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


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.


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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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Correspondence to Elizabeth A Jares-Erijman or Thomas M Jovin.

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The authors declare no competing financial interests.

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)


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 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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