title: Fluorescence imaging of amyloid formation in living cells by a functional, tetracysteine-tagged alpha-synuclein.
abstract: Alpha-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 alpha-synuclein (alpha-synuclein-C4) bearing a tetracysteine target for fluorogenic biarsenical compounds. The biophysical, biochemical and aggregation properties of alpha-synuclein-C4 matched those of the wild-type protein in vitro and in living cells. We observed aggregation of alpha-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 alpha-synuclein-C4 molecules. Alpha-synuclein-C4 offers the means for directly probing amyloid formation and interactions of alpha-synuclein with other proteins in living cells, the response to cellular stress and screening drugs for Parkinson disease.
authors.affiliation: Department of Molecular Biology, Max Planck Institute for Biophysical Chemistry, 37077 Göttingen, Germany.
authors.forename: María J
authors.initials: MJ
authors.lastname: Roberti
authors.forename: Carlos W
authors.initials: CW
authors.lastname: Bertoncini
authors.forename: Reinhard
authors.initials: R
authors.lastname: Klement
authors.forename: Elizabeth A
authors.initials: EA
authors.lastname: Jares-Erijman
authors.forename: Thomas M
authors.initials: TM
authors.lastname: Jovin
mesh_terms: D000682:Amyloid / Q000032:analysis*; D015374:Biosensing Techniques / Q000379:methods*; D045744:Cell Line, Tumor; D003545:Cysteine / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000737:chemistry*; D005822:Genetic Vectors; D006801:Humans; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D018384:Oxidative Stress; D017382:Reactive Oxygen Species / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D051844:alpha-Synuclein / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D000682:Amyloid; D005456:Fluorescent Dyes; D017382:Reactive Oxygen Species; D011993:Recombinant Fusion Proteins; D051844:alpha-Synuclein; D003545:Cysteine