Spatiotemporal modulations in heterotypic condensates of prion and α-synuclein control phase transitions and amyloid conversion

Abstract

Biomolecular condensation via liquid-liquid phase separation of proteins and nucleic acids is associated with a range of critical cellular functions and neurodegenerative diseases. Here, we demonstrate that complex coacervation of the prion protein and α-synuclein within narrow stoichiometry results in the formation of highly dynamic, reversible, thermo-responsive liquid droplets via domain-specific electrostatic interactions between the positively-charged intrinsically disordered N-terminal segment of prion and the acidic C-terminal tail of α-synuclein. The addition of RNA to these coacervates yields multiphasic, vesicle-like, hollow condensates. Picosecond time-resolved measurements revealed the presence of transient electrostatic nanoclusters that are stable on the nanosecond timescale and can undergo breaking-and-making of interactions on slower timescales giving rise to a liquid-like behavior in the mesoscopic regime. The liquid-to-solid transition drives a rapid conversion of complex coacervates into heterotypic amyloids. Our results suggest that synergistic prion-α-synuclein interactions within condensates provide mechanistic underpinnings of their physiological role and overlapping neuropathological features.

Fig. 1. Heterotypic phase separation of α-Syn and PrP.

a An overlay of 576 conformations obtained from the ensemble structure of α-Syn (PED ID: PED00024e001) generated using PyMOL (Schrödinger, LLC, New York). b Domain architecture and the amino acid sequence of α-Syn. Positively and negatively charged amino acids are shown in blue and red, respectively. c An overlay of 20 conformations obtained from the NMR structure of human PrP (90–231) (PDB ID: 2LSB) generated using PyMOL. d Schematic representation of PrP (23–231) indicating the N-terminal disordered and the C-terminal globular domains. Positively and negatively charged amino acids are shown in blue and red, respectively. Prediction of the LLPS propensity using FuzDrop for e α-Syn and f PrP (23–231). g LLPS upon mixing of homogenous solutions of α-Syn (45 µM) and PrP (30 µM) at pH 6.8 at 37 °C. h, i Confocal images of mixed homogeneous phases of PrP and α-Syn and complex coacervates of PrP (red) and α-Syn (green) performed using Alexa-594-labeled PrP (Cys 31) and Alexa-488-labeled α-Syn (Cys 90) indicating their complete colocalization (yellow) within droplets (Scale bar: 10 µm). See Supplementary Movie 1 for droplet fusion events. j FRAP kinetics of multiple droplets (~1% Alexa-488-labeled protein) for PrP (red) and α-Syn (olive). The FRAP experiments were performed using Alexa-488-labeled α-Syn and PrP independently. The data represent mean ± s.d for n = 5 independent experiments. Source data are provided as a Source Data file. k Fluorescence images of droplets during FRAP measurements. PrP and α-Syn concentrations were 20 and 30 µM, respectively. See Methods for details. The imaging was performed thrice with similar observations (h, i, k).

Fig. 2. Charge neutralization drives heterotypic LLPS of α-Syn and PrP.

a Solution turbidity plot at fixed PrP concentration (20 µM) as a function of increasing α-Syn concentrations showing reentrant phase behavior. The data represent mean ± s.d. for n = 4 independent experiments. The solid line is for eye guide only. b Confocal microscopy images of Alexa-594-labeled PrP (red) and Alexa-488-labeled α-Syn (green) at different stoichiometries as indicated. Scale bar: 10 µm. c Electrophoretic mobility (µ) measurements reveal charge inversion with the increase in the α-Syn:PrP ratio. The data represent mean ± s.d. for n = 3 independent experiments (corresponding data points are shown using black dot plots). d Confocal images of Alexa-594-labeled PrP (20 µM) and Alexa-488-labeled α-Syn (30 µM) droplets with increasing salt concentrations. Scale bar: 10 µm. The imaging was performed thrice with similar observations (b, d). Source data are provided as a Source Data file.

Fig. 3. Domain-specific heterotypic interactions and the presence of electrostatic clusters within PrP-α-Syn condensates.

a Schematic representation of different α-Syn constructs used. Phase diagram for different α-Syn constructs at fixed PrP concentration (20 µM) as a function of increasing α-Syn concentrations created from mean turbidity values. b Schematic representation of different PrP constructs used. Phase diagram for different PrP constructs (20 µM) as a function of increasing α-Syn concentrations created from mean turbidity values. c Steady-state fluorescence anisotropy of single-Cys α-Syn labeled at different positions using F5M in the mixed monomer (olive) and droplets (cyan). The data represent mean ± s.d. for n = 6 independent experiments (corresponding data points are shown as black dot plots). d Steady-state fluorescence anisotropy of PrP labeled at different positions using F5M in the monomer (olive) and droplets (cyan). The data represent mean ± s.d. for n = 3 independent experiments (corresponding data points are shown as black dot plots). e, f Time-resolved anisotropy decays of F5M-labeled α-Syn in dispersed α-Syn monomers and complex coacervate of PrP-α-Syn. g, h Time-resolved anisotropy decays of F5M-labeled PrP in dispersed PrP monomer and complex coacervate of PrP-α-Syn. The solid lines are fits obtained using the biexponential and triexponential decay analysis for monomers and droplets, respectively. See Methods, for details of picosecond time-resolved anisotropy decays measurements, analysis, and the estimation of R_h. Source data are provided as a Source Data file.

Fig. 4. RNA participates in a competitive multicomponent coacervation.

a Solution turbidity plot as a function of increasing polyU RNA at a fixed α-Syn:PrP ratio showing a reentrant phase behavior. The inset shows charge inversion at different RNA concentrations. The data represent mean ± s.d. for n = 3 independent experiments (corresponding data points are shown as black dot plots) b Confocal fluorescence images for different regions of the phase diagram with RNA concentrations as indicated. Before the maximum (C_m), the ternary complex exhibits miscibility, whereas, beyond the maximum (C_m), the droplets start dispersing by transitioning into multiphasic, vesicle-like, hollow condensates. The imaging was performed thrice with similar observations. Top panel: PrP (20 µM) + α-Syn (30 µM) + RNA (25 ng/µL). Scale bar: 10 µm. Bottom panel: PrP (60 µM) + α-Syn (90 µM) + RNA (150 ng/µL). Scale bar: 5 µm. The inset shows a DIC image for a single hollow condensate (Scale bar: 10 µm). See Supplementary Movie 2 for hollow condensates. c Steady-state fluorescence anisotropy for F5M-labeled α-Syn at residue 124 indicating its displacement from the condensates with increasing RNA concentrations. The data represent mean ± s.d. for n = 3 independent experiments (corresponding data points are shown as black dot plots). d Steady-state fluorescence anisotropy for F5M-labeled PrP residue 31 indicating an increase in the order within hollow condensates with the increase in the RNA concentration. The data represent mean ± s.d. for n = 3 independent experiments (corresponding data points are shown as black dot plots). e FRAP kinetics of multiple droplets (~1% Alexa-488-labeled protein) for PrP at different RNA concentrations (25 ng/µL: blue; 150 ng/µL: red). The data represent mean ± s.d. for n = 3 independent experiments. f Fluorescence images of droplets during FRAP measurements. Source data are provided as a Source Data file.

Fig. 5. Synergistic heterotypic interactions promote a liquid-to-solid amyloid transition.

a ThT kinetics for de novo aggregation α-Syn (30 µM) and PrP (20 µM) (separately) and LLPS-mediated aggregation via a liquid-to-solid transition of complex coacervates of α-Syn and PrP completely bypassing the lag phase. The data represent mean ± s.d. for n = 3 independent experiments. The black solid lines are fits for isodesmic (olive colored plot) and nucleation-dependent polymerization kinetics (blue colored plot). Source data are provided as a Source Data file. b Vibrational Raman spectra of PrP-α-Syn aggregates indicating their heterotypic nature. c Amide I is shown for comparison between PrP-α-Syn heterotypic aggregates formed via LLPS and α-Syn homotypic aggregates formed via de novo aggregation. d AFM image of LLPS-mediated heterotypic aggregates showing the presence of typical amyloid fibrils. The inset shows the height profile (~10 nm). e Two-color confocal fluorescence images showing colocalization of α-Syn and PrP within these heterotypic aggregates. f Confocal fluorescence image of a ThT-positive fibril. Scale bar: 10 µm. The imaging was performed twice with similar observations (d, f).

Fig. 6. A schematic of PrP-α-Syn-RNA multicomponent condensates.

Complex coacervation of PrP and α-Syn drives the formation of partially ordered transient electrostatic nanoclusters. The addition of salt results in a monotonic condensate dissolution, whereas, the addition of RNA results in a non-monotonic dissolution via multiphasic hollow condensates. PrP-α-Syn condensates undergo a liquid-to-solid transition into heterotypic amyloids.