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Mass photometric detection and quantification of nanoscale α-synuclein phase separation

Nature Chemistry volume 15, pages 1306–1316 (2023)Cite this article

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Abstract

Protein liquid–liquid phase separation can lead to disease-related amyloid fibril formation. The mechanisms of conversion of monomeric protein into condensate droplets and of the latter into fibrils remain elusive. Here, using mass photometry, we demonstrate that the Parkinson’s disease-related protein, α-synuclein, can form dynamic nanoscale clusters at physiologically relevant, sub-saturated concentrations. Nanoclusters nucleate in bulk solution and promote amyloid fibril formation of the dilute-phase monomers upon ageing. Their formation is instantaneous, even under conditions where macroscopic assemblies appear only after several days. The slow growth of the nanoclusters can be attributed to a kinetic barrier, probably due to an interfacial penalty from the charged C terminus of α-synuclein. Our findings reveal that α-synuclein phase separation occurs at much wider ranges of solution conditions than reported so far. Importantly, we establish mass photometry as a promising methodology to detect and quantify nanoscale precursors of phase separation. We also demonstrate its general applicability by probing the existence of nanoclusters of a non-amyloidogenic protein, Ddx4n1.

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Fig. 1: In vitro α-Syn phase separation.
Fig. 2: Characterization of α-Syn nanoclusters in mass photometry.
Fig. 3: Ageing of α-Syn nanoclusters into larger assemblies.
Fig. 4: Effect of C terminus on α-Syn LLPS and clustering.
Fig. 5: Nanoscale LLPS accounts for a large dense-phase fraction above Capp.
Fig. 6: Ddx4n1 nanocluster formation.

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Data availability

All the data supporting the findings of this study are available within the paper and the supplementary information. All numerical datasets are provided as source data files. All the data analysis was performed using published tools and packages and has been cited in the paper and supplementary information. No data have been excluded. The amino-acid sequences of the proteins/peptides used in this study can be found in the supplementary information. Source data are provided with this paper.

Code availability

RCF (×g) centrifugal acceleration related analysis was carried out using a Python script in Matplotlib v3.5.0. The code (https://doi.org/10.11583/DTU.22336624) is provided in the supplementary information.

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Acknowledgements

We thank N. Lorenzen for introducing us to mass photometry. We thank C. Galvagnion-Buell (Department of Drug Design and Pharmacology) at Copenhagen University for gifting us the Alexa488-140C-α-Syn protein. We thank K. Lindorff-Larsen at Copenhagen University for valuable feedback on our preprint and for sharing his ideas, which we experimentally test in this paper. The DTU bio-imaging core at DTU Bioengineering is acknowledged for confocal/fluorescence imaging and FRAP experiments. Funding from Novo Nordisk Foundation (grant no. NNF19OC0055625) for the infrastructure ‘Imaging microbial language in biocontrol (IMLiB)’ is acknowledged. DTU Nanolabs is acknowledged for TEM imaging. We thank L. K. Klausen and K. Mielec for technical assistance with α-Syn expression and purification. S.R. would like to acknowledge a Horizon MSCA individual postdoctoral (grant no. 110361) fellowship for funding. A.K.B. would like to acknowledge VILLUM FONDEN for financial support (grant no. 35823). T.O.M. and A.K.B. would like to acknowledge the Novo Nordisk Foundation (grant no. NNFSA170028392) and the Lundbeck Foundation (grant no. R400-2022-911) for funding. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.

Author information

Authors and Affiliations

  1. Department of Biotechnology and Biomedicine, Technical University of Denmark, Lyngby, Denmark

    Soumik Ray, Thomas O. Mason, Lars Boyens-Thiele, Azad Farzadfard, Jacob Aunstrup Larsen, Rasmus K. Norrild & Alexander K. Buell

  2. Novo Nordisk A/S, Novo Nordisk Park, Måløv, Denmark

    Nadin Jahnke

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Contributions

S.R., T.O.M., A.F., L.B.-T., J.A.L. and R.K.N. designed and performed the experiments and analysed data. N.J. analysed data and trained S.R. on the mass photometry instrument. T.O.M. generated microemulsion droplets and performed experiments. A.F. purified α-Syn oligomers and Crev α-Syn. L.B.-T. generated the codes. J.A.L. performed the Ddx4n1 LLPS experiments. R.K.N. expressed and purified Ddx4n1. A.K.B. acquired funding, conceived and supervised the study, designed experiments and analysed the data. S.R. and A.K.B. wrote the paper. S.R. prepared all schematics and illustrations. All authors commented on the paper.

Corresponding author

Correspondence to Alexander K. Buell.

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Extended data

Extended Data Fig. 1 LLPS regime of α-Syn inside microemulsion droplets.

a. (Left) Schematic of a 3-component microemulsion droplet maker (materials and methods). The inlets for protein, PEG and FC-40 + surfactant are marked in blue. The outlet for microemulsion droplet collection is marked in grey. The arrows indicate direction of flow. (Right) A representative bright-field (BF) microscopy image of the region where the individual components mix is shown. b. Representative fluorescence microscopy images of the spontaneous LLPS regime of α-Syn inside the microemulsion droplets. The blue dashed circles mark the boundary of the microemulsion droplets. The white triangular pointer and the enlarged inset (right) indicate spherical phase-separated α-Syn assemblies inside the microemulsion droplets. The experiment is repeated n = 2 independent times with similar observations. c. Comparison of LLPS regimes of α-Syn using the sealed well-plate method (left) and inside microemulsion droplets (right). The dashed box (left) represents the conditions which are replicated inside microemulsion droplets. The blue circles indicate LLPS and the red circles indicate no LLPS/soluble. PEG: PEG-8000 unless mentioned otherwise.

Extended Data Fig. 2 Effect of NaCl on α-Syn LLPS.

a. Representative fluorescence microscopy images of the spontaneous LLPS regime of α-Syn at 0 mM (left), 110 mM (middle) and 250 mM (right) NaCl concentrations. The experiments are performed in a sealed well-plate setup at 25 °C. The green rectangles indicate samples where macroscopic phase separation is observed. b. Representative fluorescence microscopy images of 200 μM α-Syn + 20% (w/v) PEG samples prepared in 20 mM sodium phosphate buffer, pH 7.4 at increasing (0–500 mM) NaCl concentrations (at 25 °C). Sticking/clumping of droplets without actual merging immediately after their formation is observed at 500 mM NaCl concentration due to altered material properties (more gel-like) (indicated with a white triangular pointer). Therefore, at ≥500 mM NaCl, the phase transition is no longer accurately described as liquid to liquid; but rather as a liquid to gel transition. The experiments (a-b) are performed n = 2 independent times with similar observations. c. (Left) Normalized fluorescence intensity profiles of α-Syn droplets post photobleaching at 200, 400 and 500 mM NaCl concentration are shown. Values represent mean±SD for n = 3 independent experiments. The downward arrow indicates increased solidity of the droplets with increasing NaCl concentration. (Right) Representative fluorescence microscopy images of droplets at 0 s, 10 s and 20 s after photobleaching for samples containing 200, 400 and 500 mM NaCl. The bleached droplets are indicated with white squares. d. Schematic depicting the effect of NaCl on spontaneous α-Syn phase separation. PEG: PEG-8000 unless mentioned otherwise.

Source data

Extended Data Fig. 3 Amyloid aggregation pathways in a phase-separated α-Syn solution.

a. (Left) Representative fluorescence microscopy images of 200 μM α-Syn + 20% (w/v) PEG samples labeled with 100 nM Alexa488-α-Syn (upper panel) and 50 μM ThT (lower panel) at 0, 48 and 72 h are shown. The inset is an enlarged image of the droplets at 0 h. (Right) Fluorescence microscopy images (thermal LUT) of a 48 h old α-Syn droplet at different times (0–40 s) post-bleaching showing no recovery of fluorescence. b. (Left) Fluorescence microscopy image of a 72 h old microemulsion droplet containing 200 μM α-Syn + 20% (w/v) PEG (250 mM NaCl) is shown. The sample contains 50 μM ThT as a reporter for aggregation. The red dashed circle indicates the boundary of the microemulsion droplet (Right) Fluorescence microscopy images of 240 h old microemulsion droplets containing 200 μM α-Syn + 20% (w/v) PEG (0 mM NaCl) are shown. The samples contain 100 nM Alexa488-α-Syn (upper panel) and 50 μM ThT (lower panel) as reporters. c. (Upper panel) Bulk ThT aggregation kinetics of 200 μM α-Syn + 20% (w/v) PEG (250 mM NaCl) under quiescent conditions at 25 °C. (Lower panel) The first phase (marked with a rectangle) is magnified for better visualization. d. ThT fluorescence intensity (background corrected) from microscopic measurements of individual phase-separated droplets. Values represent mean±SD calculated from 45 individual droplets (n = 2 independent experiments). The slight decrease of the intensity over the first 5 h arises from passive bleaching. Representative snapshots of a droplet at different time-points are shown (upper panel). e. Representative fluorescence microscopy images after addition of 200 nM Alexa488-α-Syn monomer to an aged (~48 h) LLPS sample is shown. Subsequent incubation (~72–96 h) of this sample leads to aggregation of the labeled monomer on the surface of unlabeled droplets (indicated with white triangular pointers). A fluorescence signal intensity plot across a droplet showing no partitioning of the Alexa488-α-Syn inside the solidified droplet at ~48 h. f. Bulk ThT aggregation kinetics of freshly made 200 μM α-Syn + 20% (w/v) PEG (250 mM NaCl) under quiescent conditions, at 25 °C and in presence of 10% (v/v) isolated droplets (aged for ~48 h) and 10% (v/v) 50 μM α-Syn amyloid fibrils (pre-made in solution). The inset shows a schematic of the experimental approach. g. ThT aggregation kinetics of 200 μM α-Syn + 20% (w/v) PEG in absence of NaCl. h. Schematic depicting the three amyloid aggregation processes that can concurrently occur in an α-Syn LLPS sample. i. Representative TEM images of 200 μM α-Syn + 20% (w/v) PEG (250 mM NaCl) at 0 h, 72 h, 96 h and 168 h. Fibrillar aggregates and typical amyloid fibrils are indicated with yellow triangular pointers. The inset shows presence of amyloid fibrils even in the dilute phase after ageing for 168 h. The experiments (a-i) are performed n = 2 independent times with similar results. PEG: PEG-8000 unless mentioned otherwise. Detailed results are provided in the supplementary information.

Source data

Extended Data Fig. 4 α-Syn nanoclusters are kinetically and thermodynamically less stable than oligomers.

(Left) MWapp histograms obtained from mass photometry measurements of 60 μM α-Syn + 20% (w/v) PEG before (blue) and after (red) 2x dilution are shown. MWapp histograms of the same sample after addition of 1 M urea (orange) are also reported. Note that addition of 1 M urea leads to a final concentration of 50 μM α-Syn + 17% (w/v) PEG which is still in the nanocluster regime (Supplementary Fig. 6). Therefore, disappearance of clusters indicated urea-induced destabilization/dissolution. Representative mass photometry images (ratiometric) of the nanoclusters before and after dilution are shown. (Right) MWapp histograms obtained with 1 μM α-Syn oligomers, purified by size exclusion chromatography, before dilution (green), after 2x dilution (grey) and after addition of 1 M urea (orange). Representative mass photometry images (ratiometric) of the oligomers before dilution, after dilution and after addition of 1 M urea are shown. All the experiments are performed in 20 mM sodium phosphate buffer, 250 mM NaCl, 5 mM KCl, pH 7.4 and at 25 °C. The observed persistence of oligomers even after urea treatment is in stark contrast to the dynamic nature of the nanoclusters we describe here and therefore we believe that the clusters do genuinely represent a hitherto unseen assembly state of α-Syn. The experiments are performed n = 2 independent times with similar results. PEG: PEG-8000 unless mentioned otherwise.

Source data

Supplementary information

Supplementary Information

Materials and Methods, Results, Figs. 1–22, Tables 1–4, video legends 1–8, amino-acid sequences for proteins used in this study, code used in this study, two uncropped SDS–PAGE gels (shown in Supplementary Fig. 22) as additional supplementary data.

Reporting Summary

Supplementary Video 1

Phase-separated α-Syn droplets inside microemulsion droplets: video showing micrometre-scale α-Syn phase-separated assemblies inside a microemulsion droplet. The sample (200 μM α-Syn + 20% (wt/vol) PEG-8000 in 20 mM sodium phosphate buffer, 250 mM NaCl, 5 mM KCl, pH 7.4) is labelled with 100 nM Alexa488-α-Syn for visualization under a fluorescence microscope. The video is captured for 46 s at an interval of 500 ms.

Supplementary Video 2

ThT fluorescence intensity from individual α-Syn droplet while ageing: video showing emergence of ThT fluorescence from α-Syn condensates sedimented on the surface of a sealed 96-well clear-bottom plate. The sample (200 μM α-Syn + 20% (wt/vol) PEG-8000 in 20 mM sodium phosphate buffer, 250 mM NaCl, 5 mM KCl, pH 7.4) is spiked with 50 μM ThT. The video is recorded over a timescale of ⁓20 h with an interval of 12 min and at 800-ms exposure.

Supplementary Video 3

Delayed α-Syn assemblies are morphologically distinct: video showing aggregation of 700 μM α-Syn in 20 mM PBS, pH 7.4 after 168 h of incubation in bulk solution followed by a few hours of incubation on a coverslip surface at 25 °C and imaged under BF microscopy. The video is captured with an interval of 10 min for ⁓10 h. The appearance of aggregates within 3–5 h of incubation in this sample was due to the presence of the glass surface promoting clumping of the already formed fibrils independent of phase separation.

Supplementary Video 4

Mass photometric detection of α-Syn nanoclusters below the apparent saturation concentration (Capp): a mass photometry measurement of 10 μM α-Syn + 20% (wt/vol) PEG-8000 (in 20 mM sodium phosphate buffer, 250 mM NaCl, 5 mM KCl, pH 7.4 at 25 °C) is shown at both native (upper panel) and ratiometric (lower panel) modes. Note that the smallest nanoclusters are only visible in the ratiometric mode. The video is captured with an interval of 0.01 s for 1 min.

Supplementary Video 5

α-Syn nanoclusters very close to the apparent saturation concentration (Capp): a mass photometric measurement showing large α-Syn nanoclusters (however, still below the detection limit of conventional microscopes) very close to the saturation concentration. For this experiment, 100 μM α-Syn in the presence of 15% (wt/vol) PEG-8000 is used (in 20 mM sodium phosphate buffer, 250 mM NaCl, 5 mM KCl, pH 7.4 at 25 °C). The video is captured in the native mode with an interval of 0.01 s for 1 min. No mass calculations could be performed from this particular dataset due to progressive saturation of the slide surface with high density protein assemblies.

Supplementary Video 6

Mass photometric measurement of α-Syn nanoclusters at relatively lower concentrations: a mass photometry measurement of 100 μM α-Syn + 5% (wt/vol) PEG-8000 (in 20 mM sodium phosphate buffer, 250 mM NaCl, 5 mM KCl, pH 7.4 at 25 °C) is shown at both native (left) and ratiometric (right) modes. Note that the smallest nanoclusters are only visible in the ratiometric mode. The video is captured with an interval of 0.01 s for 1 min. The apparent mass of the nanoclusters could be reliably quantified from this sample.

Supplementary Video 7

Mass photometric measurement of α-Syn nanoclusters at high concentrations: a mass photometry measurement of 300 μM α-Syn + 5% (wt/vol) PEG-8000 (in 20 mM sodium phosphate buffer, 250 mM NaCl, 5 mM KCl, pH 7.4 at 25 °C) is shown at both native (left) and ratiometric (right) mode. Note that the nanoclusters are visible both in native and ratiometric modes. The video is captured with an interval of 0.01 s for 1 min. The apparent mass of the nanoclusters could not be reliably quantified from this sample due to increased heterogeneity (a range of masses is given).

Supplementary Video 8

Surface wetting by α-Syn nanoclusters as observed in mass photometry: a mass photometric measurement showing gradual sedimentation/surface wetting of α-Syn nanoclusters. For this experiment, 300 μM α-Syn in the presence of 5% (wt/vol) PEG-8000 (in 20 mM sodium phosphate buffer, 250 mM NaCl, 5 mM KCl, pH 7.4 at 25 °C) is used. The video is captured in the native channel with an interval of 0.01 s for 1 min. The video is captured after 2 min of nanocluster formation (by addition of PEG) to allow enough time for the nanoclusters to sediment.

Supplementary Data

Source data for graphs shown in Supplementary Figs. 1–22.

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Data underlying figures that include graphs.

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Source Data Extended Data Fig. 2

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Source Data Extended Data Fig. 3

Data underlying figures that include graphs.

Source Data Extended Data Fig. 4

Data underlying figures that include graphs.

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Ray, S., Mason, T.O., Boyens-Thiele, L. et al. Mass photometric detection and quantification of nanoscale α-synuclein phase separation. Nat. Chem. 15, 1306–1316 (2023). https://doi.org/10.1038/s41557-023-01244-8

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