Dissecting aggregation and seeding dynamics of α-Syn polymorphs using the phasor approach to FLIM

Tittelmeier, Jessica; Druffel-Augustin, Silke; Alik, Ania; Melki, Ronald; Nussbaum-Krammer, Carmen

doi:10.1038/s42003-022-04289-6

Cited by 6 publications

(3 citation statements)

References 69 publications

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“…We tested the effects of the generated polymorphs on cells. As reported 24,41 , fibrils strongly induce aggregation in treated cells compared to ribbons, however, we did not notice differences in toxicity between the polymorphs, based on measuring the levels of lactate dehydrogenase at days 4, 7 and 10. There was no significant difference in cytotoxicity between HS/LS treated cells and nontreated cells, indicating the HS/LS did not induce cytotoxicity on D4/7/10.…”

Section: Cellular Effects Of Fibrils and Ribbonssupporting

confidence: 59%

Single molecule fingerprinting reveals different growth mechanisms in seed amplification assays for different polymorphs of αSynuclein fibrils

Lau,

Tang,

Kenche

et al. 2024

Preprint

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Alpha-synuclein (aSyn) aggregates, detected in the biofluids of patients with Parkinsons disease, have the ability to catalyze their own aggregation, leading to an increase in the number and size of aggregates. This self-templated amplification is used by newly developed assays to diagnose Parkinsons disease and turned the presence of aSyn aggregates into a biomarker of the disease. It has become evident that aSyn can form fibrils with slightly different structures, called strains or polymorphs, but little is known about their differential reactivity in diagnostic assays. Here we compared the properties of two well-described aSyn polymorphs. Using single molecule techniques, we observed that one of the polymorphs had an increased tendency to undergo secondary nucleation and we showed that this could explain the differences of reactivity observed in in vitro seed amplification assay and cellular assays. Simulations and high-resolution microscopy suggest that a 100-fold difference in apparent rate of growth can be generated by a surprisingly low number of secondary nucleation points (1 every 2,000 monomers added by elongation). When both strains are present in the same seeded reaction, secondary nucleation displaces proportions dramatically and causes a single strain to dominate the reaction as the major end-product.

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Section: Cellular Effects Of Fibrils and Ribbonssupporting

confidence: 59%

Single molecule fingerprinting reveals different growth mechanisms in seed amplification assays for different polymorphs of αSynuclein fibrils

Lau,

Tang,

Kenche

et al. 2024

Preprint

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“…In our case, the fluorescence lifetime decreases in correlation with the diffusion coefficient for G3BP1 both in the WT and in the mutated cell conditions, indicating a rearrangement of the dye molecules during the SG formation. A decrease in fluorescence lifetime has already been reported during amyloid fibril formation 45 . The fluorescence lifetime reaches a plateau at a value around 2.55 ns, which corresponds well to the fluorescence lifetime value of eGFP (between 2.4 and 2.6 ns 46 ), suggesting that there is no influence on the fluorescence lifetime before oxidative stress when G3BP1 is diffusing almost freely in the cytoplasm.…”

Section: Resultsmentioning

confidence: 58%

Single-photon microscopy to study biomolecular condensates

Perego,

Zappone,

Castagnetti

et al. 2023

Nat Commun

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Biomolecular condensates serve as membrane-less compartments within cells, concentrating proteins and nucleic acids to facilitate precise spatial and temporal orchestration of various biological processes. The diversity of these processes and the substantial variability in condensate characteristics present a formidable challenge for quantifying their molecular dynamics, surpassing the capabilities of conventional microscopy. Here, we show that our single-photon microscope provides a comprehensive live-cell spectroscopy and imaging framework for investigating biomolecular condensation. Leveraging a single-photon detector array, single-photon microscopy enhances the potential of quantitative confocal microscopy by providing access to fluorescence signals at the single-photon level. Our platform incorporates photon spatiotemporal tagging, which allowed us to perform time-lapse super-resolved imaging for molecular sub-diffraction environment organization with simultaneous monitoring of molecular mobility, interactions, and nano-environment properties through fluorescence lifetime fluctuation spectroscopy. This integrated correlative study reveals the dynamics and interactions of RNA-binding proteins involved in forming stress granules, a specific type of biomolecular condensates, across a wide range of spatial and temporal scales. Our versatile framework opens up avenues for exploring a broad spectrum of biomolecular processes beyond the formation of membrane-less organelles.

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Section: Fluorescence Lifetime Reveals Protein Confinementmentioning

confidence: 58%

Content-enriched fluorescence lifetime fluctuation spectroscopy to study bio-molecular condensate formation

Perego¹,

Zappone²,

Castagnetti³

et al. 2023

Preprint

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Quantitative fluorescence microscopy is experiencing an important revolution thanks to single-photon array detectors. These detectors provide users with so far inaccessible specimen information: The distribution of the specimen's fluorescence emission at single-photon level and high spatiotemporal sampling. In laser-scanning microscopy, this photon-resolved measurement has enabled robust fluorescence lifetime imaging at sub-diffraction spatial resolution, thus opening new perspectives for structural and functional imaging. Despite these significant advances in imaging, studying the time evolution of biological processes remains a considerable challenge. Here we present a comprehensive framework of live-cell spectroscopy methodologies -- compatible with imaging -- to investigate bio-molecular processes at various spatiotemporal scales. We use photon-resolved spatial and temporal measurements granted by a single-photon array detector to boost the information content of a unified fluorescence fluctuation spectroscopy and fluorescence lifetime experiment. To demonstrate the potential of this approach, we investigate the phase transition of liquid-like condensates during oxidative stress inside living cells. These condensates are generally found in several cellular processes and exhibit substantial variations in molecular composition, size, and kinetics, posing a significant challenge for quantifying their underlying molecular dynamics. This study demonstrates how the proposed approach reveals the mutual dynamics of different RNA-binding proteins involved in the stress granules formation -- inaccessible to imaging alone. We observe condensate formation by performing time-lapse super-resolved imaging of the cellular macro-environment while simultaneously monitoring the molecular mobility, the sub-diffraction environment organization, interactions, and nano-environment properties through fluorescence lifetime fluctuation spectroscopy. We are confident that our framework offers a versatile toolkit for investigating a broad range of bio-molecular processes -- not limited to liquid-liquid phase transition -- and we anticipate their widespread application in future life-science research.

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Dissecting aggregation and seeding dynamics of α-Syn polymorphs using the phasor approach to FLIM

Cited by 6 publications

References 69 publications

Single molecule fingerprinting reveals different growth mechanisms in seed amplification assays for different polymorphs of αSynuclein fibrils

Single molecule fingerprinting reveals different growth mechanisms in seed amplification assays for different polymorphs of αSynuclein fibrils

Single-photon microscopy to study biomolecular condensates

Content-enriched fluorescence lifetime fluctuation spectroscopy to study bio-molecular condensate formation

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