title: A FRET sensor for non-invasive imaging of amyloid formation in vivo.
abstract: Misfolding and aggregation of amyloidogenic polypeptides lie at the root of many neurodegenerative diseases. Whilst protein aggregation can be readily studied in vitro by established biophysical techniques, direct observation of the nature and kinetics of aggregation processes taking place in vivo is much more challenging. We describe here, however, a Förster resonance energy transfer sensor that permits the aggregation kinetics of amyloidogenic proteins to be quantified in living systems by exploiting our observation that amyloid assemblies can act as energy acceptors for variants of fluorescent proteins. The observed lifetime reduction can be attributed to fluorescence energy transfer to intrinsic energy states associated with the growing amyloid species. Indeed, for a-synuclein, a protein whose aggregation is linked to Parkinson's disease, we have used this sensor to follow the kinetics of the self-association reactions taking place in vitro and in vivo and to reveal the nature of the ensuing aggregated species. Experiments were conducted in vitro, in cells in culture and in living Caenorhabditis elegans. For the latter the readout correlates directly with the appearance of a toxic phenotype. The ability to measure the appearance and development of pathogenic amyloid species in a living animal and the ability to relate such data to similar processes observed in vitro provides a powerful new tool in the study of the pathology of the family of misfolding disorders. Our study confirms the importance of the molecular environment in which aggregation reactions take place, highlighting similarities as well as differences between the processes occurring in vitro and in vivo, and their significance for defining the molecular physiology of the diseases with which they are associated.
authors.affiliation: Department of Chemical Engineering and Biotechnology, University of Cambridge, Pembroke Street, Cambridge CB2 3RA (U.K.).
authors.forename: Gabriele S
authors.initials: GS
authors.lastname: Kaminski Schierle
authors.affiliation: Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge, CB2 1EW (U.K.).|Laboratory of Molecular Biophysics, Institute for Research in Biomedicine, Baldiri Reixac 10-12, 08028, Barcelona (Spain).
authors.forename: Carlos W
authors.initials: CW
authors.lastname: Bertoncini
authors.affiliation: Department of Chemical Engineering and Biotechnology, University of Cambridge, Pembroke Street, Cambridge CB2 3RA (U.K.).
authors.forename: Fiona T S
authors.initials: FTS
authors.lastname: Chan
authors.affiliation: Department of Genetics, University Medical Centre Groningen and University of Groningen 9700 RB Groningen (The Netherlands).
authors.forename: Annemieke T
authors.initials: AT
authors.lastname: van der Goot
authors.affiliation: Physikalische Chemie I, Fakultät für Chemie Universität Bielefeld, Universitätsstr. 25, 33615, Bielefeld (Germany).
authors.forename: Stefanie
authors.initials: S
authors.lastname: Schwedler
authors.affiliation: Department of Physiology, Development and Neuroscience University of Cambridge, Downing Street, Cambridge CB2 3DY (U.K.).
authors.forename: Jeremy
authors.initials: J
authors.lastname: Skepper
authors.affiliation: Department of Chemical Engineering and Biotechnology, University of Cambridge, Pembroke Street, Cambridge CB2 3RA (U.K.).
authors.forename: Simon
authors.initials: S
authors.lastname: Schlachter
authors.affiliation: Department of Genetics, University Medical Centre Groningen and University of Groningen 9700 RB Groningen (The Netherlands).
authors.forename: Tjakko
authors.initials: T
authors.lastname: van Ham
authors.affiliation: Department of Chemical Engineering and Biotechnology, University of Cambridge, Pembroke Street, Cambridge CB2 3RA (U.K.).
authors.forename: Alessandro
authors.initials: A
authors.lastname: Esposito
authors.affiliation: Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge, CB2 1EW (U.K.).
authors.forename: Janet R
authors.initials: JR
authors.lastname: Kumita
authors.affiliation: Department of Genetics, University Medical Centre Groningen and University of Groningen 9700 RB Groningen (The Netherlands).
authors.forename: Ellen A A
authors.initials: EAA
authors.lastname: Nollen
authors.affiliation: Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge, CB2 1EW (U.K.).
authors.forename: Christopher M
authors.initials: CM
authors.lastname: Dobson
authors.affiliation: Department of Chemical Engineering and Biotechnology, University of Cambridge, Pembroke Street, Cambridge CB2 3RA (U.K.).|Friedrich-Alexander University of Erlangen Nürnberg 91052 Erlangen (Germany).
authors.forename: Clemens F
authors.initials: CF
authors.lastname: Kaminski
mesh_terms: D000682:Amyloid / Q000737:chemistry*; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017173:Caenorhabditis elegans / Q000378:metabolism; D045744:Cell Line, Tumor; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D007700:Kinetics; D008164:Luminescent Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008856:Microscopy, Fluorescence; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D000682:Amyloid; D001426:Bacterial Proteins; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; C054562:yellow fluorescent protein, Bacteria