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Article Dans Une Revue Scientific Reports Année : 2017

Polarized super-resolution structural imaging inside amyloid fibrils using Thioflavine T

Cesar Valades Cruz
Sophie Brasselet
Julien Savatier
  • Fonction : Auteur

Résumé

Thioflavin T (ThT) is standardly used as a fluorescent marker to detect aggregation of amyloid fibrils by conventional fluorescence microscopy, including polarization resolved imaging that brings information on the orientational order of the fibrils. These techniques are however diffraction limited and cannot provide fine structural details at the fibrils scales of 10–100 nm, which lie beyond the diffraction limit. In this work, we evaluate the capacity of ThT to photoswitch when bound to insulin amyloids by adjusting the redox properties of its environment. We demonstrate that on-off duty cycles, intensity and photostability of the ThT fluorescence emission under adequate buffer conditions permit stochastic super-resolution imaging with a localization precision close to 20 nm. We show moreover that signal to noise conditions allow polarized orientational imaging of single ThT molecules, which reveals ultra-structure signatures related to protofilaments twisting within amyloid fibrils. Amyloid aggregates, which originate from protein misfolding as a starting point for aggregation and plaque formations , are known to be at the origin of crucial processes responsible for neurodegenerative diseases. Thioflavin T (ThT), a small benzothiazole fluorescent compound, is commonly used for imaging amyloid fibrils by exploiting its capacity to generate high fluorescence emission when bound to amyloid in specific sites 1. Fluorescence-based optical imaging techniques have used ThT to study the formation and growth processes of amyloid fibrils 2,3 , as well as for the diagnosis of plaque formation in diseases related to protein disorder 4,5. Moreover, fluorescence ani-sotropy and fluorescence polarized microscopy have evidenced the high orientational order of ThT in amyloids and its ability to report indirectly the organization and structure of fibrils in vitro 6–8. Although fluorescence optical imaging is a powerful tool to gain insight into the formation and the aggrega-tion of amyloids, it is still limited to spatial scales of the order of the optical diffraction limit, e.g. about 200 nm. This resolution scale is however inappropriate for monitoring the supra-molecular architecture of the amyloid aggregates, which size is around 10–100 nm from electron and atomic force microscopies 9,10. Individual filaments made of repeated β-sheets are known in particular to arrange into protofilaments that twist around each other in a helical ribbon, which pitch size varies depending on the number of filaments involved 8. Accessing such scales in non-invasive and ideally in-vivo conditions is essential for understanding the molecular mechanisms responsible for the formation of amyloids and their functional consequences in neurodegenerative diseases. Super resolution imaging based on single molecule localization has recently brought a determining step to overcome this limitation, using the stochastic nature of emission from isolated molecules localized with nano-metric accuracy 11,12. Direct Stochastic Optical Reconstruction Microscopy (dSTORM) uses in particular the capacity of fluorophores to photoswitch from dark to bright fluorescence states, at a rate accessible in imaging conditions 13. This method has been used to study the high resolution morphology and aggregates formation of different types of amyloids such as amyloid beta (Aβ) 14,15 ,α-synuclein 16–18 and Huntingtin protein 19–21 , in both in vivo conditions and in fixed cells, with a resolution down to 20 nm. These approaches however have so far relied on either immunolabeling of the amyloid fibrils or the covalent binding of monomeric proteins by fluorescent organic dye. Immunolabeling reports only apparent fibrils organization due to the non-negligible size of the
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Dates et versions

hal-01643496 , version 1 (12-04-2018)

Identifiants

Citer

Haitham Ahmed H A Shaban, Cesar Valades Cruz, Sophie Brasselet, Julien Savatier. Polarized super-resolution structural imaging inside amyloid fibrils using Thioflavine T. Scientific Reports, 2017, 7 (1), pp.12482 - 12482. ⟨10.1038/s41598-017-12864-9⟩. ⟨hal-01643496⟩
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