Abstract
Many RNA-binding proteins undergo liquid–liquid phase separation, which underlies the formation of membraneless organelles, such as stress granules and P-bodies. Studies of the molecular mechanism of phase separation in vitro are hampered by the coalescence and sedimentation of organelle-sized droplets interacting with glass surfaces. Here, we demonstrate that liquid droplets of fused in sarcoma (FUS)—a protein found in cytoplasmic aggregates of amyotrophic lateral sclerosis and frontotemporal dementia patients—can be stabilized in vitro using an agarose hydrogel that acts as a cytoskeleton mimic. This allows their spectroscopic characterization by liquid-phase NMR and electron paramagnetic resonance spectroscopy. Protein signals from both dispersed and condensed phases can be observed simultaneously, and their respective proportions can be quantified precisely. Furthermore, the agarose hydrogel acts as a cryoprotectant during shock-freezing, which facilitates pulsed electron paramagnetic resonance measurements at cryogenic temperatures. Surprisingly, double electron–electron resonance measurements revealed a compaction of FUS in the condensed phase.
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Data availability
Data supporting the findings of this study are available within the paper and its Supplementary Information files.
Code availability
MATLAB code used for analysis of the DEER data and ImageJ script used for analysis of the fluorescence microscopy experiments are available from the authors upon reasonable request.
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Acknowledgements
This work was supported by the Swiss National Science Foundation with Sinergia grant no. CR-SII5_170976, NCCR RNA & Disease and an EMBO long-term postdoctoral fellowship. E.K. acknowledges support from Volkswagen Foundation Experiment! grant no. 95664. We thank K. Weis and M. Hondele for sharing microscope instruments and expertise, and A. Gossert and I. Ritsch for valuable discussions.
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Contributions
L.E. and L.E.-H. designed the project. L.E. performed the NMR, turbidity and microscopy experiments. L.E.-H. performed the EPR and microscopy experiments. F.F.D. and M.Y. assisted with the experimental design for NMR and EPR, respectively. T.d.V. and C.K.X.N. provided protein samples. L.F.I. assisted with DEER data analysis. S.M. and E.K. performed fluorescence microscopy and analysis. G.J. and F.H.-T.A. provided infrastructure, financial support and overall supervision of the project. L.E. and L.E.-H. wrote the manuscript with support from all authors.
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Extended data
Extended Data Fig. 1 FUS NTD droplets.
Light microscope image of FUS NTD droplets formed upon dilution of urea. Representative image from four independent experiments. Scale bar: 10 μm.
Extended Data Fig. 2 FUS NTD droplets in presence and absence of agarose.
a,b) Upfield region of 1H 1D and 1H-15N HSQC NMR spectra of FUS NTD in absence and presence of 0.5% agarose hydrogel showing comparable linewidths. c) Unattenuated DOSY signal of FUS NTD biphasic sample at different agarose concentrations.
Extended Data Fig. 3 FUS NTD droplet fraction quantification correlates with turbidity.
a) Aliphatic proton region of 1H 1D spectra in the presence and in absence of urea. Color scale (magenta → blue → red) corresponds to spectra recorded with increasing diffusion gradient strength. b) Integrals of the spectral region in (a) as a function of diffusion gradient strength normalized to the integral at the lowest gradient strength. The fraction of the slowly diffusing component obtained by averaging data points obtained at >30 G/cm is 19%. c) Droplet size comparison among different total protein concentrations determined by fluorescence microscopy. d) Correlation of final percentage of unattenuated signal from the DOSY experiments with sample turbidity as function of protein concentration. Error bars indicate standard deviation and dots the mean from three independent experiments.
Extended Data Fig. 4 Protein phase exchange during NMR DOSY experiment.
Estimation of protein molecules remaining in the same phase during the diffusion time of our diffusion NMR experiments (0.08 s). In vitro half time, defined as ln(2)/(exchange rate), of FUS NTD in the droplet phase, as measured by FRAP experiments14, is marked with a yellow dashed line. The range of accessible experimental diffusion times in NMR experiments is highlighted with diagonal stripes.
Extended Data Fig. 5 Effect of agarose hydrogel on stability of liquid droplet of FUS, DDX4, PTBP1 and SRSF1.
Brightfield microscope images of liquid droplets from different proteins hours post preparation in the presence and absence of agarose hydrogel. Representative images from three independent experiments. (Scale bar: 10 μm).
Extended Data Fig. 6 DDX4 droplets in 0.5% agarose hydrogel.
a) Time progression of sample turbidity in the absence (black) and presence (red) of agarose hydrogel. Error bars indicate standard deviation and dots the mean from three independent experiments. b) Overlay of 1H 1D DOSY spectra of dispersed (top) and biphasic (bottom) sample in agarose. Increasing gradient strength is visualized by a color gradient magenta → blue → red. c) Integral of the spectral region shown in (b) normalized to the integral at the lowest gradient strength as a function of the gradient strength. Black and red data denote dispersed and biphasic sample in agarose, respectively. d) Integrals of 1H 1D DOSY spectra as function of gradient strength at different DDX4 concentrations. e) Correlation of final percentage of unattenuated signal from the DOSY experiments shown in (e) with sample turbidity as function of protein concentration. Error bars indicate standard deviation and dots the mean from three independent experiments.
Extended Data Fig. 7 Diffusion NMR experiments on SRSF1 and PTBP1.
a, b) Integrated normalized aliphatic spectral region plotted vs. gradient strength for dispersed (black) and biphasic (red) PTBP1 and SRSF1 respectively. c, d) Amide and aliphatic regions of 1H 1D NMR spectra of biphasic PTBP1 and SRSF1 in agarose. e, f) 1H-15N HSQC spectrum of biphasic PTBP1 and SRSF1 in agarose.
Extended Data Fig. 8 Brightfield microscope images of FUS NTD.
FUS NTD in a) 50% glycerol and in b) 50% PEG shows no phase separation under buffer conditions where liquid phase separation is otherwise observed, as reflected in c). Representative images from three independent experiments. Scale bar: 30 μm.
Extended Data Fig. 9 DEER experiments on the NTD of FUS.
Primary DEER data and corresponding distance distributions using a model-free fit with Tikhonov regularization (black) and a Gaussian distribution (red) of A10C S29C R1 and A105C G128C R1 in a,b) the dispersed state with 3 M urea, in c, d) the dispersed state with 0.6 M urea, and e, f) in the bulk phase. The experimental data are displayed as black dots, the fit as a solid line and the 95% confidence interval obtained via 1000 bootstrap samples as shaded area. The Tikhonov regularization and fit with unimodal Gaussian function with variable mean and width lead to very similar distribution shapes. Total modulation depth Δ as a function of nominal inversion efficiency λnominal for A10C S29C R1 in g) the biphasic state and h) the bulk phase, and A105C G128C R1 in i) the biphasic state and j) the bulk phase. Good fits of the experimental data (black dots) are obtained using a model for two spins (solid red line), which confirms that the spin dilution employed is sufficient to avoid intermolecular distance contributions in the DEER experiment.
Extended Data Fig. 10 Schematics of the fitting algorithm employed for the analysis of the biphasic DEER measurements.
Red corresponds to spin-labeled protein in the dispersed phase, blue is spin-labeled protein in the condensed phase, and gray represents unlabeled, and therefore EPR-silent, protein. The total signal of the biphasic sample is displayed in black.
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Emmanouilidis, L., Esteban-Hofer, L., Damberger, F.F. et al. NMR and EPR reveal a compaction of the RNA-binding protein FUS upon droplet formation. Nat Chem Biol 17, 608–614 (2021). https://doi.org/10.1038/s41589-021-00752-3
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DOI: https://doi.org/10.1038/s41589-021-00752-3
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