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FUS fibrillation occurs through a nucleation-based process below the critical concentration required for liquid-liquid phase separation - PubMed

  • ️Sun Jan 01 2023

FUS fibrillation occurs through a nucleation-based process below the critical concentration required for liquid-liquid phase separation

Emilie Bertrand et al. Sci Rep. 2023.

Abstract

FUS is an RNA-binding protein involved in familiar forms of ALS and FTLD that also assembles into fibrillar cytoplasmic aggregates in some neurodegenerative diseases without genetic causes. The self-adhesive prion-like domain in FUS generates reversible condensates via the liquid-liquid phase separation process (LLPS) whose maturation can lead to the formation of insoluble fibrillar aggregates in vitro, consistent with the appearance of cytoplasmic inclusions in ageing neurons. Using a single-molecule imaging approach, we reveal that FUS can assemble into nanofibrils at concentrations in the nanomolar range. These results suggest that the formation of fibrillar aggregates of FUS could occur in the cytoplasm at low concentrations of FUS, below the critical ones required to trigger the liquid-like condensate formation. Such nanofibrils may serve as seeds for the formation of pathological inclusions. Interestingly, the fibrillation of FUS at low concentrations is inhibited by its binding to mRNA or after the phosphorylation of its prion-like domain, in agreement with previous models.

© 2023. The Author(s).

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1

FUS is able to produce nanofibrils at nanomolar concentration. (A) AFM images of FUS at different incubation times in AFM buffer. Size 1.5 × 1.5 µm. Inset: zooms on specific structures. Z scale: 10 nm. (B) Circularity measurements of the FUS structures adsorbed on mica surface for different incubation time and protein concentration. Red line: mean value. *, p < 0.05; **, p < 0.01. (C) Length measurements of nanofibrils formed after the incubation of FUS (540 nM) for different incubation time. Red line: mean value. *, p < 0.05; **, p < 0.01; ns, not significant. (D) AFM images of FUS assemblies (540 nM) after 1 h incubation in AFM buffer (left) followed by 10 min exposure to SDS at 0.5%. Z scale: 10 nm. (E) Height measurements of FUS assemblies after incubation and co-incubation with non-aggregated FUS at different times and concentrations. A threshold of 4 nm in height was applied. US samples indicate a sonication of the FUS assemblies for 1 min before addition of non-aggregated FUS. The protocol is detailed in supplementary Fig. S2B. Red line: mean value. ***, p < 0.005.

Figure 2
Figure 2

RNA slows down the kinetics of nanofibril assembly. (A) AFM images of RNA:FUS complexes ([RNA] = 2.5 nM) at different incubation times in AFM buffer. Size 2 × 2 µm. Z scale: 9 nm. (B) Height measurements of RNA:FUS complexes ([RNA] = 2.5 nM) for different FUS concentrations and incubation times. Red line: mean value. *, p < 0.05; **, p < 0.01; ns, not significant. (C) Circularity measurements of RNA:FUS complexes ([RNA] = 2.5 nM) for different FUS concentrations and after 3 days incubation in AFM buffer. Red line: mean value. *, p < 0.05; **, p < 0.01. (D) Top: AFM images of RNA:FUS complexes ([FUS] = 540 nM, ) after 3 days incubation in AFM buffer at different RNA concentrations (left: 0.8 nM, middle: 2.5 nM, right: 8 nM). Size 2 × 2 µm. Z scale: 9 nm. Bottom: zoom on FUS:RNA assembly produced with a low RNA concentration, revealing fibrillar structures in the dense core of the granule. Size: 1 × 1 µm. (E) AFM images of FUS:RNA complexes ([RNA] = 2.5 nM, [FUS] = 540 nM, incubation time: 3 days) before (top) and after (bottom) RNAse treatment (1 h). Size 2.5 × 2.5 µm. Inset: zooms on specific structures. Z scale: 9 nm.

Figure 3
Figure 3

Phosphomimetic FUS is unable to trigger the formation of nanofibrils. (A) Top: large scale AFM image of FUS12E (60 mM) for 5 min of incubation in AFM buffer. Size 15 × 15 µm. Inset: zoom on the shell of the granule. Bottom: AFM images of FUS12E assemblies after incubation for 5 min-3 h in AFM buffer (size 4 × 4 µm, z scale: 10 nm). Inset: zoom on the core of the granule with a z scale of 20 nm to evidence the granular shape of the core. (B) Height profiles of FUS12E assemblies according to the red line on AFM image on Fig. 3A (bottom). (C) AFM images of FUS12E (540 nM) after incubation for 10 min or 1 day in AFM buffer. (D) Circularity measurements of FUS 12E (540 nM) assemblies adsorbed on mica for different incubation time. Red line: mean value. **, p < 0.01.

Figure 4
Figure 4

FUS12E distributes along RNA and forms large and compact assemblies. (A) AFM images of RNA:FUS12E complexes ([RNA] = 5 nM) after different incubation times in AFM buffer. Z scale: 10 nm. (B) Diagram representing the proportion of molecules with a maximum height higher than 3.5 nm for RNA alone, RNA:FUS12E and RNA:FUS WT assemblies with [RNA] = 5 nM and [protein] = 540 nM after incubation for 1 h. Each point corresponds to the analysis of an area of 9 µm2 (total of 90 µm2 for each condition). Red line: mean value. *, p < 0.05; ***, p < 0.005. (C) Circularity measurements of RNA:FUS12E (5 nM : 540 nM respectively) complexes adsorbed on mica for different incubation times. Red line: mean value. *, p < 0.05; **, p < 0.01; ***, p < 0.005.

Figure 5
Figure 5

FUS nanofibril formation proceeds independently of the LLPS. In the LLPS process, FUS condensate formation is triggered when a sufficiently high protein concentration is reached above which the system separates in two phases (left). Protein clusters produced at low concentrations could be the crucible for the dense phase (condensate) formation which could evolve toward fibrillar structure. FUS fibrillar assemblies (nanofibrils) could be formed also at low protein concentrations with a sufficient long incubation time (right) and independently of the LLPS process. In this context, FUS incubation in the presence of RNA and/or FUS phosphorylation impedes the formation of nanofibrils.

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