Biomolecular condensates can both accelerate and suppress aggregation of α-synuclein

Abstract

Biomolecular condensates present in cells can fundamentally affect the aggregation of amyloidogenic proteins and play a role in the regulation of this process. While liquid-liquid phase separation of amyloidogenic proteins by themselves can act as an alternative nucleation pathway, interaction of partly disordered aggregation-prone proteins with preexisting condensates that act as localization centers could be a far more general mechanism of altering their aggregation behavior. Here, we show that so-called host biomolecular condensates can both accelerate and slow down amyloid formation. We study the amyloidogenic protein α-synuclein and two truncated α-synuclein variants in the presence of three types of condensates composed of nonaggregating peptides, RNA, or ATP. Our results demonstrate that condensates can markedly speed up amyloid formation when proteins localize to their interface. However, condensates can also significantly suppress aggregation by sequestering and stabilizing amyloidogenic proteins, thereby providing living cells with a possible protection mechanism against amyloid formation.

Fig. 1.. Amyloidogenic αSyn variants used in this study.

(A) Variants of αSyn used in the study and predicted disorder along the protein chain (PrDOS, in gray) (75) and distribution of charged residues (in color). For comparison of predicted disorder using different online tools, please see the Supplementary Materials and fig. S1. (B) Aggregation traces (normalized ThT fluorescence intensity) for various αSyn variants (recorded for 40 μM concentration of FL-αSyn and αSyn-108 and for 160 μM concentration of NACore), without coacervates. (C) Schematic depiction of the basic protein aggregation cycle model used in this study. (D) TEM images of fibrils formed by studied variants. Scale bars, 200 nm. (E) FL-αSyn conformation when bound to lipids [left, Protein Data Bank (PDB) ID: 1XQ8] and stacked in amyloid fibrils (right, PDB ID: 2N0A); relevant residues are indicated.

Fig. 2.. Coacervate systems and interactions with αSyn variants.

(A) Schematic depiction of coacervate systems used in the study. (B) Confocal microscopy images of coacervate systems with labeled αSyn variants [Alexa Fluor 647–labeled S9C-FL-αSyn and S9C-αSyn-108 and carboxytetramethylrhodamine (TAMRA)–labeled NACore]. Scale bar, 20 μm. (C) Partition coefficient and transfer free energy (dilute phase-coacervate) determined from microscopy experiments. (D) Gray value profiles (angular averaging) of coacervate droplets from selected systems.

Fig. 3.. FL-αSyn aggregation in the presence of coacervates.

(A) Aggregation traces for FL-αSyn: without coacervates (reference), with RP3/polyU supernatant, and with RP3/polyU coacervates. (B) TEM images of aggregates formed in the presence of RP3/polyU coacervates. Scale bars, 1 μm (images on the left side) and 200 nm (images on the right side).

Fig. 4.. Analysis of aggregation kinetics.

(A) Distribution of the lag times (t_lag) for all protein variants and all coacervate systems (s, supernatant; c, coacervate) and for the reference sample. Symbols at the top indicate localization of the corresponding variant as determined using fluorescence microscopy. Differences between samples were tested for statistical significance (Student’s t test) in coacervate droplet–supernatant control pairs. “ns” indicates values above 0.05, single asterisk indicates α < 0.05, double asterisk indicates α < 0.01, triple asterisk indicates α < 0.001, and quadruple asterisk indicates α < 0.0001. Violin plots were prepared using Gaussian kernels with bandwidth determined automatically using the Scott’s method; density plots were cut at two bandwidth units past the extreme data points; violins are scaled to have the same area in supernatant-coacervate pairs. (B) Distribution of the maximum aggregation rates (v_max) for all protein variants and all coacervate systems [colors and symbols as in (A)].

Fig. 5.. Aggregation monitored by FRET.

(A) Positions of FRET labels in the FL-αSyn chain (PDB ID: 2N0A). (B) FRET maps of coacervate samples incubated with FRET-labeled FL-αSyn. Scale bar, 20 μm. Insets show three times enlarged part of the image at 60 hours; the experiment was performed in the presence of the FRET probe and 10 μM (RP3/polyU and pLys/ATP) or 40 μM (pLys/pGlu) concentration of nonlabeled FL-αSyn. (C) Changes in FRET intensity in different areas of the coacervate systems over time. (D) FRET intensity radial profiles for coacervate droplets after 60 hours of incubation (distance from the droplet center normalized by the droplet diameter, angular averaging).

Fig. 6.. Fitting of the aggregation models.

(A) Schematic depiction of the aggregation-in-droplets model. (B) Schematic depiction of the interface-aggregation model. (C) Fits to the experimental data for RP3/polyU with FL-αSyn for the aggregation-in-droplets model and fits to the experimental data for pLys/pGlu and pLys/ATP with FL-αSyn for the interface-aggregation model. (D) Resulting aggregation kinetic rate constants, for the diluted-supernatant phase (s) and for the coacervate/interface phase (c/i), normalized by values for reference sample (without coacervate components). Violin plots were prepared analogously to plots in Fig. 4.