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.
Highlights
Protein misfolding and aggregation has been linked to many neurodegenerative diseases including Alzheimer’s disease (AD), Parkinson’s disease (PD), and Huntington’s disease (HD)
Various approaches have been employed to reveal the morphology of individual amyloid fibrils or clusters directly, for instance, electron microscopy (EM),[2,5,8,12,13] atomic-force microscopy (AFM),[11,12,14−16] and super-resolution fluorescence microscopy.[17−22] such methods are slow to perform and can require elaborate sample preparation protocols, making them impractical to perform for screening applications or the analysis of large data sets
We demonstrate that the fluorescence lifetime of synthetic dyes covalently attached to amyloid proteins informs on the density and morphological properties of amyloid clusters, and conclude that this is affected by fluorescence self-quenching of the dye molecules upon aggregation
Summary
These phasors almost overlap because the local dye densities in the amyloid clusters are similar Taken together, these results provide firm evidence that self-quenching is the main mechanism by which the lifetime sensor reports on aggregation. We note that the SROS-SIM experiments revealed that for incubation times less than 30 h, aggregates consisted mostly of low density clusters of loosely assembled amyloid fibrils This suggests that the lifetime sensor does not sensitively report on the conversion of labeled peptide into single amyloid fibrils at labeling ratios below 30% for K18. Both aggregation rate constant and the average sink formation time did not vary significantly for different labeling ratios, providing firm evidence that labeling ratios up to 30% do not significantly influence the aggregation kinetics of the K18 peptide
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