 

  17. Pellegrino, T. et al. Quantum dot-based cell motility assay. Differentiation 71, 542–548 (2003).

    Article  Google Scholar 

  18. Dubertret, B. et al. In vivo imaging of quantum dots encapsulated in phospholipid micelles. Science 298, 1759–1762 (2002).

    Article  CAS  Google Scholar 

  19. Dahan, M. et al. Diffusion dynamics of glycine receptors revealed by single-quantum dot tracking. Science 302, 442–445 (2003).

    Article  CAS  Google Scholar 

  20. Groc, L. et al. Differential activity-dependent regulation of the lateral mobilities of AMPA and NMDA receptors. Nat. Neurosci. 7, 695–696 (2004).

    Article  CAS  Google Scholar 

  21. Lidke, D.S. et al. Quantum dot ligands provide new insights into erbB/HER receptor-mediated signal transduction. Nat. Biotechnol. 22, 198–203 (2004).

    Article  CAS  Google Scholar 

  22. Pinaud, F., King, D., Moore, H.P. & Weiss, S. Bioactivation and cell targeting of semiconductor CdSe/ZnS nanocrystals with phytochelatin-related peptides. J. Am. Chem. Soc. 126, 6115–6123 (2004).

    Article  CAS  Google Scholar 

  23. Charrier, C., Ehrensperger, M.V., Dahan, M., Levi, S. & Triller, A. Cytoskeleton regulation of glycine receptor number at synapses and diffusion in the plasma membrane. J. Neurosci. 26, 8502–8511 (2006).

    Article  CAS  Google Scholar 

  24. Luccardini, C., Tribet, C., Vial, F., Marchi-Artzner, V. & Dahan, M. Size, charge, and interactions with giant lipid vesicles of quantum dots coated with an amphiphilic macromolecule. Langmuir 22, 2304–2310 (2006).

    Article  CAS  Google Scholar 

  25. Giepmans, B.N., Deerinck, T.J., Smarr, B.L., Jones, Y.Z. & Ellisman, M.H. Correlated light and electron microscopic imaging of multiple endogenous proteins using Quantum dots. Nat. Methods 2, 743–749 (2005).

    Article  CAS  Google Scholar 

  26. Sekine-Aizawa, Y. & Huganir, R.L. Imaging of receptor trafficking by using alpha-bungarotoxin-binding-site-tagged receptors. Proc. Natl. Acad. Sci. USA 101, 17114–17119 (2004).

    Article  CAS  Google Scholar 

  27. Howarth, M., Takao, K., Hayashi, Y. & Ting, A.Y. Targeting quantum dots to surface proteins in living cells with biotin ligase. Proc. Natl. Acad. Sci. USA 102, 7583–7588 (2005).

    Article  CAS  Google Scholar 

  28. Fujiwara, T., Ritchie, K., Murakoshi, H., Jacobson, K. & Kusumi, A. Phospholipids undergo hop diffusion in compartmentalized cell membrane. J. Cell Biol. 157, 1071–1081 (2002).

    Article  CAS  Google Scholar 

  29. Bonneau, S., Cohen, L. & Dahan, M. A multiple target approach for single quantum dot tracking. Proc. IEEE Int. Symp. Biol. Imag. 664 (2004).

  30. Bonneau, S., Dahan, M. & Cohen, L.D. Single quantum dot tracking based on perceptual grouping using minimal paths in a spatiotemporal volume. IEEE Trans. Image Process. 14, 1384–1395 (2005).

    Article  Google Scholar 

  31. Courty, S., Luccardini, C., Bellaiche, Y., Cappello, G. & Dahan, M. Tracking individual kinesin motors in living cells using single quantum-dot imaging. Nano Lett. 6, 1491–1495 (2006).

    Article  CAS  Google Scholar 

  32. Hanus, C., Ehrensperger, M.V. & Triller, A. Activity-dependent movements of postsynaptic scaffolds at inhibitory synapses. J. Neurosci. 26, 4586–4595 (2006).

    Article  CAS  Google Scholar 

  33. Yao, J., Larson, D.R., Vishwasrao, H.D., Zipfel, W.R. & Webb, W.W. Blinking and nonradiant dark fraction of water-soluble quantum dots in aqueous solution. Proc. Natl. Acad. Sci. USA 102, 14284–14289 (2005).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank S. Bonneau and M.-V. Ehrensperger (Ecole Normale Supérieure, Paris, France) for writing the custom-made software for data analysis and T. Inoue and K. Mikoshiba (University of Tokyo, Japan) for the custom-made software TI Workbench and for valuable discussions on data analysis. We also thank Quantum Dot Corporation for providing the composition of the QD binding buffer. This work was supported by grants from INSERM, the CNRS, the Ministère de la Recherche and the FRM to S.L., M.D. and A.T. and Toyobo Biotechnology Foundation, Hayashi Memorial Foundation for Female Natural Scientists and the Japan Society for the Promotion of Science to H.B.

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Authors and Affiliations

  1. INSERM U789, Biologie Cellulaire de la Synapse N&P, Ecole Normale Supérieure Paris, 46, Rue d'Ulm, 75005, Paris, France

    Hiroko Bannai, Sabine Lévi, Claude Schweizer & Antoine Triller

  2. Physics Department, CNRS UMR8552, Laboratoire Kastler Brossel, Ecole Normale Supérieure Paris, 24, rue Lhomond, 75005, Paris, France

    Maxime Dahan

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  1. Hiroko Bannai
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Correspondence to Antoine Triller.

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Supplementary information

Supplementary Video 1

Example of QD recording. (MOV 1574 kb)

Supplementary Video 2

Example of simultaneous QDs and calcium indicator imaging. (MOV 2325 kb)

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Bannai, H., Lévi, S., Schweizer, C. et al. Imaging the lateral diffusion of membrane molecules with quantum dots. Nat Protoc 1, 2628–2634 (2006). https://doi.org/10.1038/nprot.2006.429

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