title: Fluorescence Self-Quenching from Reporter Dyes Informs on the Structural Properties of Amyloid Clusters Formed in Vitro and in Cells.
abstract: The characterization of the aggregation kinetics of protein amyloids and the structural properties of the ensuing aggregates are vital in the study of the pathogenesis of many neurodegenerative diseases and the discovery of therapeutic targets. In this article, we show that the fluorescence lifetime of synthetic dyes covalently attached to amyloid proteins informs on the structural properties of amyloid clusters formed both in vitro and in cells. We demonstrate that the mechanism behind such a "lifetime sensor" of protein aggregation is based on fluorescence self-quenching and that it offers a good dynamic range to report on various stages of aggregation without significantly perturbing the process under investigation. We show that the sensor informs on the structural density of amyloid clusters in a high-throughput and quantitative manner and in these aspects the sensor outperforms super-resolution imaging techniques. We demonstrate the power and speed of the method, offering capabilities, for example, in therapeutic screenings that monitor biological self-assembly. We investigate the mechanism and advantages of the lifetime sensor in studies of the K18 protein fragment of the Alzheimer's disease related protein tau and its amyloid aggregates formed in vitro. Finally, we demonstrate the sensor in the study of aggregates of polyglutamine protein, a model used in studies related to Huntington's disease, by performing correlative fluorescence lifetime imaging microscopy and structured-illumination microscopy experiments in cells.
authors.affiliation: Department of Chemical Engineering and Biotechnology, University of Cambridge , Pembroke Street, Cambridge CB2 3RA, United Kingdom.
authors.forename: WeiYue
authors.identifier: 0000-0001-5004-4376
authors.initials: W
authors.lastname: Chen
authors.affiliation: Department of Chemical Engineering and Biotechnology, University of Cambridge , Pembroke Street, Cambridge CB2 3RA, United Kingdom.
authors.forename: Laurence J
authors.identifier: 0000-0002-0269-7548
authors.initials: LJ
authors.lastname: Young
authors.affiliation: Department of Chemical Engineering and Biotechnology, University of Cambridge , Pembroke Street, Cambridge CB2 3RA, United Kingdom.
authors.forename: Meng
authors.initials: M
authors.lastname: Lu
authors.affiliation: Department of Chemical Engineering and Biotechnology, University of Cambridge , Pembroke Street, Cambridge CB2 3RA, United Kingdom.
authors.forename: Alessio
authors.initials: A
authors.lastname: Zaccone
authors.affiliation: Department of Chemical Engineering and Biotechnology, University of Cambridge , Pembroke Street, Cambridge CB2 3RA, United Kingdom.
authors.forename: Florian
authors.initials: F
authors.lastname: Ströhl
authors.affiliation: Department of Chemical Engineering and Biotechnology, University of Cambridge , Pembroke Street, Cambridge CB2 3RA, United Kingdom.
authors.forename: Na
authors.initials: N
authors.lastname: Yu
authors.affiliation: Department of Chemical Engineering and Biotechnology, University of Cambridge , Pembroke Street, Cambridge CB2 3RA, United Kingdom.
authors.forename: Gabriele S
authors.initials: GS
authors.lastname: Kaminski Schierle
authors.affiliation: Department of Chemical Engineering and Biotechnology, University of Cambridge , Pembroke Street, Cambridge CB2 3RA, United Kingdom.
authors.forename: Clemens F
authors.initials: CF
authors.lastname: Kaminski
mesh_terms: D000544:Alzheimer Disease / Q000000981:diagnostic imaging / Q000378:metabolism; D000682:Amyloid / Q000737:chemistry* / Q000378:metabolism*; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000737:chemistry; D057809:HEK293 Cells; D006801:Humans; D006816:Huntington Disease / Q000000981:diagnostic imaging / Q000378:metabolism; D007700:Kinetics; D061848:Optical Imaging; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D066329:Protein Aggregates; D011487:Protein Conformation
chemical_list: D000682:Amyloid; D005456:Fluorescent Dyes; D010455:Peptides; D066329:Protein Aggregates; C097188:polyglutamine
keywords: FLIM; SIM; Self-quenching; amyloid aggregation; super-resolution