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Fluorescence imaging of amyloid formation in living cells by a functional, tetracysteine-tagged α-synuclein

Nature Methods volume 4, pages 345–351 (2007)Cite this article

Abstract

α-synuclein is a major component of intraneuronal protein aggregates constituting a distinctive feature of Parkinson disease. To date, fluorescence imaging of dynamic processes leading to such amyloid deposits in living cells has not been feasible. To address this need, we generated a recombinant α-synuclein (α-synuclein-C4) bearing a tetracysteine target for fluorogenic biarsenical compounds. The biophysical, biochemical and aggregation properties of α-synuclein-C4 matched those of the wild-type protein in vitro and in living cells. We observed aggregation of α-synuclein-C4 transfected or microinjected into cells, particularly under oxidative stress conditions. Fluorescence resonance energy transfer (FRET) between FlAsH and ReAsH confirmed the close association of fibrillized α-synuclein-C4 molecules. α-synuclein-C4 offers the means for directly probing amyloid formation and interactions of α-synuclein with other proteins in living cells, the response to cellular stress and screening drugs for Parkinson disease.

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Figure 1: Characterization of tetracysteine-tagged α-synuclein-C4.
Figure 2: α-synuclein-C4 aggregation in vitro.
Figure 3: Direct visualization of α-synuclein-C4 aggregation in microinjected HeLa cells by fluorescence microscopy.
Figure 4: Fluorescence imaging of α-synuclein-C4 aggregates in transfected SH-SY5Y cells; effect of oxidative stress.
Figure 5: Characterization of aggregated α-synuclein-C4 in vitro and in cells by apbFRET microscopy.

References

  1. Dobson, C.M. Protein folding and misfolding. Nature 426, 884–890 (2003).

    CAS  Google Scholar 

  2. Dawson, T.M. & Dawson, V.L. Molecular pathways of neurodegeneration in Parkinson's disease. Science 302, 819–822 (2003).

    Article  CAS  Google Scholar 

  3. Shults, C.W. Lewy bodies. Proc. Natl. Acad. Sci. USA 103, 1661–1668 (2006).

    Article  CAS  Google Scholar 

  4. Reixach, N., Deechongkit, S., Jiang, X., Kelly, J.W. & Buxbaum, J.N. Tissue damage in the amyloidoses: Transthyretin monomers and nonnative oligomers are the major cytotoxic species in tissue culture. Proc. Natl. Acad. Sci. USA 101, 2817–2822 (2004).

    Article  CAS  Google Scholar 

  5. Lashuel, H.A. et al. Alpha-synuclein, especially the Parkinson's disease-associated mutants, forms pore-like annular and tubular protofibrils. J. Mol. Biol. 322, 1089–1102 (2002).

    Article  CAS  Google Scholar 

  6. Chandra, S., Gallardo, G., Fernandez-Chacon, R., Schluter, O.M. & Sudhof, T.C. Alpha-synuclein cooperates with CSPalpha in preventing neurodegeneration. Cell 123, 383–396 (2005).

    Article  CAS  Google Scholar 

  7. Bertoncini, C.W. et al. Release of long-range tertiary interactions potentiates aggregation of natively unstructured alpha-synuclein. Proc. Natl. Acad. Sci. USA 102, 1430–1435 (2005).

    Article  CAS  Google Scholar 

  8. Uversky, V.N., Li, J. & Fink, A.L. Metal-triggered structural transformations, aggregation, and fibrillation of human alpha-synuclein. A possible molecular NK between Parkinson's disease and heavy metal exposure. J. Biol. Chem. 276, 44284–44296 (2001).

    Article  CAS  Google Scholar 

  9. Hoyer, W. et al. Dependence of alpha-synuclein aggregate morphology on solution conditions. J. Mol. Biol. 322, 383–393 (2002).

    Article  CAS  Google Scholar 

  10. Stefanova, N., Klimaschewski, L., Poewe, W., Wenning, G.K. & Reindl, M. Glial cell death induced by overexpression of alpha-synuclein. J. Neurosci. Res. 65, 432–438 (2001).

    Article  CAS  Google Scholar 

  11. Sherer, T.B. et al. An in vitro model of Parkinson's disease: linking mitochondrial impairment to altered alpha-synuclein metabolism and oxidative damage. J. Neurosci. 22, 7006–7015 (2002).

    Article  CAS  Google Scholar 

  12. Vila, M. et al. Alpha-synuclein up-regulation in substantia nigra dopaminergic neurons following administration of the parkinsonian toxin MPTP. J. Neurochem. 74, 721–729 (2000).

    Article  CAS  Google Scholar 

  13. Lee, H.J., Shin, S.Y., Choi, C., Lee, Y.H. & Lee, S.J. Formation and removal of alpha-synuclein aggregates in cells exposed to mitochondrial inhibitors. J. Biol. Chem. 277, 5411–5417 (2002).

    Article  CAS  Google Scholar 

  14. Bence, N.F., Sampat, R.M. & Kopito, R.R. Impairment of the ubiquitin-proteasome system by protein aggregation. Science 292, 1552–1555 (2001).

    Article  CAS  Google Scholar 

  15. Kim, S., Nollen, E.A., Kitagawa, K., Bindokas, V.P. & Morimoto, R.I. Polyglutamine protein aggregates are dynamic. Nat. Cell Biol. 4, 826–831 (2002).

    Article  CAS  Google Scholar 

  16. Outeiro, T.F. & Lindquist, S. Yeast cells provide insight into alpha-synuclein biology and pathobiology. Science 302, 1772–1775 (2003).

    Article  CAS  Google Scholar 

  17. Fortin, D.L. et al. Neural activity controls the synaptic accumulation of alpha-synuclein. J. Neurosci. 25, 10913–10921 (2005).

    Article  CAS  Google Scholar 

  18. Pandey, N., Schmidt, R.E. & Galvin, J.E. The alpha-synuclein mutation E46K promotes aggregation in cultured cells. Exp. Neurol. 197, 515–520 (2006).

    Article  CAS  Google Scholar 

  19. McLean, P.J., Kawamata, H. & Hyman, B.T. Alpha-synuclein-enhanced green fluorescent protein fusion proteins form proteasome sensitive inclusions in primary neurons. Neuroscience 104, 901–912 (2001).

    Article  CAS  Google Scholar 

  20. Adams, S.R. et al. New biarsenical ligands and tetracysteine motifs for protein labeling in vitro and in vivo: synthesis and biological applications. J. Am. Chem. Soc. 124, 6063–6076 (2002).

    Article  CAS  Google Scholar 

  21. Gaietta, G. et al. Multicolor and electron microscopic imaging of connexin trafficking. Science 296, 503–507 (2002).

    Article  CAS  Google Scholar 

  22. Arhel, N. et al. Quantitative four-dimensional tracking of cytoplasmic and nuclear HIV-1 complexes. Nat. Methods 3, 817–824 (2006).

    Article  CAS  Google Scholar 

  23. Martin, B.R., Giepmans, B.N., Adams, S.R. & Tsien, R.Y. Mammalian cell-based optimization of the biarsenical-binding tetracysteine motif for improved fluorescence and affinity. Nat. Biotechnol. 23, 1308–1314 (2005).

    Article  CAS  Google Scholar 

  24. Croisier, E. et al. Comparative study of commercially available anti-alpha-synuclein antibodies. Neuropathol. Appl. Neurobiol. 32, 351–356 (2006).

    Article  CAS  Google Scholar 

  25. Heise, H. et al. Molecular-level secondary structure, polymorphism, and dynamics of full-length alpha-synuclein fibrils studied by solid-state NMR. Proc. Natl. Acad. Sci. USA 102, 15871–15876 (2005).

    Article  CAS  Google Scholar 

  26. Malik, Z. et al. Fourier transform multipixel spectroscopy and spectral imaging for quantitative cytology. J. Microsc. 182, 133–140 (1996).

    Article  CAS  Google Scholar 

  27. Fernandez, C.O. et al. NMR of alpha-synuclein-polyamine complexes elucidates the mechanism and kinetics of induced aggregation. EMBO J. 23, 2039–2046 (2004).

    Article  CAS  Google Scholar 

  28. Matsuzaki, M. et al. Histochemical features of stress-induced aggregates in alpha-synuclein overexpressing cells. Brain Res. 1004, 83–90 (2004).

    Article  CAS  Google Scholar 

  29. Jares-Erijman, E.A. & Jovin, T.M. Imaging molecular interactions in living cells by FRET microscopy. Curr. Opin. Chem. Biol. 10, 409–416 (2006).

    Article  CAS  Google Scholar 

  30. Egner, A. & Hell, S.W. Fluorescence microscopy with super-resolved optical sections. Trends Cell Biol. 15, 207–215 (2005).

    Article  CAS  Google Scholar 

  31. Spagnuolo, C.C., Vermeij, R.J. & Jares-Erijman, E.A. Improved photostable FRET-competent biarsenical-tetracysteine probes based on fluorinated fluoresceins. J. Am. Chem. Soc. 128, 12040–12041 (2006).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank R. Rasia for NMR acquisition and analysis, G. Heim for the AFM images, K. Lidke and B. Rieger for the DIPimage routines, J. Post for fruitful discussions, and R. Vermeij for the synthesis of FlAsH and ReAsH. E.A.J.-E. thanks the Volkswagen Foundation (Grants I/79986, I/179987), Max Planck Society (Partner Group grant), ANPCyT, CONICET and UBACyT for financial support. This work was supported by the Deutsche Forschungsgemeinschaft (DFG) Center for Molecular Physiology of the Brain (DFG CMPB) in Göttingen (grant to T.M.J.) and by the Max Planck Society. M.J.R. and C.W.B. were recipients of fellowships from the DFG CMPB at the time of this work.

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Authors and Affiliations

  1. Department of Molecular Biology, Max Planck Institute for Biophysical Chemistry, Göttingen, 37077, Germany

    María J Roberti, Carlos W Bertoncini, Reinhard Klement & Thomas M Jovin

  2. Departamento de Química Orgánica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires, 1428, Argentina

    María J Roberti & Elizabeth A Jares-Erijman

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  1. María J Roberti
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  2. Carlos W Bertoncini
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Correspondence to Elizabeth A Jares-Erijman or Thomas M Jovin.

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Supplementary information

Supplementary Fig. 1

NMR spectroscopy of 15N-labeled alpha;-synuclein-C4-FlAsH and α-synuclein. (PDF 2126 kb)

Supplementary Fig. 2

Size exclusion chromatography of samples containing α-synuclein, α-synuclein-C4, α-synuclein-C4-FlAsH, and α-synuclein-C4-ReAsH. (PDF 417 kb)

Supplementary Fig. 3

ThioS staining of HeLa cells microinjected with 200 μM α-synuclein-C4-ReAsH. (PDF 2562 kb)

Supplementary Video 1

Confocal imaging 3D reconstruction of a SH-SY5Y cell, with α-synuclein-C4-FlAsH aggregates distributed along the cytoplasm, and α-synuclein-C4-FlAsH also present in the nuclear region. (MOV 1848 kb)

Supplementary Methods (PDF 92 kb)

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Roberti, M., Bertoncini, C., Klement, R. et al. Fluorescence imaging of amyloid formation in living cells by a functional, tetracysteine-tagged α-synuclein. Nat Methods 4, 345–351 (2007). https://doi.org/10.1038/nmeth1026

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