Abstract
The self-assembly of normally soluble proteins into fibrillar amyloid structures is associated with a range of neurodegenerative disorders, such as Parkinson’s and Alzheimer’s diseases. In the present study, we show that specific events in the kinetics of the complex, multistep aggregation process of one such protein, α-synuclein, whose aggregation is a characteristic hallmark of Parkinson’s disease, can be followed at the molecular level using optical super-resolution microscopy. We have explored in particular the elongation of preformed α-synuclein fibrils; using two-color single-molecule localization microscopy we are able to provide conclusive evidence that the elongation proceeds from both ends of the fibril seeds. Furthermore, the technique reveals a large heterogeneity in the growth rates of individual fibrils; some fibrils exhibit no detectable growth, whereas others extend to more than ten times their original length within hours. These large variations in the growth kinetics can be attributed to fibril structural polymorphism. Our technique offers new capabilities in the study of amyloid growth dynamics at the molecular level and is readily translated to the study of the self-assembly of other nanostructures.
Highlights
Considerable insight has been gained from both experimental and theoretical studies into the events that contribute to the overall conversion of soluble proteins to their aggregated states, much remains to be ascertained about the individual molecular steps involved in such processes.[3−5] Of particular importance in this context is the ability to probe at high resolution the various structural mechanisms involved in the initiation and growth of different fibrillar states
Fibril growth kinetics are most commonly studied in vitro, using assays based on the enhancement of fluorescence of dyes, such as Thioflavin-T (ThT) or by using surface-based biosensing assays,[4] both of which measure the growth of large numbers of aggregates
Studies in which individual aggregates can be visualized using techniques such as TIRF-ThT6−9 and in situ atomic force microscopy (AFM)[10−15] shed new light into the complex nature of the individual molecular steps involved in the kinetics of aggregation reactions, including the way in which fibrils can elongate by addition of further soluble molecular species, providing information that is not available from ensemble measurements
Summary
Reconstruction Microscopy (dSTORM) as a powerful means of elucidating the nature and the kinetics of the growth of individual fibrils in vitro, through its ability to distinguish by color those regions of fibrils formed at different stages of the reaction, providing details on the molecular level of the process. De novo formation of α-synuclein amyloid fibrils via primary nucleation occurs at a negligible rate relative to fibril growth, confirming elongation of seeds as the only significant process during the aggregation reactions presented here. In order to investigate possible variations in growth kinetics for individual fibrils, we analyzed multiple dSTORM images of growing fibrils, recorded at different time points during the aggregation reaction and in different regions of the glass coverslips
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