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High-speed nanoscopic tracking of the position and orientation of a single virus

Nature Methods volume 6, pages 923–927 (2009)Cite this article

Abstract

Optical studies have revealed that, after binding, virions move laterally on the plasma membrane, but the complexity of the cellular environment and the drawbacks of fluorescence microscopy have prevented access to the molecular dynamics of early virus-host couplings, which are important for cell infection. Here we present a colocalization methodology that combines scattering interferometry and single-molecule fluorescence microscopy to visualize both position and orientation of single quantum dot–labeled Simian virus 40 (SV40) particles. By achieving nanometer spatial and 8 ms temporal resolution, we observed sliding and tumbling motions during rapid lateral diffusion on supported lipid bilayers, and repeated back and forth rocking between nanoscopic regions separated by 9 nm. Our findings suggest recurrent swap of receptors and viral pentamers as well as receptor aggregation in nanodomains. We discuss the prospects of our technique for studying virus-membrane interactions and for resolving nanoscopic dynamics of individual biological nano-objects.

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Figure 1: iSCAT experimental approach.
Figure 2: Determination of localization accuracy and orientation.
Figure 3: iSCAT control experiments.
Figure 4: Diffusion at low (0.05 mol%) GM1 concentration.
Figure 5: Diffusion at high (1 mol%) GM1 concentration.

References

  1. Betzig, E. Proposed method for molecular optical imaging. Opt. Lett. 20, 237–239 (1995).

    Article  CAS  Google Scholar 

  2. Hell, S.W. Far-field optical nanoscopy. Science 316, 1153–1158 (2007).

    Article  CAS  Google Scholar 

  3. Hettich, C. et al. Nanometer resolution and coherent optical dipole coupling of two individual molecules. Science 298, 385–389 (2002).

    Article  CAS  Google Scholar 

  4. Yildiz, A. et al. Myosin V walks hand-over-hand: Single fluorophore imaging with 1.5-nm localization. Science 300, 2061–2065 (2003).

    Article  CAS  Google Scholar 

  5. Betzig, E. et al. Imaging intracellular fluorescent proteins at nanometer resolution. Science 313, 1642–1645 (2006).

    Article  CAS  Google Scholar 

  6. Rust, M.J., Bates, M. & Zhuang, X. Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nat. Methods 3, 793–795 (2006).

    Article  CAS  Google Scholar 

  7. Hess, S.T. et al. Dynamic clustered distribution of hemagglutinin resolved at 40 nm in living cell membranes discriminates between raft theories. Proc. Natl. Acad. Sci. USA 104, 17370–17375 (2007).

    Article  CAS  Google Scholar 

  8. Moerner, W.E. & Orrit, M. Illuminating single molecules in condensed matter. Science 283, 1670–1676 (1999).

    Article  CAS  Google Scholar 

  9. Weiss, S. Fluorescence spectroscopy of single biomolecules. Science 283, 1676–1683 (1999).

    Article  CAS  Google Scholar 

  10. Thompson, R.E., Larson, D. & Webb, W. Precise nanometer localization analysis for individual fluorescent probes. Biophys. J. 82, 2775–2783 (2002).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  12. Seisenberger, G. et al. Real-time single-molecule imaging of the infection pathway of an adeno-associated virus. Science 294, 1929–1932 (2001).

    Article  CAS  Google Scholar 

  13. Lindfors, K., Kalkbrenner, T., Stoller, P. & Sandoghdar, V. Detection and spectroscopy of gold nanoparticle using supercontinuum white light confocal microscopy. Phys. Rev. Lett. 93, 037401 (2004).

    Article  CAS  Google Scholar 

  14. Jacobsen, V., Stoller, P., Brunner, C., Vogel, V. & Sandoghdar, V. Interferometric optical detection and tracking of very small gold nanoparticles at a water-glass interface. Opt. Express 14, 405–414 (2006).

    Article  CAS  Google Scholar 

  15. Ewers, H. et al. Label-free optical detection and tracking of single virions bound to their receptor in supported membrane bilayers. Nano Lett. 7, 2263–2266 (2007).

    Article  CAS  Google Scholar 

  16. Liddington, R.C. et al. Structure of Simian Virus-40 at 3.8-A resolution. Nature 354, 278–284 (1991).

    Article  CAS  Google Scholar 

  17. Tsai, B. et al. Gangliosides are receptors for murine polyoma virus and SV40. EMBO J. 22, 4346–4355 (2003).

    Article  CAS  Google Scholar 

  18. Neu, U., Woellner, K., Gauglitz, G. & Stehle, T. Structural basis of GM1 ganglioside recognition by Simian Virus 40. Proc. Natl. Acad. Sci. USA 105, 5219–5224 (2008).

    Article  CAS  Google Scholar 

  19. Churchman, L.S., Okten, Z., Rock, R.S., Dawson, J.F. & Spudich, J.A. Single molecule high-resolution colocalization of Cy3 and Cy5 attached to macromolecules measures intramolecular distances through time. Proc. Natl. Acad. Sci. USA 102, 1419–1423 (2005).

    Article  CAS  Google Scholar 

  20. Kukura, P., Celebrano, M., Renn, A. & Sandoghdar, V. Imaging a single quantum dot when it is dark. Nano Lett. 9, 926–929 (2009).

    Article  CAS  Google Scholar 

  21. Einstein, A. A new determination of the molecular dimensions. Ann. Phys. 19, 289–306 (1906).

    Article  CAS  Google Scholar 

  22. Ewers, H. et al. Single-particle tracking of murine polyoma virus-like particles on live cells and artificial membranes. Proc. Natl. Acad. Sci. USA 102, 15110–15115 (2005).

    Article  CAS  Google Scholar 

  23. Shi, J. et al. GM1 clustering inhibits cholera toxin binding in supported phospholipid membranes. J. Am. Chem. Soc. 129, 5954–5961 (2007).

    Article  CAS  Google Scholar 

  24. Frey, S. et al. Condensing and fluidizing effects of ganglioside GM1 on phospholipid films. Biophys. J. 94, 3047–3064 (2008).

    Article  CAS  Google Scholar 

  25. Fujita, A. et al. Gangliosides GM1 and GM3 in the living cell membrane form clusters susceptible to cholesterol depletion and chilling. Mol. Biol. Cell 18, 2112–2122 (2007).

    Article  CAS  Google Scholar 

  26. Eggeling, C. et al. Direct observation of the nanoscale dynamics of membrane lipids in a living cell. Nature 457, 1159–1162 (2009).

    Article  CAS  Google Scholar 

  27. Jacobsen, V., Klotzsch, E. & Sandoghdar, V. Interferometric detection and tracking of nanoparticles. in Nano Biophotonics (eds., Masuhara, H., Kawata, S. & Tokunaga, F.) 143–160 (Elsevier, Amsterdam, 2007).

  28. Damm, E.M. et al. Clathrin- and caveolin-1independent endocytosis: entry of Simian Virus 40 into cells devoid of caveolae. J. Cell Biol. 168, 477–488 (2005).

    Article  CAS  Google Scholar 

  29. Kusumi, A. et al. Paradigm shift of the plasma membrane concept from the two-dimensional continuum fluid to the partitioned fluid: High-speed single-molecule tracking of membrane molecules. Annu. Rev. Biophys. Biomol. Struct. 34, 351–378 (2005).

    Article  CAS  Google Scholar 

  30. Kalb, E., Frey, S. & Tamm, L.K. Formation of supported planar bilayers by fusion of vesicles to supported phospholipid monolayers. Biochim. Biophys. Acta 1103, 307–316 (1992).

    Article  CAS  Google Scholar 

  31. Kosukegawa, A. et al. Purification and characterization of virus-like particles and pentamers by the expression of SV40 capsid proteins in insect cells. Biochim. Biophys. Acta 1290, 37–45 (1996).

    Article  Google Scholar 

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Acknowledgements

We thank the Swiss Ministry of Education and Research for financial support (EU Integrated project Molecular Imaging), J. Helenius for comments, R. Mancini for providing electron micrographs of quantum dots, A. Oppenheim (Department of Hematology, Hebrew University, Hadassah Medical School and Hadassah Hospital, Jerusalem) for providing SV40 VLPs and Gunter Schwarzmann (Kekule-Institut für Organische Chemie, Universität Bonn) for NBD-GM1. A.H. thanks the Swiss National Science Foundation (SNF) for financial support.

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Author notes

  1. Christian Müller

    Present address: Present address: Laboratory of Organic Chemistry, ETH Zurich, Zurich, Switzerland.,

Authors and Affiliations

  1. Laboratory of Physical Chemistry, Eidgenössische Technische Hochschule (ETH) Zurich, Zurich, Switzerland

    Philipp Kukura, Helge Ewers, Christian Müller, Alois Renn & Vahid Sandoghdar

  2. Institute of Biochemistry, Eidgenössische Technische Hochschule (ETH) Zurich, Zurich, Switzerland

    Helge Ewers & Ari Helenius

Authors
  1. Philipp Kukura
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  6. Vahid Sandoghdar
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Contributions

P.K. designed the experimental setup, performed SV40 experiments and analyzed data. H.E. performed FRAP experiments and purified and labeled SV40. H.E. and C.M. prepared supported lipid bilayers. V.S. conceived and supervised the project in collaboration with A.H.; V.S., P.K. and H.E. wrote the manuscript; and P.K., H.E., C.M., A.R., A.H. and V.S. discussed the results and commented on the manuscript.

Corresponding author

Correspondence to Vahid Sandoghdar.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–2 (PDF 257 kb)

Supplementary Video 1

Sequence of 500 consecutive iSCAT (left) and fluorescence (right) images acquired at a frame rate of 130 Hz. The frame rate has been reduced to 25 frames s−1 for clarity. (MOV 3496 kb)

Supplementary Video 2

Illustrative 3D rendering of a 200 frame selection from simultaneously acquired iSCAT and fluorescence trajectories. (MOV 3305 kb)

Supplementary Video 3

Sequential illustration of Figure 5b from the manuscript at the experimental acquisition speed of 25 frames s−1. (MOV 74 kb)

Supplementary Video 4

Sequential illustration of Figure 5c from the manuscript at the experimental acquisition speed of 25 frames s−1. (MOV 25 kb)

Supplementary Video 5

Sequential illustration of Figure 5d from the manuscript at the experimental acquisition speed of 25 frames s−1. (MOV 135 kb)

Supplementary Video 6

Sequential illustration of Figure 5e from the manuscript at the experimental acquisition speed of 25 frames s−1. (MOV 217 kb)

Supplementary Video 7

Sequential illustration of Figure 5f from the manuscript at the experimental acquisition speed of 25 frames s−1. (MOV 154 kb)

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Kukura, P., Ewers, H., Müller, C. et al. High-speed nanoscopic tracking of the position and orientation of a single virus. Nat Methods 6, 923–927 (2009). https://doi.org/10.1038/nmeth.1395

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