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Heat-shock chaperone HSPB1 regulates cytoplasmic TDP-43 phase separation and liquid-to-gel transition

Abstract

While acetylated, RNA-binding-deficient TDP-43 reversibly phase separates within nuclei into complex droplets (anisosomes) comprised of TDP-43-containing liquid outer shells and liquid centres of HSP70-family chaperones, cytoplasmic aggregates of TDP-43 are hallmarks of multiple neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). Here we show that transient oxidative stress, proteasome inhibition or inhibition of the ATP-dependent chaperone activity of HSP70 provokes reversible cytoplasmic TDP-43 de-mixing and transition from liquid to gel/solid, independently of RNA binding or stress granules. Isotope labelling mass spectrometry was used to identify that phase-separated cytoplasmic TDP-43 is bound by the small heat-shock protein HSPB1. Binding is direct, mediated through TDP-43’s RNA binding and low-complexity domains. HSPB1 partitions into TDP-43 droplets, inhibits TDP-43 assembly into fibrils, and is essential for disassembly of stress-induced TDP-43 droplets. A decrease in HSPB1 promotes cytoplasmic TDP-43 de-mixing and mislocalization. HSPB1 depletion was identified in spinal motor neurons of patients with ALS containing aggregated TDP-43. These findings identify HSPB1 to be a regulator of cytoplasmic TDP-43 phase separation and aggregation.

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Fig. 1: Cytoplasmic TDP-43 phase separation and liquid-to-gel/solid transition are induced by oxidative stress and a reduction in proteasome activity.
Fig. 2: Slow depletion of nuclear TDP-43 by cytoplasmic TDP-43 phase separation is enhanced by stress-induced liquid-to-gel/solid transition.
Fig. 3: RNA-binding domains, especially RRM1, are crucial for gelation of TDP-43 droplets.
Fig. 4: Combined APEX proximity labelling, quantitative mass spectrometry using isotopically labelled TMTs and co-expression and immunofluorescence identify the small heat-shock protein HSPB1 to bind cytoplasmic TDP-43 and de-mix with it into droplets/gels after arsenite stress.
Fig. 5: HSPB1 de-mixes in vitro into liquid droplets of full-length TDP-43, the TDP-43 LCD alone or TDP-43 without its LCD, and acts to inhibit TDP-43 LCD assembly into amyloid fibrils.
Fig. 6: Activities of HSPB1, HSP70 and BAG2—the NEF for HSPA1A—are essential for disassembly of stress-induced de-mixing and aggregation of TDP-43.
Fig. 7: HSPB1 is expressed at high levels in normal motor neurons but its accumulation is sharply decreased in spinal motor neurons with TDP-43 pathology in patients with ALS.
Fig. 8: TDP-43 de-mixes and mislocalizes to cytoplasm when HSPB1 levels are depleted.

Data availability

Quantitative mass spectrometry datasets have been deposited to the ProteomeXchange Consortium with the dataset identifier PXD035001. BacTrap RNA-seq data from mice are available under the accession code GSE74724. Single-nucleus RNA-seq transcriptome data of mouse spinal cord are available under the accession code GSE161621. The human genome project datasets analysed are available from the Project MinE77 (http://databrowser.projectmine.com/), ALS Variant Server78 (http://als.umassmed.edu/), ALS Data Browser79 (http://alsdb.org/) and ALS Knowledge Portal80 (http://alskp.org/). Further information on the post-mortem samples analysed are available from the UCSD ALS tissue repository (contact information at https://health.ucsd.edu/specialties/neuro/specialty-programs/als-clinic/pages/default.aspx). Source data are provided with this paper. All other data supporting the findings of this study are available from the corresponding author on reasonable request.

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Acknowledgements

We thank J. Santini at the UCSD Microscopy Core, E. Griffis at the UCSD Nikon Imaging Center and D. Jenkins from the SMD group of the Ludwig Institute for assistance with imaging and image analysis. We thank Y. Jones at Electron Microscopy Core Facility of UCSD for epoxy resin embedding and sectioning. We thank Z. Melamed, C. Chen, M. S. Beccari, J. Lopez-Erauskin, D. H. Kim and P. Trivedi for their helpful discussions, and Y. Jin and N. Monther for their help with experiments. D.W.C. acknowledges support from the NIH (grant no. R01 NS27036) and the Nomis Foundation; J.R.Y. acknowledges support from the NIH (grant no. P41 GM103533); J.R. acknowledges grants from Target ALS (grant no. 20134792), the National Institute of Neurological Diseases and Stroke (grant nos NIH R01NS088578 and NS047101), Kraatz Family/Nicholas Martin Jr Family Foundation and Pam Golden; O.A.A. acknowledges a National Science Foundation Graduate Research Fellowship (grant no. DGE-1650112) and S.V.-S. acknowledges the ALS Association (grant no. 21-PDF-583). We thank the UCSD School of Medicine Microscopy Core grant no. P30 NS047101. We thank the staff members of the National Facility for Protein Science in Shanghai, Zhangjiang Laboratory, China for providing technical support and assistance in the NMR data collection.

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Authors and Affiliations

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Contributions

S.L. and D.W.C. conceived the project. S.L., D.W.C., C.L. and J.R.Y. planned the experiments. S.L. performed the in vivo experiments and proximity labelling. J.H. and J.G. purified all proteins and performed the in vitro phase separation and NMR experiment. O.A.A. performed the patient-tissue staining. A.G. performed the correlative electron and light microscopy experiment. J.D. ran the mass spectrometry samples. S.V.-S. helped by providing neuronal cultures. J.B. plotted the expression of HSPB1 in single-cell RNA-seq data from mouse spinal cord. H.Y. and Q.Y. provided reagents. All authors interpreted data. S.L. and J.H. prepared figures. S.L. and D.W.C. wrote the manuscript with input from C.L., J.H., O.A.A., A.G., S.V.-S., H.Y., S.O., J.R. and J.R.Y.

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Correspondence to Don W. Cleveland.

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Nature Cell Biology thanks Philipp Kahle, and the other, anonymous reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 HSP70 inhibition, proteasome inhibition, or arsenite-mediated stress induces cytoplasmic TDP-43 de-mixing independent of stress granule.

(a) Schematic of experimental design to characterize stress induced TDP-43 de-mixing droplets independent of stress granules or RNA binding. (b) Representative images of induced expression of cytoplasmic TDP-43 (TDP-43∆NLS-Clover) for 1 day or 2 days in U2OS cells. (c) Boxplot of relative mean fluorescence intensity of diffuse TDP-43 in the U2OS cells. Number of cells quantified are 70 and 41, respectively. The cells are from one experiment. (d) Fluorescence intensity curve of recombinant TDP-43Clover at different concentration. (e) Concentration of TDP-43∆NLS/5FL-Clover in the diffuse pool of cells with de-mixing droplets calculated based on the standard curve. Seven cells are analysed. (f) Representative images of induction of cytoplasmic RNA binding deficient TDP-43 (TDP-43∆NLS/2KQ-Clover) droplets by 10 μM proteasome inhibitor (MG132), 50 μM HSP70 inhibitor (VER155008) or 250 μM NaAsO2 treatment. (g) Representative images of stress granules (EIF3η) and cytoplasmic TDP-43∆NLS-Clover or TDP-43∆NLS/2KQ-Clover de-mixing droplets under NaAsO2, NaAsO2/cycloheximide, VER155008 or MG132 treatment. Percent of TDP-43 droplets showing no recruitment of EIF3η was labelled on the top of merged images. (h) Representative fluorescence images of TDP-43∆NLS-Clover de-mixing droplets (green) and Poly-A RNA (oligo-dT FISH; red). Percent of TDP-43 droplets showing no enrichment of Poly-A RNA was labelled on the top of merged images. (i) Representative images of the induction of TDP-43∆NLS/5FL-Clover (green) de-mixing droplets and stress granules (red) by live-cell imaging. (j-k) Circularity of TDP-43 droplets formed after 1 hr, 2 hr, 3 hr and 4 hr of 250 μM NaAsO2 treatment. j: TDP-43∆NLS-Clover; k: TDP-43∆NLS/2KQ-Clover (l-m) Area of TDP-43 droplets formed after 1 hr, 2 hr, 3 hr and 4 hr of sodium arsenite treatment. l: TDP-43∆NLS-Clover; m: TDP-43∆NLS/2KQ-Clover. Number of droplets quantified are indicated on the figures. Images are from one live-cell imaging experiment. (c,e,l,m) Medians, 25th and 75th percentiles are shown in the boxes; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles.

Source data

Extended Data Fig. 2 Proteasome inhibition, or arsenite-mediated stress rapidly converts liquid droplets of cytoplasmic TDP-43 into gels/solids.

(a-c) Representative images of FRAP analysis of cytoplasmic TDP-43∆NLS/5FL-Clover droplets under (a) no stress but at higher accumulated level, (b) proteasome inhibition, and (c) arsenite stress. (d) FRAP curves of cytoplasmic TDP-43∆NLS/5FL-Clover droplets in (a-c). Light colour lines, s.d.; Number of droplets analysed in no stress, proteasome inhibition and arsenite stress conditions are 5, 3 and 11, respectively, from three independent experiments. (e) Schematic of experimental design for TDP-43 droplet dissolution assay by mild cell permeabilization. (f-h) Representative images of U2OS cells containing TDP-43∆NLS-Clover droplets which recruit nuclear TDP-43mRuby2 under no stress (f, h) or 2 h of 250 μM NaAsO2 treatment (g) after permeabilization with 50 μg/mL digitonin. (i) Relative level of TDP-43 in U2OS cells that did or did not form TDP-43∆NLS-Clover de-mixing droplets after 2 h of 250 μM NaAsO2 treatment comparing to the endogenous TDP-43 level in naïve U2OS nucleus. Number of cells are 211, 106, 51, respectively, from an experiment. (j) Representative image of dynamical arrest of liquid TDP-43 into droplets after arsenite treatment. (k) Examples of U2OS cells that form large, elongated droplets when cytoplasmic TDP-43∆NLS/5FL-Clover is accumulated with time. (l) Area of the droplets increased with time. Number of droplets: 32, 150, 390, 488. Data are from one live-cell imaging experiment. (m) Circularity of the droplets in different sizes. Number of droplets: 244, 158, 28, 15, 13, 11. Data are from an experiment. (n) FRAP of small and big TDP-43 droplets. FRAP curve are from three big droplets formed after three days of expression. (o) Circularity of RRM-del TDP-43 droplets were not changed by size of the droplets. Numbers quantified are indicated in the figure. Data are from an experiment (p) Solubility of TDP-43 variants after arsenite-induced phase separation. Fluorescence images of TDP-43 variants in lysates from U2OS cells treated with 250 μM sodium arsenite and lysed with RIPA buffer. Western blot of the solution and insoluble fractions. Images were taken at 10 frames with similar results in two independent experiments. (l,m,o) Medians, 25th and 75th percentiles are shown in the boxes; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles.

Source data

Extended Data Fig. 3 Proximity labelling of cytoplasmic TDP-43 de-mixing structures and verification of HSPB1 partition into cytoplasmic TDP-43 de-mixing structures in iPSC-cortical and motor neurons.

(a) Representative images of proximity labelling by cytoplasmic TDP-43∆NLS-Clover-APEX2 in diffuse (no stress) and de-mixed state (sodium arsenite, MG132). Images represents 10 independent images taken for each condition. (b) Representative images of proximity labelling by Clover-APEX2NES under no stress, sodium arsenite and MG132 treatment conditions. Images represents 10 independent images taken for each condition. (c) Volcano plots of statistical significance against fold-change (TDP-43∆NLS-Clover-APEX v.s. Clover-APEX2NES) of each protein under no stress, sodium arsenite and MG132 treatment conditions. P-value is calculated by one-sample t-test. (d) Representative immunofluorescence images of HSPB1 enriched in cytoplasmic TDP-43∆NLS-Clover droplets induced by sodium arsenite. Images represents 10 independent images taken for each condition. (e) Representative immunofluorescence images of HSPB1 enriched in cytoplasmic TDP-43∆NLS/2KQ-Clover droplets in iPSC-derived cortical neurons and motor neurons. Images represents 5 independent images taken for each condition.

Source data

Extended Data Fig. 4 HSPB1 inhibits TDP-43 phase separation at higher molecular ratio.

(a) SDS-PAGE analysis of all TDP-43 variants and HSPB1 variants used for in vitro phase separation assay and NMR analysis in Fig. 4 and supplementary fig. 4, 5. Images represent analysis from three independent runs. (b) SDS-PAGE analysis of TDP-43 and HSPB1 phase separation samples after adding TEV protease to cleave MBP tag off. Images represent analysis from three independent runs. (c) Fluorescence images of in vitro phase separated droplets of untagged TDP-43 (2% NHS-Alexa 488 labelled) and HSPB1 (2% NHS-Alexa 555 labelled). (d) DIC images of mixtures of TDP-43 and HSPB1 at different concentrations. (e) Measurement of the size of de-mixed droplets in different conditions. Data of over 20 droplets from three independent experiments are presented as mean values ± SD. *** p < 0.001 one-way ANOVA analysis. (f) Phase diagram of TDP-43 in (e). The size of dots represents the size of droplets formed at that condition.

Source data

Extended Data Fig. 5 HSPB1 binds to TDP-43 LCD through the conserved transient α-helix region (320-340 aa) and binds to RRM1 domain.

(a) Fluorescence images of in vitro phase separated TDP-43 (2% NHS-Alexa 488 labelled) droplets with or without phosphor-mimetic HSPB13SD (2% NHS-Alexa 555 labelled). Phase separation of 50 μM TDP-43-MBP was conducted by adding 7.5% dextran with or without 10 μM HSPB13SD. (b) Fluorescence images of in vitro phase separated TDP-43 LCD (50 μM, 2% NHS-Alexa 488 labelled) droplets with or without HSPB13SD (50 μM, 2% NHS-Alexa 555 labelled). (c) Thioflavin T aggregation assay to monitor the TDP-43 LCD (10 μM) amyloid assembly over time in the presence or absence of HSPB13SD at different molecular ratios. Data are collected from three biological replicates. Data are presented as mean values ± SD. (d) The 2D 1H15N HSQC spectra of 15N-labelled TDP-43 LCD titrated with increasing concentrations of HSPB1 (left). The representative residues that are markedly attenuated by HSPB1 titration are shown in right panel. (e-f) Profiles of the intensity changes (top) and chemical shift perturbations (bottom) of 20 μM 15N-labelled TDP-43 LCD in the presence of 20 (e) and 10 μM (f) HSPB13D, respectively. (g) Intensity changes of signals in the 2D 1H15N HSQC spectra of 20 μM 15N-labelled TDP-43 LCDA326P with 20 μM or 10 μM HSPB1. Data represents analysis from three independent runs. (h-i) Representative fluorescence images of (h) TDP-43∆NLS/∆RRM1-Clover (green) and (i) TDP-43∆NLS/∆RRM2-Clover (green) and HSPB1 (red) in U2OS cells. Images represent 10 independent images for each condition. (j) Immunoprecipitation of HSPB1 by full-length TDP-43 and RRM1-containing variant TDP-43∆NLS/∆RRM2-Clover but not RRM1-deletion variant TDP-43∆NLS/∆RRM1-Clover.

Source data

Extended Data Fig. 6 HSPB1, HSP70/HSC70 and BAG2 are partitioned into the sodium arsenite-induced TDP-43 gels/solids.

(a) Live imaging of TDP-43∆NLS-Clover and HSPB1mCherry in U2OS cells treated by NaAsO2. (b-j) Immunofluorescence images of TDP-43∆NLS-Clover and (b) HSPB1, (c) HSP70/HSC70, (d) BAG2, (e) HSPB8, (f) HSPH1, (g) BAG3, (h) DNAJA1, (i) DNAJB1 and (j) BAG1 in U2OS cells treated with sodium arsenite for 80 min or 120 min. Images represent 10 independent images for each condition. (k) Summary of the result in (b-j).

Extended Data Fig. 7 Reduction in HSPB1, HSPA1A, BAG2 or mild inhibition of HSP70 activity inhibits or delays the disassembly of cytoplasmic TDP-43 de-mixed droplets.

(a) Immunofluorescence images of HSPB1 and HSP70 in U2OS cells transfected with control siRNA, siHSPB1 or siHSPA1A. (b) Immunofluorescence images of BAG2 in U2OS cells transfected with control siRNA, siBAG2. (c) Quantification of fluorescence intensity of HSPB1, HSP70 and BAG2 in cells transfected with control siRNA, siRNA to HSPB1, HSPA1A/HSPA1B or BAG2. Number of cells for quantification are indicated in the figure (633, 73, 633, 511, 481, 297, respectively). Images are from one experiment. P < 0.0001 (student t-test, two-tailed). Medians, 25th and 75th percentiles are labelled as lines in the plots. (d-g) Time lapse images of the disassembly of cytoplasmic TDP-43∆NLS/2KQ-Clover de-mixing droplets in U2OS cells transfected with control siRNA (d), siRNA to HSPB1(siHSPB1; e), siRNA to HSPA1A/HSPA1B (siHSPA1A/HSPA1B; f), siRNA to BAG2(siBAG2; g). (h) Time lapse images of TDP-43∆NLS/2KQ-Clover in U2OS cells after 10 μM VER155008 treatment. Quantification of U2OS cells containing cytoplasmic TDP-43∆NLS/2KQ-Clover de-mixing droplets after 10 μM VER155008 treatment. The number of cells for quantification is 117. (i) Time lapse images of the disassembly of cytoplasmic TDP-43∆NLS-Clover de-mixing droplets in U2OS cells after washing off sodium arsenite with or without 10 μM VER155008 in the medium.

Source data

Extended Data Fig. 8 Reduction in HSC70/HSPA8 induces the expression of the HSP70 family member HSPA1A, inhibits the arsenite-induced de-mixing of cytoplasmic TDP-43, and promotes droplet/gel disassembly.

(a) Immunofluorescence images of HSC70 (HSPA8) and HSP70 (HSPA1A/HSPA1B) in U2OS cells transfected with control siRNA, siRNA to HSPA8(siHSPA8) siRNA to HSPA1A/HSPA1B (siHSPA1A/HSPA1B) or co-transfected with siRNA to HSPA8 and siRNA to HSPA1A/HSPA1B. (b-c) Quantification of fluorescence intensity of (b) HSP70 and (c) HSC70 (HSPA8) in (a). Each dot represents a single cell and orange lines represent mean value and standard error (S.E.M.) of fluorescence intensity. The number of cells quantified are 407, 261, 220 and 149, respectively. Images are taken from one experiment. (d,e) Time lapse imaging of TDP-43∆NLS-Clover de-mixing droplets induction by arsenite stress and disassembly after removal of sodium arsenite in cells transfected with (d) siHSPA8 or (e) control siRNA. (f) Quantification of U2OS cells containing cytoplasmic TDP-43∆NLS/2KQ-Clover de-mixing droplets after arsenite treatment (left) and quantification of U2OS cells that have TDP-43 de-mixing droplets disassembled after stress removal (right) in (d,e). Cells are quantified from four independent replicates. N: 346, 425, 309, 354 for siRNA control group; 86,88, 86, 69 for siHSPA8 group. (g) Fluorescence intensity of TDP-43∆NLS-Clover in U2OS cells transfected with siRNA control or siHSPA8 (up) and percentage of TDP-43∆NLS-Clover in de-mixing droplets (bottom). ***P < 0.0001, n.s. P = 0.2159 (student t-test, two-tailed). Data are presented as mean values ± SEM. Number of cells quantified in siRNA control group are 42 and in siHSPA8 group are 39. Cells are pooled from four independent experiments. (h) Time lapse images of TDP-43∆NLS/2KQ-Clover expressing U2OS cells transfected with siHSPA8 after sodium arsenite treatment and wash-off. (i) Representative images of U2OS cells expressing TDP-43∆NLS-Clover together with HSPA1AmRuby2 or HSPA8mRuby2 after 2 h of sodium arsenite treatment. Images represent 10 independent images.

Source data

Extended Data Fig. 9 Enhanced recruitment of HSP70 and its co-chaperones DNAJB1 and BAG3 to phase separated cytoplasmic TDP-43 droplets after stress removal and assembled microtubule array is required for disassembly of TDP-43 droplets.

(a) Fluorescence images of TDP-43∆NLS-Clover (green) with BAG3 (red), HSP70/HSC70 (red), DNAJB1 (red), and DNAJA1 (red), respectively, in U2OS cells treated with sodium arsenite for 1 h followed by 4-h wash-off. Images represent 10 independent images for each condition. (b-c) Microtubule disassembly by nocodazole treatment. Microtubule structures are imaged by sir-Tubulin dye. (d) Schematic design of testing if microtubule disassembly affects TDP-43 droplets fuse/aggregate/coalesce. (e) Microtubule disassembly does not strongly affect TDP-43 droplet fusion/aggregation but affect the resolution of the droplets.

Extended Data Fig. 10 Reduction in HSPB1 induces cytoplasmic TDP-43 de-mixing and mislocalization.

(a) Experimental design for testing the effect of HSPB1 depletion on cytoplasmic TDP-43 de-mixing in cell cycle arrested U2OS cells. (b) DNA content analysis by FACS. U2OS cells are treated by reduced serum medium and 1 μM G1 cell cycle blocker palbociclib to block cell cycle. Line is drawn to separate 2-N and 4-N cells based on FACS plot of cell population. (c) Fluorescence images of TDP-43∆NLS/5FL-Clover (upper), TDP-43∆NLS-Clover (medium) and Clover (bottom) in cell cycle arrested U2OS cells transfected with control siRNA (siCtrl) or siRNA to HSPB1 (siHSPB1) after induction with doxycycline for 1 day or 2 days. (d) Quantification of the percentage of cells forming cytoplasmic TDP-43 de-mixing droplets in (c). Numbers of cells quantified are 4108, 3632, 3897 (TDP-43∆NLS/5FL-Clover, control siRNA, 1 day) and 2901, 2978, 2897 (TDP-43∆NLS/5FL-Clover, control siRNA, 2 day), 4035, 3788, 3673 (TDP-43∆NLS/5FL-Clover, siHSPB1, 1 day) and 3663, 3266, 3278 (TDP-43∆NLS/5FL-Clover, siHSPB1, 2 day), 4203, 4399, 4272 (TDP-43∆NLS-Clover, siRNA control, 1 day) and 3777, 3957, 3709 (TDP-43∆NLS-Clover, siRNA control, 2 day), 3913, 3803, 3829 (TDP-43∆NLS-Clover, siHSPB1, 1 day) and 3469, 3358, 3345 (TDP-43∆NLS-Clover, siHSPB1, 2 day). Data are presented as mean values ± SD. Each data are from three independent experiments. (e) Fluorescence images of endogenous TDP-43 and HSPB1 in U2OS cells transfected with siRNA control or siHSPB1 and quantification of cytoplasmic/nuclear fluorescence intensity of TDP-43 in cells expressing different levels of HSPB1. The number of cells for plotting are 114, 883, 519 and 292, respectively. <200 group V.S. 200-400 group, n.s. P = 0.2878; <200 group V.S. 400-600 group, **P = 0.0005; <200 group V.S. > 600 group, **P = 0.027; 200-400 group V.S. 400-600 group, ***P < 0.0001; 200-400 group V.S. > 600 group, ***P = 0.0001 (unpaired student t-test, two-tailed). Images are pooled from two independent experiments. (f) Fluorescence images of endogenous TDP-43 and HSPB1 in U2OS cells transfected with control siRNA or siHSPB1 and quantification of cells forming cytoplasmic de-mixing TDP-43 droplets. The number of quantified cells for control siRNA is 586 and the number of cells for siHSPB1 is 175. Data are from an experiment.

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Supplementary information

Supplementary Information

Supplementary Fig. 1.

Reporting Summary

Supplementary Table 1

Supplementary Tables 1–4

Supplementary Video 1

Live imaging of cytoplasmic TDP-43 droplets in cells.

Supplementary Video 2

Live imaging of cytoplasmic TDP-43 droplets in cells under arsenite stress.

Supplementary Video 3

Live imaging of induction of TDP-43 droplets by arsenite stress and disassembly of TDP-43 droplets after stress removal.

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Lu, S., Hu, J., Arogundade, O.A. et al. Heat-shock chaperone HSPB1 regulates cytoplasmic TDP-43 phase separation and liquid-to-gel transition. Nat Cell Biol 24, 1378–1393 (2022). https://doi.org/10.1038/s41556-022-00988-8

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