Protein solubility was predicted using CamSol intrinsic. The CamSol method is based on a combination of algorithms that assign a score to the intrinsic solubility of protein residues based on linear protein sequences. This score is tested against solubility changes that have been experimentally determined and reported in the literature. Aggregation propensity was validated using Aggrescan3D. Aggrescan 3D does not simply rely on linear protein sequence but takes into consideration protein structure as well as empirical data on aggregation derived from the cellular setting. In particular, the analysis of the aggregation profiles of a number of mutant Aβ peptides in E. Coli has allowed deriving a scale of intrinsic aggregation values for each natural amino acid, which represents the basis of the algorithm. Contrary to standard sequence-based algorithms, Aggrescan 3D uses 3-dimensional structures as inputs, i.e. PDB files. This enables accurate predictions especially in the case of structured proteins, where aggregation-prone regions may be naturally masked inside the hydrophobic core of the protein. To analyze the aggregation propensity of the FUS C-terminus, we used the PDB entry "4FDD", with a distance of aggregation of 10 Å and static mode. Results referred to chain B, corresponding to the 498–526 fragment of FUS (GPGKMDSRGEHRQDRRERPY). FoldX measurements relative to the free energy of the FUS C-terminus were obtained in conjunction with Aggrescan 3D results. Intrinsic disorder and secondary structure were predicted with s2D, which exploits information from NMR measurements to predict the probability distributions of secondary-structures in disordered proteins using their linear sequence as an input. AGDIR was used to predict the helical content of the GPGKMDSRGEHRQDRRERPY and GPGKMDSRGEHRQDRRERLY peptides, respectively.
Purification of recombinant proteins
Full-length recombinant human MBP-FUS-eGFP-His protein was isolated from eukaryotic Sf9 cells cultured in suspension in Insect Cell Medium (ESF921, Expression Systems). For protein over-expression, Sf9 cells were infected with recombinant baculoviruses at a density of 1×106 cells/mL. After 4 days of incubation at 27°C in agitation, cells were harvested for protein purification. To this end, infected Sf9 cells were centrifuged at 4000 × g for 30 min. Pellets were resuspended in lysis buffer (50 mM Tris-HCl pH 7.4, 1 M KCl, 0.1% CHAPS, 5% glycerol, 1 mM DTT, and protease inhibitors) and disrupted mechanically. The lysate was ultra-centrifuged at 40,000 rpm for 20 min at room temperature (RT). Following resin equilibration in 50 mM Tris-HCl pH 7.4, 1 M KCl, 5% glycerol, and 1 mM DTT, the clear supernatant was poured through a nickel (Ni)-Excel column to allow for FUS-eGFP binding via its His-tag. The protein was then washed with the same buffer containing 20 mM imidazole and eventually eluted with 250 mM imidazole. Next, the eluate was loaded onto an amylose column (E8021L, NEB) for further protein selection. The bound protein was eluted with a buffer containing 10 mM maltose. Following protein concentration, the eluate was eventually loaded onto an AKTA system for size-exclusion chromatography and the fractions containing FUS-eGFP were pooled. To allow for P525L FUS protein purification, all buffers were supplemented with 2 M urea. Purified proteins were concentrated using 3–50 kDa Amicon Ultra 0.5–15 Centrifugal filters (Sigma). Concentrations were determined to measure their absorbance at 280 nm using theoretical extinction coefficients calculated with Expasy ProtParam. Proteins were aliquoted, flash-frozen in liquid nitrogen, and stored at -80°C.
SDS page and Coomassie
Proteins were diluted in 4X Lämmli buffer, boiled for 5 min at 95°C, loaded onto an SDS-page gel, and run for 25 min at 200 V. Gels were incubated in Coomassie blue Fast Stain (GeneCopoeia) for band revelation.
Phase separation experiments
Unless otherwise stated, phase separation was induced by diluting the protein stock (1 M KCl) to 40 mM KCl and a working concentration of 1.5 µM FUS (1X) in the presence of 5% dextran. After vortexing, the mixture was pipetted onto slides (Ibidi), and FUS-eGFP droplet evolution was monitored over time using an Axiovert 200M microscope (Zeiss) at 100X magnification. Droplet size and circularity were calculated using Fiji after removing background noise using the Process -> Subtract Background function. The phase-separation time course reported in "Supplementary Video" displays images acquired at 0 min, 10 min, 30 min, 45 min, 1 h, 1.5 h, 2 h, 3 h, 4 h, 5 h, 6 h, 8 h and 24 h post-induction of phase separation.
Following induction of phase-separation as described above, samples were pipetted onto 96-well black/clear bottom plates (6005182, PerkinElmer) and absorbance was measured at 405 nm using a Spectra Max M5 plate reader (Molecular Devices) endowed with a SoftMax Pro 7.1 software. Turbidity measurements of Sf9 cell lysates were performed by sampling a fraction of the lysate prior to column purification and recording absorbance values over time while leaving the plate at RT.
Structural properties of FUS assemblies
AmytrackerTM dyes are molecules that become highly fluorescent when bound to their target, i.e. pre-fibrillary and fibrillary states of amyloids. To stain FUS aggregates, the AmytrackerTM 680 (Ebba Biotech) stock was diluted 1:10,000 in the phase-separation mixture. Images were acquired using the red channel.
NSC34 cell culture and transfection
Cells were cultured in T75 flasks using DMEM medium (D5796, Sigma) supplemented with 10% FBS (A15-751, PAA) and split with trypsin at a 1:3 dilution when 80% confluent. Transfection was performed on 6-well plates. Fully confluent wells were supplemented with 1 µM aggregated α-synuclein protein added directly to the medium. After 24 h, cells were incubated with AmytrackerTM 680 diluted 1:500 in fresh medium for 30 min prior to fixation. Cells were fixed with 4% PFA, stained with Hoechst, and mounted for imaging. Images were acquired using an Axiovert 200M microscope (Zeiss) at 100X magnification.
Generation of α-synuclein seed fibrils
Seed fibrils were produced as previously described by Buell et al., with minor modifications. Briefly, 500 µL aliquots of α-synuclein at concentrations ranging from 500 to 800 µM were incubated in 20 mM phosphate buffer, pH 6.5 for 48–72 h at 40°C and stirred at 1500 rpm with a Teflon bar on an RCT Basic Heat Plate (IKA). Fibrils were diluted to a monomer equivalent concentration of 200 µM, divided into aliquots, flash-frozen in liquid N2 and stored at -80°C. Prior to use, the 200 µM fibril stock was sonicated for 15 sec using a probe sonicator (Bandelin, Sonopuls HD2070), set at 10% maximum power and 50% cycle.