Fluorescence imaging of amyloid formation in living cells by a functional, tetracysteine-tagged alpha-synuclein.
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.
Roberti
María J
MJ
Department of Molecular Biology, Max Planck Institute for Biophysical Chemistry, 37077 Göttingen, Germany.
Bertoncini
Carlos W
CW
Klement
Reinhard
R
Jares-Erijman
Elizabeth A
EA
Jovin
Thomas M
TM
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
D000682:Amyloid; D005456:Fluorescent Dyes; D017382:Reactive Oxygen Species; D011993:Recombinant Fusion Proteins; D051844:alpha-Synuclein; D003545:Cysteine