Hsp40 Proteins Phase Separate to Chaperone the Assembly and Maintenance of Membraneless Organelles

Hsp40 proteins phase separate to chaperone the assembly and maintenance of membraneless organelles

Jinge Gua,b,1, Zhenying Liua,b,1, Shengnan Zhanga,b, Yichen Lic, Wencheng Xiaa,b, Chen Wanga,b, Huaijiang Xianga,b, Zhijun Liud, Li Tana,b, Yanshan Fanga,b, Cong Liua,b,2, and Dan Lic,e,2

aInterdisciplinary Research Center on Biology and Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai 201210, China; bUniversity of the Chinese Academy of Sciences, Shijingshan District, Beijing 100049, China; cBio-X-Renji Hospital Research Center, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200240, China; dNational Facility for Protein Science in Shanghai, Zhangjiang Laboratory, Shanghai Advanced Research Institute, Chinese Academy of Sciences, Shanghai 201210, China; and eKey Laboratory for the Genetics of Developmental and Neuropsychiatric Disorders (Ministry of Education), Bio-X Institutes, Shanghai Jiao Tong University, Shanghai 200030, China

Edited by Roy Parker, University of Colorado Boulder, Boulder, CO, and approved October 14, 2020 (received for review February 11, 2020) Membraneless organelles contain a wide spectrum of molecular in SGs and directly influence the assembly, dynamics and clear- chaperones, indicating their important roles in modulating the ance of SGs (17). Human Hsp40 proteins, e.g., Hdj1 (DNAJB1) metastable conformation and biological function of membraneless and Hdj2 (DNAJA1), cannot only inhibit the aggregation of a organelles. Here we report that class I and II Hsp40 (DNAJ) proteins variety of amyloid proteins including α-synuclein, Τau, and polyQ possess a high ability of phase separation rendered by the flexible in vitro (18–20) and rescue the cytotoxicity of polyQ and SG- G/F-rich region. Different Hsp40 proteins localize in different mem- associated RNA-binding proteins including fused in sarcoma braneless organelles. Specifically, human Hdj1 (DNAJB1), a class II (FUS) and TAR DNA-binding protein 43 (TDP-43) in cellular Hsp40 protein, condenses in ubiquitin (Ub)-rich nuclear bodies, and animal disease models (21–23). However, the mechanism of while Hdj2 (DNAJA1), a class I Hsp40 protein, condenses in nucle- how Hsp40s rescue the cytotoxicity of these proteins remains oli. Upon stress, both Hsp40 proteins incorporate into stress gran- unknown. Moreover, genetic defects of Hsp40 proteins have been ules (SGs). Mutations of the G/F-rich region not only markedly

identified in neurodegenerative diseases including ALS (24), BIOCHEMISTRY impaired Hdj1 phase separation and SG involvement and disrup- ’ ted the synergistic phase separation and colocalization of Hdj1 and Parkinson s disease (25), cerebellar ataxia (26), and distal hered- fused in sarcoma (FUS) in cells. Being cophase separated with FUS, itary motor neuron neuropathy (27). These evidences suggest that Hdj1 stabilized the liquid phase of FUS against proceeding into Hsp40 proteins play an important role in maintaining the neuro- amyloid aggregation in vitro and alleviated abnormal FUS aggre- nal proteostasis by regulating protein assembly and preventing gation in cells. Moreover, Hdj1 uses different domains to chaper- pathological amyloid aggregation. one FUS phase separation and amyloid aggregation. This paper In this paper, we find that human class I and II Hsp40 suggests that phase separation is an intrinsic property of Hsp40 proteins—Hdj2 and Hdj1 localize differently in nucleoli and proteins, which enables efficient incorporation and function of Hsp40 in membraneless organelles and may further mediate the Significance buildup of chaperone network in membraneless organelles. Protein phase separation is a progressive process of high-ordered Hsp40 | stress granule | ALS | FUS | LLPS assembly in vitro, which can experience dynamic liquid–liquid phase separation (LLPS) followed by maturation into patho- embraneless organelles in cells are highly diverse and logical solid fibrils. The latter is closely associated with neuro- Mfulfill important biological functions under both normal degenerative disorders, such as amyotrophic lateral sclerosis and stress conditions (1, 2). Protein phase separation can drive and frontotemporal dementia. In cells, protein phase separa- the assembly of membraneless organelles, which incorporates tion is tightly regulated to maintain a metastable state of hundreds of different proteins and nucleic acids (3–6). Mem- molecular condensates and avoid further aberrant aggrega- braneless organelles feature a physical property in between liq- tion. However, this regulation process is largely unknown. Our uid and solid and present as a metastable state of complex paper demonstrates that Hsp40 (DNAJ) proteins, e.g., Hdj1 supermolecular assembly (7). The maintenance of membraneless (DNAJB1) and Hdj2 (DNAJA1), have a strong ability of LLPS, organelles in the liquidlike state rather than spontaneous solid- which enables Hsp40 to incorporate into stress granules, and ification or dissociation is essential for membraneless organelles stabilize the amyloid-forming proteins, e.g., FUS, in the con- to fulfill their biological functions (8, 9). Failure of the confor- densed phase-separated state rather than proceeding to toxic mational maintenance of membraneless organelles results in aggregation. pathological protein aggregation, which is closely associated with various neurodegenerative diseases, such as amyotrophic lateral Author contributions: J.G., C.L., and D.L. designed research; J.G., Zhenying Liu, S.Z., Y.L., W.X., and C.W. performed research; H.X., L.T., and Y.F. contributed new reagents/analytic sclerosis (ALS) and frontotemporal dementia (FTD) (10–14). tools; J.G., Zhenying Liu, S.Z., Zhijun Liu, C.L., and D.L. analyzed data; and J.G. and D.L. Molecular chaperones are key components of the quality wrote the paper. control system in cells to maintain protein homeostasis (pro- The authors declare no competing interest. teostasis). They assist client proteins in folding and translocation This article is a PNAS Direct Submission. and maintain clients in the native conformation avoiding mis- Published under the PNAS license. folding and aggregation (15). Proteomic analysis of SGs has 1J.G. and Zhenying Liu contributed equally to this work. identified a wide spectrum of molecular chaperones, including 2To whom correspondence may be addressed. Email: [email protected] or liulab@ Hsp40 (DNAJ), Hsp70, Hsp90, and small Hsps (5, 16), which sioc.ac.cn. indicate the importance of chaperones in the assembly and This article contains supporting information online at https://www.pnas.org/lookup/suppl/ maintenance of membraneless organelles. Studies on yeast doi:10.1073/pnas.2002437117/-/DCSupplemental. Hsp40 proteins (e.g., Sis1 and Ydj1) reveal that they accumulate

www.pnas.org/cgi/doi/10.1073/pnas.2002437117 PNAS Latest Articles | 1of11 Downloaded by guest on September 24, 2021 Ub-rich nuclear bodies, respectively, under normal conditions. importance for Hdj1 LLPS. Thus, we deleted the G/F-rich region Under stress, both chaperones condense in SGs in the cytoplasm. (Fig. 2A, ΔG/F), and the result showed that ΔG/F indeed dras- We show that the close association of Hdj1 and Hdj2 with var- tically diminished the LLPS of Hdj1 (Fig. 2 B and C). ious membraneless organelles attributes to their phase separa- We next sought to examine the role of the J domain in Hdj1 tion property via the flexible G/F-rich region, which we show is a LLPS. As we removed the J domain (ΔJ), the solubility of Hdj1 common property shared by class I and II Hsp40 proteins in drastically decreased, and in the presence of crowding agents, ΔJ different organisms. Furthermore, we find that Hdj1 synergisti- aggregated rather than LLPS (SI Appendix, Fig. S2B). As we cally phase separates with FUS and maintains the phase- removed the entire N-terminal domain (NTD) including the J separated state of FUS from proceeding to amyloid aggrega- domain and the G/F-rich region, the resulting construct, termed tion. Interestingly, Hdj1 employs distinctive mechanisms to ΔNTD, showed further weakened LLPS and tended to aggregate chaperone the different states (i.e., soluble and phase-separated (Fig. 2 B and C). These results indicate that the J domain is states) of FUS. This paper suggests that phase separation ren- important for the solubility and stability of Hdj1, which may ders the localization and function of Hsp40 in various mem- indirectly influence the LLPS. braneless organelles, which may further recruit cochaperones When we truncated the DD at the C terminus (Fig. 2A, ΔDD), (e.g., Hsp70) for the function and regulation of membraneless the result showed that ΔDD weakened the LLPS of Hdj1, while organelles. the degree is significantly less severe than that caused by ΔG/F (Fig. 2 B and C). Intriguingly, even as we truncated the whole Results CTD (Fig. 2A, ΔCTD), the remaining segment still exhibited a Hdj1 Condenses in Ub-Rich Nuclear Bodies and Redistributes to Stress moderate ability of LLPS (Fig. 2B). Taken together, the results Granules upon Stress. To investigate the intracellular localization of domain analysis indicate that the G/F-rich region is primary of Hdj1, we used immunostaining to visualize endogenous Hdj1 for the LLPS of Hdj1, meanwhile the J and DD domains may in HeLa cells. We found that under the normal growth condition, also contribute to this process. Hdj1 extraordinarily condensed in the nucleus (Fig. 1A), al- To investigate the molecular mechanism underlying the LLPS though diffused Hdj1 can be also observed in the cytoplasm. To of the G/F-rich region, we mutated the Phe residues of the identify which nuclear membraneless organelles Hdj1 resides in, G/F-rich region to Ala and Tyr, respectively (Fig. 2A, F-A/F-Y). we sought for the colocalization of Hdj1 with any marker protein Phe has been found to mediate protein LLPS via π–π and of the nuclear membraneless organelles. The result showed that π-cation interactions, and mutation of Phe to Tyr can strengthen the Hdj1 condensates were separated from many known nucleic protein LLPS (28, 29). Indeed, our result showed that the F-A membraneless organelles including nucleolus, Cajal body, para- mutations impaired the LLPS of Hdj1; in contrast, F-Y enhanced speckle, nuclear speckle, and promyelocytic leukemia protein the process (Fig. 2 D and E). We further randomly shuffled the (PML) body but colocalized with a type of Ub-rich nuclear body sequence of the G/F-rich region (Fig. 2A, G/F shuffle) and ob- (Fig. 1 A and B). served no obvious influence on the LLPS of Hdj1 (Fig. 2 D Intriguingly, as we stressed the cells with sodium arsenite, we and E). observed a decrease in Hdj1 in the Ub-rich nuclear body and the We noted that the G/F-rich region contains five Arg residues, incorporation of Hdj1 in the cytoplasmic SGs (Fig. 1C and SI and there are four additional Arg residues forming a positive Appendix, Fig. S1A), indicating redistribution of Hdj1 upon patch on the J domain (SI Appendix, Fig. S2 A). Since Arg may stress. Taken together, these cellular observations indicate that form a π-cation interaction with Phe, we wondered whether these Hdj1 may play a role in the function of various membraneless Arg residues may influence Hdj1 LLPS. Thus, we mutated Arg to organelles in cells under both normal and stress conditions. Ala (Fig. 2A, R-A). The result showed that, although the R-A mutations showed discernible impairment on the LLPS of Hdj1 Hdj1 Features a High Ability of Liquid–Liquid Phase Separation. The as we mutated both Phe and Arg to Ala (Fig. 2A, F/R-A), Hdj1 close association of Hdj1 with membraneless organelles invoked nearly completely lost its ability of LLPS (Fig. 2 D and E). In us to test its phase-separation ability. We found that many addition, for the above truncations and mutations, we confirmed conditions can stimulate Hdj1 liquid–liquid phase separation their conformations and molecular sizes in solution by multi- (LLPS), such as low salt concentration, crowding agents, hepa- angle light scattering with size exclusion chromatography and rin, and polyuridylic acid, and the LLPS can be reversed by in- circular dichroism spectroscopy (SI Appendix, Fig. S3). Taken creasing NaCl concentration (Fig. 1D and SI Appendix, Fig. S1 B together, these results demonstrate that the G/F-rich region and C). We mapped the phase diagram of Hdj1 in the presence plays an essential role in the LLPS of Hdj1 in which interactions, of polyethylene glycol (PEG) 3,350 and observed that as the such as the π–π and π-cations mediated by the Phe and Arg concentration of Hdj1 increased, and its LLPS increased (Fig. 1E residues provide important contributions. and SI Appendix, Fig. S1D). On the contrary, as the concentra- E tion of NaCl increased, the LLPS of Hdj1 decreased (Fig. 1 ). Hdj1 LLPS Is Essential for Its SG Incorporation. To assess the biological We further performed fluorescence recovery after photo- significance of the LLPS property of Hdj1, we transfected HeLa bleaching (FRAP) to assess the mobility of Hdj1 within the cells with myc-tagged wild type (WT) and variants including droplets. The result showed that the fluorescence signal in the F/R-A, F-Y, G/F shuffle, and ΔDD, respectively. Upon stress, droplets rapidly recovered to ∼80% within 130 s after photo- immunostaining showed WT Hdj1 in SGs in ∼75% of the SG- bleaching (Fig. 1F and SI Appendix, Fig. S1E). Together, these positive transfected cells (Fig. 2F). In contrast, F/R-A and ΔDD, results demonstrate a high ability of reversible LLPS of Hdj1. which are disabled or deficient to LLPS, showed markedly decreased ability of entering SGs (Fig. 2F). F-Y, which has an in- The G/F-Rich Region Is Essential to Mediate Hdj1 LLPS. We next sought creased ability of LLPS, consistently showed an enhanced to decipher which domain is important for Hdj1 LLPS. Hdj1 as incorporation in SG (Fig. 2F). G/F shuffle, which showed no well as other class I and II Hsp40 proteins, contains a N-terminal influence on LLPS, also showed no significant influence on SG J domain followed by a flexible G/F-rich region, client-binding incorporation of Hdj1 (Fig. 2F). These results were validated by C-terminal domains (CTDs 1 and 2), and a dimerization domain analyzing Hdj1-positive SG area per cell (Fig. 2F). In addition, (DD) (Fig. 2A). The G/F-rich region is predicted to be intrinsi- Western blot confirmed the overexpression of Hdj1 (SI Appen- cally disordered and exposed to the solvent based on its primary dix, Fig. S4A) and a similar expression level of WT and variant sequence (SI Appendix, Fig. S2A), which indicates its potential Hdj1 in the HeLa cells (Fig. 2G). Note that the F/R-A band

2of11 | www.pnas.org/cgi/doi/10.1073/pnas.2002437117 Gu et al. Downloaded by guest on September 24, 2021 A Endo-Hdj1DAPI Merge

B C Nuclear body Endo-Hdj1 DAPI Merge Endo-Hdj1 DAPI G3BP1 Merge Ub (PBS) No stress 1h) NPM1 Ars, M Stress μ (500 Coilin *** 2.0 2.0 Hdj1 Hdj1 f 1.5 1.5 o μm) nrb

1.0 er 1.0 p54 SG ( 0.5 0.5 BIOCHEMISTRY

Diamet 0.0 Diameter of nuclear body(μm) 0.0 No stress Stress No stress Stress SC35 Nuclear Stress body granule Stress PML

D E 50 μM Hdj1, pH 7.5, 50 mM NaCl Droplets 90 μM Hdj1, pH7.5 25 10% PEG 3,350 100 μM heparin 300 ng/μl polyU 10 mM NaCl No droplets 20 μM) 15 10 Hdj1 ( 5 0 100 300 500 F NaCl (mM)

Pre-bleach After-bleach 13 s 20 s 28 s 101 s ) 100 ce % 80 60 scen ery (

v 40 20 luore reco

F 0 0 40 80 120 time (s)

Fig. 1. Condensation of Hdj1 in the Ub-rich nuclear body and SG in cells and LLPS of Hdj1 in vitro. (A) Immunostaining of endogenous Hdj1 (endo-Hdj1) in HeLa cells. Hdj1 forms condensates in the nucleus. The 4′,6-diamidino-2-phenylindole (DAPI) is used to stain the nucleus. (Scale bar, 10 μm.) (B) Immunos- taining of endo-Hdj1 (green) and the marker proteins (red) of various nuclear bodies, including NPM1 for the nucleolus, coilin for the Cajal body, p54nrb for the paraspeckle, SC35 for the nuclear speckle, PML for the PML body, and Ub for the Ub-rich nuclear body. (Scale bar, 10 μm.) (C) Immunostaining images of endo-Hdj1 with or without stress (Top). The solid arrows indicate the colocalization of Hdj1 and G3BP1. G3BP1 marks stress granules (red). The empty arrows indicate the Hdj1-positive nuclear bodies. Ars: sodium arsenite. (Scale bar, 10 μm.) The fluorescence changes of Hdj1 in the nuclear bodies and SGs in the imaging data are analyzed and shown in the Middle. Student’s t test, n > 70 cells, ***P < 0.001. Error bars correspond to ± SD. Schematic of the redistribution of Hdj1 upon stress is shown at the Bottom.(D) Differential interference contrast (DIC) images of Hdj1 LLPS under indicated conditions. (Scale bar, 7.5 μm.) (E) Phase diagram of Hdj1 with 10% PEG 3,350. (F) FRAP measurement of Hdj1 droplets in 100 μM Hdj1, pH 7.5, 50 mM NaCl, and 10% dextran 70. (Scale bar, 2 μm.) Montages on the left show the processes of FRAP of a droplet. The graph on the right shows the recovery fraction as the function of time. Data shown are means ± SD, n = 3.

Gu et al. PNAS Latest Articles | 3of11 Downloaded by guest on September 24, 2021 Fig. 2. Molecular mechanism of Hdj1 LLPS. (A) Domain architecture of Hdj1 variants. (B) DIC imaging for the LLPS of Hdj1 domain-truncated variants under the indicated condition. (Scale bars, 10 μm.) Quantitative analysis of the droplet areas is shown on the right. Error bars correspond to mean ± SD, with n = 3. (C) LLPS turbidity of the Hdj1 variants under the same condition in B. Error bars correspond to mean ± SD with n = 3. Student’s t test was used in B and C; ***P < 0.001. (D) DIC imaging for the LLPS of Hdj1 amino acid-mutation or shuffle variants under the indicated condition. (Scale bars, 5 μm.) Quantitative analysis of the droplet areas is shown on the right. Error bars correspond to mean ± SD with n = 3. (E) LLPS turbidity of the Hdj1 variants under the same condition in (D). Error bars correspond to mean ± SD with n = 3. Student’s t test was used in (D and E), *P < 0.05; **P < 0.01; ***P < 0.001; N.S., not significant. (F) Confocal images of HeLa cells transfected with myc-Hdj1 WT or variants. Cells were treated with 0.1 mM sodium arsenite for 4 h and immunostained with anti-myc, anti-G3BP1, and DAPI. Arrows indicate Hdj1-positive stress granules. (Scale bar, 10 μm.) Quantitative analyses are shown on the right. Values of the percentage of cells with Hdj1-positive SGs over SG-positive cells are means ± SD and n > 450 cells from three repeats. Values of the Hdj1-positive SG area per cell are means ± SD and n > 100 cells from three repeats, normalized to the WT. Student’s t test, *P < 0.05; **P < 0.01; ***P < 0.001; N.S. (G) Western blot for the expression of Hdj1 variants in the transfected HeLa cells. GAPDH serves as a loading control.

4of11 | www.pnas.org/cgi/doi/10.1073/pnas.2002437117 Gu et al. Downloaded by guest on September 24, 2021 appeared higher than the others (Fig. 2G). Given the similarly compare the LLPS of FUS, Hdj1, and their mixture under the higher band of the F/R-A protein purified from Escherichia coli same condition, we used crowding agents, in the presence of (SI Appendix, Fig. S4B), this observation might not reflect which both FUS-EGFP and Hdj1 underwent LLPS individually posttranslational modifications but a probably more extended monitored by confocal microscopy and turbidity measurement conformation of F/R-A due to the mutations. Taken together, (Fig. 3 A and B). Remarkably, as mixing the two proteins to- these results indicate that the LLPS property of Hdj1 is impor- gether, we found that they not only cophase separated, but also tant for its incorporation into SGs. exhibited a markedly enhanced LLPS behavior (Fig. 3 A and B). Enhanced co-LLPS of Hdj1 and FUS was also observed in the LLPS Is a General Property of Class I and II Hsp40 Proteins. Since class absence of crowding agents (SI Appendix,Fig.S6A). Measure- I and II Hsp40 proteins generally contain a flexible G/F-rich ment of the amounts of proteins in the droplets showed that the region, we asked whether phase separation is a common feature co-LLPS led to significantly more Hdj1 involved in the droplets shared by class I and II Hsp40s. We tested human Hdj2, a (SI Appendix,Fig.S6B), indicating a synergistic phase separa- member of class I Hsp40. The result showed that similar to Hdj1, tion of Hdj1 and FUS. Furthermore, FRAP experiment showed Hdj2 spontaneously underwent phase separation in the presence that both proteins exhibited high mobility in the co-LLPS C of the crowding agent or polyarginine (peptide CR20), which was droplets (Fig. 3 ). Consistent with the co-LLPS of Hdj1 and reversed by the increase in salt concentration (SI Appendix, Fig. FUS, we observed colocalization of myc-Hdj1 and red fluo- S5A). Unlike the liquidlike droplets formed by Hdj1, Hdj2 rescent protein (RFP)-FUS R521H (a FUS mutant that is ready formed gel-like droplets with low mobility detected by FRAP (SI to involve in SGs, ref. 30) in SGs in HeLa cells upon stress Appendix, Fig. S5A). Moreover, Hdj2 mainly localized in the (Fig. 3D). nucleolus rather than the Ub-rich nuclear body as Hdj1 (SI To know whether Hdj1 specifically undergoes synergistic co- Appendix, Fig. S5B). While, similar to Hdj1, Hdj2 also transfers LLPS with FUS, we examined the co-LLPS of Hdj1 with to SGs upon stress (SI Appendix, Fig. S5C). hnRNPA1, another RNA-binding protein in SGs. The result In addition, we examined Hdj1 and Hdj2 homologs Sis1 and showed that although Hdj1 and hnRNPA1 underwent co-LLPS, Ydj1 from yeast, which also contain a flexible G/F-rich region. synergistic effect was not obvious by confocal microscopy (SI We observed phase-separation phenomena of Sis1 and Ydj1, Appendix, Fig. S6C), neither could Hdj1 enhance the phase similar to their human homologs (SI Appendix, Fig. S5D). These transition of hnRNPA1 by the turbidity measurement (SI Ap- results suggest that phase separation may represent a common pendix, Fig. S6D). Taken together, these results suggest that the

feature shared by class I and II Hsp40 proteins from different co-LLPS of Hdj1 with RNA-binding proteins, especially FUS, BIOCHEMISTRY organisms, and they may localize in different membraneless or- may enable an efficient SG incorporation of Hdj1. ganelles to fulfill their functions. Hdj1 Protects Phase-Separated FUS against Amyloid Aggregation and Synergistic Phase Separation and Colocalization of Hdj1 and FUS in Alleviates Abnormal FUS Aggregation in Cells. FUS can form path- SGs. Since many SG-associated RNA-binding proteins possess ological amyloid fibrils mainly mediated by its N-terminal LC the ability of LLPS, we asked whether there is synergistic inter- domain (FUS-low complexity [LC]) (31, 32), which is associated action between the LLPS of Hdj1 and the RNA-binding pro- with ALS and FTD (9, 33). As we observed in vitro, both FUS- teins. We prepared FUS with enhanced green fluorescent EGFP and FUS-LC domains N-terminally fused with cyan fluo- protein (EGFP) fused at the C terminus (FUS-EGFP). To rescent protein (CFP) (CFP-FUS-LC) formed amyloid fibrils

Fig. 3. Synergistic LLPS and colocalization of Hdj1 and FUS in vitro and in SGs. (A) Fluorescence images of co-LLPS of Hdj1 and FUS. Protein concentrations and LLPS conditions are indicated. (Scale bar, 20 μm.) (B) Turbidity measurement of LLPS of Hdj1 and FUS. Data show mean ± SD, n = 3. (C) FRAP of Alexa647- labeled Hdj1 or FUS-EGFP in co-LLPS droplets. Error bars correspond to mean ± SD with n = 7. (D) Representative confocal images of HeLa cells transfected with RFP-FUS-R521H and myc-Hdj1 WT. Cells were treated with phosphate-buffered saline (no stress) or 500 μM sodium arsenite (stress) for 1 h and immunostained with anti-G3BP1 and anti-myc. Insets are shown to clarify colocalization. (Scale bar, 10 μm.)

Gu et al. PNAS Latest Articles | 5of11 Downloaded by guest on September 24, 2021 Fig. 4. Hdj1 stabilizes FUS liquid droplets against proceeding to solid aggregation. (A) Representative images of the morphological changes of full-length FUS-EGFP droplets in the absence or presence of Hdj1 over time are shown on the left. (Scale bar, 25 μm.) Images of CFP-FUS-LC (residues 1–214) droplets are shown on the right. (Scale bar, 15 μm.) (B) FRAP of CFP-FUS-LC with or without Hdj1. Data are shown as mean ± SD with n = 3. (C) The ThT fluorescence assay of 25 μM FUS-LC (residues 1–163) with different concentrations of Hdj1. Data correspond to mean ± SEM, n = 3. Transmission electron microscropy (TEM) images of ThT samples visualized by TEM at 40 h are shown on the right. (Scale bar, 200 nm.) (D) Images of CFP-FUS-P525L aggregation puncta in cells in the presence of Hdj1 WT or F/R-A visualized by CFP fluorescence for FUS-P525L and pFTAA staining for amyloid aggregation. (Scale bar, 20 μm.) Quantification of the aggregation area per transfected cell at each image is shown (right). Student’s t test, n > 450 cells and 40 images from four repeats, ***P < 0.001; N.S. Error bars, S.D. (E) Fluorescence images of HeLa cells transfected with CFP-FUS-P525L and myc-Hdj1 WT or F/R-A. Hdj1 proteins were immunostained with anti- myc. The arrows show the Hdj1-positive FUS puncta. (Scale bar, 10 μm.) The percentage of transfected cells with Hdj1-positive FUS puncta is counted. Student’s t test, n > 250 cells from three repeats, and **P < 0.01. Values are means ± SD. Western blot shows the expression of the myc-Hdj1 WT and F/R-A in the transfected cells. GAPDH serves as a loading control.

growing out of the phase separated droplets over time (Fig. 4A). A FUS P525L is an ALS-associated mutation which disrupts the similar phenomenon was observed in G156E mutant FUS (11). nuclear localization of FUS and aggregates in the cytoplasm Intriguingly, we found that this so-called maturation process can (34–37). We transfected HeLa cells with N-terminally CFP be disrupted by Hdj1. In the presence of Hdj1, the maturation fused FUS P525L (CFP-FUS P525L) and probed its aggrega- processes of both FUS and FUS-LC were markedly delayed, in- tion in the cytoplasm by CFP fluorescence and pFTAA staining dicating that FUS proteins were maintained in the liquidlike (Fig. 4D). Note that no stress, such as sodium arsenite was phase-separated state rather than proceeding to fibril formation applied, and no typical SGs were observed in this experiment (Fig. 4A). Analysis of the FUS fluorescence confirmed that the (SI Appendix,Fig.S7B). As we transfected myc-Hdj1 into the addition of Hdj1 did not influence the concentration of FUS in cells, myc-Hdj1 entered the FUS P525L aggregation puncta the droplets (SI Appendix,Fig.S7A). Consistently, the FRAP and significantly mitigated the aggregation of FUS P525L experiment showed that the presence of Hdj1 maintained the (Fig. 4D and SI Appendix,Fig.S7C and D). To correlate the mobility of the CFP-FUS-LC droplets (Fig. 4B). ThT kinetic assay cellular observation with the biochemical data, we measured also showed that Hdj1 strongly inhibited the amyloid aggregation the ratio of FUS and Hdj1 in the cells (SI Appendix,Fig.S7E). of FUS-LC in a dose-dependent manner (Fig. 4C). The result showed that the amount of total Hdj1 in the cellular To assess the chaperone activity of Hdj1 against FUS aggre- lysate is 0.126 μM and that of FUS is 0.053 μM. Thus, the gation in cells, we used a cell model with FUS P525L aggregation. molar ratio of FUS to Hdj1 is ∼1: 2.4. At this ratio, Hdj1 can

6of11 | www.pnas.org/cgi/doi/10.1073/pnas.2002437117 Gu et al. Downloaded by guest on September 24, 2021 effectively inhibit FUS aggregation according to the biochemical it has been shown that FUS-RGG can enhance the LLPS of data (Fig. 4 A–C). FUS-LC (28, 38). Thus, Hdj1 and FUS-RGG act analogously in In contrast, Hdj1 F/R-A mutant showed no significant inhi- promoting the LLPS of FUS-LC. bition to FUS P525L aggregation in cells (Fig. 4D and SI Ap- We next examined the co-LLPS of Hdj1 and FUS-RGG. pendix, Fig. S7 C and D), although it exhibited a high inhibitory Confocal microscopy showed that at the concentration of 50 μM activity in vitro (SI Appendix, Fig. S8A). Immunostaining showed FUS-RGG and 250 μM Hdj1, neither of the individual proteins that Hdj1 F/R-A was deficient in localizing with the FUS P525L underwent obvious LLPS; while as mixing them together, LLPS puncta (Fig. 4E and SI Appendix, Fig. S8B). Thus, these results occurred with both proteins condensed in the droplets (Fig. 5E). indicate that, although the chaperone activity of Hdj1 on amyloid Both proteins exhibited a high mobility in the droplets as shown inhibition does not rely on the LLPS property of Hdj1, the LLPS by FRAP (Fig. 5E). Similar to that observed in the co-LLPS of property is essential for the proper localization of Hdj1 in cells to the Hdj1 and FUS-LC, Hdj1 ΔG/F diminished the co-LLPS of fulfill its biological function efficiently. Hdj1 and FUS-RGG (Fig. 5F and SI Appendix, Fig. S12); while unlike that of the Hdj1 and FUS-LC co-LLPS, the F-A muta- Hdj1 Uses Different Mechanisms to Chaperone FUS Phase Separation tions of Hdj1 were more harmful than the R-A mutations on promoting the co-LLPS of Hdj1 and FUS-RGG (Fig. 5F and SI and Amyloid Aggregation. FUS contains two major intrinsically dis- Appendix ordered regions (IDRs), i.e., a N-terminal LC domain (FUS-LC, , Fig. S12). Given that FUS-RGG is rich in Arg, this residues 1–163) and a C-terminal RGG-rich domain (FUS-RGG, result indicates that Hdj1 cophase separates with FUS-RGG mainly via the π-cation interactions between the Phe of Hdj1 residues 371–526), which drive the LLPS of full-length FUS (38). NTD and the Arg of FUS-RGG. To understand the molecular mechanism underlying the inter- In contract as for the amyloid inhibition activity of Hdj1, we play between Hdj1 and FUS, we investigated the two FUS IDRs found that Hdj1 ΔCTD nearly completely abolished the activity separately. We first examined the co-LLPS of Hdj1 and FUS-LC of Hdj1 in inhibiting the amyloid fibrillation of FUS-LC with a gradient concentration of each protein. Confocal mi- (Fig. 5G). While ΔNTD and F/R-A mutations, which showed croscopy showed that at the concentration of 50 μM of each severe impairment on the co-LLPS of Hdj1 and FUS-LC, only protein, neither of the individual FUS-LC or Hdj1 underwent moderately decreased the amyloid inhibition activity of Hdj1 LLPS; while as mixing them together, LLPS occurred with both (Fig. 5 D and G). This result indicates that the Hdj1 CTD, the proteins condensed in the droplets, and as the concentrations of client-binding domain (42, 43), plays a more important role than Hdj1 and FUS-LC increased, the complex droplets grew larger A the NTD in inhibiting FUS amyloid aggregation, which is op-

(Fig. 5 ). FRAP experiment showed that both Hdj1 and FUS- BIOCHEMISTRY B posed to the activity of Hdj1 in the co-LLPS with FUS where the LC exhibited high mobility in the droplets (Fig. 5 ). NTD is more important. Taken together, Hdj1 uses different We next sought to use NMR spectroscopy to map the inter- domains to chaperone different states of FUS. It mainly adopts action between Hdj1 and FUS-LC. Since LLPS may lead to its G/F-rich region to interact with different IDRs of FUS to signal changes in the heteronuclear single quantum coherence promote the cophase separation with FUS. In the phase- (HSQC) spectrum, to detect the interaction between Hdj1 and 15 separated state, Hdj1 further employs its CTD to tackle FUS- FUS-LC, we prepared N-labeled FUS-LC at the concentration LC against amyloid aggregation. of 40 μM in the buffer containing 25 mM 2-(N-morpholino) ethanesulfonic acid (MES), 150 mM NaCl, pH 6.6, and 10% Discussion glycerol, which is below the critical LLPS concentration of FUS- In this paper, we identified the phase-separation property of < μ SI Appendix LC alone and in the presence of Hdj1 ( 160 M) ( , class I and II Hsp40 (DNAJ) proteins and demonstrated the A Fig. S9 ). The HSQC spectra showed that upon Hdj1 titration, importance of this property to the biological function of Hsp40 the signal intensities of FUS-LC exhibited a general decrease in in chaperoning the assembly and maintenance of membraneless a concentration-dependent manner with no obvious chemical organelles. We found that human Hsp40 proteins, Hdj1 and C SI Appendix B shift perturbations (Fig. 5 and , Fig. S9 ). The Hdj2, localize in different nuclear bodies in cells under normal assignment of the HSQC spectrum of FUS-LC was archived conditions. While upon stress, both chaperones incorporate into – accordingly to previous data (39 41). We observed no distinct SGs. We further focused on Hdj1 and showed that the cophase region or residue-specific intensity attenuation of FUS-LC upon separation with FUS may mediate its SG involvement, which Hdj1 titration. These results indicate weak and transient inter- further maintains the dynamics of SGs against progressing into actions between Hdj1 and FUS-LC. solid aggregation. We deciphered the roles of the multiple do- To identify which region of Hdj1 accounts for the co-LLPS mains of Hdj1 over this process (Fig. 6, Inset). Remarkably, the with FUS-LC, we added different variants of Hdj1 to FUS-LC G/F-rich region, which is shared by class I and II Hsp40s plays an and observed that Hdj1 ΔG/F showed weakened ability on essential role in the Hdj1 LLPS, and accounts for the synergistic promoting the co-LLPS than that of the WT, and Hdj1 ΔNTD phase separation with FUS. The DD domain also provides ad- further weakened the ability (Fig. 5D and SI Appendix, Fig. S10). ditional valences for Hdj1 LLPS. As for inhibiting amyloid Relatively, Hdj1 ΔDD and ΔCTD remained significant activities aggregation, although Hdj1 mainly uses its well-folded CTD, in promoting the co-LLPS (SI Appendix, Fig. S11A). In addition, proper localization to membraneless organelles is still required NMR titration showed that ΔDD did not obviously impair the via phase separation. binding of Hdj1 with FUS-LC (SI Appendix, Fig. S11B). In- Note that class III Hsp40 proteins only contain a J domain triguingly, we found that the R-A mutations of Hdj1 resulted in with no other canonical domains (SI Appendix, Fig. S13A) and, more severe impairment than the F-A mutations on promoting thus, were not well characterized in this paper. However, we the co-LLPS of Hdj1 and FUS-LC (Fig. 5D and SI Appendix, Fig. indeed observed SG incorporation of a class III Hsp40— S10). Given that FUS-LC is rich in the Tyr residue, this result DNAJC7 under stress (SI Appendix, Fig. S13B), which indicates indicates that π-cation interactions between the Arg of Hdj1 that DNAJC7 may also be able to phase separate and play a role NTD and the Tyr of FUS-LC may play an important role in the in regulating SG assembly. This is consistent with a recent study co-LLPS of Hdj1 and FUS-LC. While the R-K mutations finding truncating variants of DNAJC7 in ALS (24). Thus, the exhibited a decreased but markedly stronger ability in promoting phase-separation property is likely to be a general property the co-LLPS than that of the R-A mutations (Fig. 5D and SI throughout different classes of Hsp40 proteins. Appendix, Fig. S10), which indicates that charge interactions also Under normal conditions, we found that Hdj1 mainly localizes contribute to the co-LLPS between Hdj1 and FUS-LC. Of note, in a type of Ub-rich nuclear body in HeLa cells. Upon stress,

Gu et al. PNAS Latest Articles | 7of11 Downloaded by guest on September 24, 2021 Fig. 5. Distinct mechanisms of Hdj1 in chaperoning FUS LLPS and fibrilization. (A) Fluorescence images of the co-LLPS of FUS-LC and Hdj1 under the indicated conditions. (Scale bar, 10 μm.) Domain organization of FUS is shown on Top.(B) FRAP of FUS-LC and Hdj1 from the droplets in A. Data represent mean ± SD, n = 3. (C) Residue-specific intensity changes of signals in the two-dimensional (2D) 1H-15N HSQC spectra of 40 μM 15N-labeled FUS-LC in the presence of Hdj1 WT (blue bars) or F/R-A (pink line). Molar ratios are indicated. Error was propagated from the signal to noise values of each spectrum. (D) Turbidity measurement of 50 μM FUS-LC with 100 μM Hdj1 and its variants as indicated at 4 °C. Data correspond to mean ± SD, n = 3. Student’s t test. **P < 0.01; ***P < 0.001. (E) Fluorescence images of the co-LLPS of Alexa555-labeled FUS-RGG (residues 371–526) and Alexa647-labeled Hdj1 under the indicated conditions. (Scale bar, 5 μm.) FRAP experiments for FUS-RGG and Hdj1 in the cophase separated droplets are shown on the right. Data correspond to mean ± SD, n = 3. (F) The turbidity measurement of 25 μM FUS-RGG with 100 μM Hdj1 variants as indicated at 8 °C. Data correspond to mean ± SD, n = 3. Student’s t test. *P < 0.05; **P < 0.01; ***P < 0.001; N.S. (G) ThT fluorescence assay of 25 μM FUS-LC with 5 μM Hdj1 variants. Data correspond to mean ± SEM with n = 3. The lag time of ThT fluorescence kinetic curves of different samples is shown in the bar graph. Data correspond to mean ± SD with n = 3. Student’s t test. **P < 0.01; ***P < 0.001.

8of11 | www.pnas.org/cgi/doi/10.1073/pnas.2002437117 Gu et al. Downloaded by guest on September 24, 2021 Fig. 6. Schematic of Hsp40 proteins in the regulation of membraneless organelles. Different Hsp40 proteins, such as Hdj1 and Hdj2, localize in different membraneless organelles in cells. Hdj1 and Hdj2 condense in SGs upon stress where they chaperone the dynamics of SGs against progressing into solid aggregation. The incorporation of Hsp40 in SG assembly may recruit cochaperones and mediate the buildup of chaperone network in SGs. Genetic defect of Hsp40 or decline of chaperone function during aging may lose the regulation of the conformation and function of membraneless organelles so that lead to protein aggregation and neurodegenerative diseases. The inset dissects the activities of Hdj1 domains.

Hdj1 condenses in SGs (Fig. 6). Our observation is discrepant the G/F-rich domain may release the J domain to attract Hsp70. BIOCHEMISTRY with an early study showing that Hdj1 is accumulated in nucleoli At the same time, condensation of Hsp40 in membraneless in heat-shocked HeLa cells (44). While we indeed observe that organelles may recruit Hsp70 and facilitate a hierarchical Hdj2 condenses in nucleoli in HeLa cells (Fig. 6 and SI Appendix, buildup of chaperone network in membraneless organelles Fig. S5B). A recent study found that the nucleolus can store (Fig. 6). misfolded proteins and avoid irreversible aggregation (45). Thus, the localization of Hdj2 in the nucleolus indicates its function in Materials and Methods protecting misfolded proteins from aggregation in this protein Protein Expression and Purification. All recombinant proteins were fused with quality control membraneless organelle. Under stress, we ob- a His tag, which were expressed in E. coli and purified using a Ni column (GE served that Hdj2 enters SGs, which is consistent with proteomic Healthcare, US) unless otherwise noted. analysis of the SG (5). A previous study has termed a type of — nuclear body a clastosome that is highly enriched in the com- DIC and Fluorescent Imaging for LLPS. Samples were loaded onto a glass slide ponents of the Ub-proteasome pathway of proteolysis (46). Thus, with a coverslip, and images were acquired on a Leica TCS SP8 microscope. the observed Ub-rich nuclear body might be clastosome, which is Temperature-dependent samples were performed using a temperature- consistent with the previous study showing that Hdj1 transfers controlled stage (TP-CHSQ-C thermal stage) at the indicated temperature. misfolded proteins to the nucleus for degradation (47). Different members in the Hsp40 family widely exist in different cellular NMR. All NMR titration experiments were performed at 298 K on a Bruker 900 localizations and are involved in diverse biological functions MHz spectrometer equipped with cryogeni probe in a NMR buffer of 25 mM (48). Shared by different Hsp40 proteins, the property of phase MES (pH 6.6), 150 mM NaCl, 10% glycerol, and 10% D2O. Each NMR sample separation may be essential for the localization of Hsp40 pro- was made with a volume of 500 μL, containing 15N-FUS-LC (40 μM) diluted teins to their functional loci to chaperone protein homeostasis from 15N-FUS-LC (∼2.0 mM) in 5 mM CAPS (pH 11.0) without/with 80 μM and maintain the dynamics of membraneless organelles. In ad- Hdj1, 160 μM Hdj1, 160 μM ΔDD, or 160 μM F/R-A. Bruker standard pulse dition, a previous study has shown that Hsp40 can rescue the sequence (hsqcetfpf3gpsi) was used to collect the 2D 1H-15N HSQC spectrum cytotoxicity of FUS in cells (22). Thus, Hsp40 proteins might be a with 256 scans. Some 2,048 × 200 complex points were used for 1H (14 ppm) promising drug target for ALS or other neurodegenerative dis- and 15N (22 ppm) dimensions, respectively. All NMR data were processed by eases. Further testing these findings in an in vivo context, such as NMRpipe (52) and analyzed by SPARK (53). a mouse or fly model would provide important information for effectively targeting Hsp40 against neurodegeneration. FUS Maturation and Solidification Assay. Samples of CFP-FUS-LC in the aging We identified that the G/F-rich region is primary to mediate buffer containing 50 mM NaCl, and 20% dextran 70 were applied to a glass the phase separation of Hdj1, truncation or mutation of which bottom cell culture dish with a coverslip. The images and FRAP recordings were markedly impaired the phase separation and SG involvement of acquired at indicated time points. As for full-length FUS, protein samples were Hdj1. Although the G/F-rich region is predicted to be exposed prepared into the 384 well microplates in 25 mM 2-amino-2-hydroxymethyl-1,3- and disordered (SI Appendix,Fig.S2A), a recent study showed propanediol·HCl, pH 7.5, 75 mM NaCl, and the plate was shaken at 900 rpm as that the conserved DI/VF motif in the G/F-rich region is in- reported previously (54). volved in the formation of a helix, which masks the J domain (49). As a cochaperone of Hsp70, Hsp40 binds to Hsp70 via its Cell Culture and Transfection. HeLa cells (Cell Bank of the Chinese Academy of J domain and regulates Hsp70 activity (50, 51). The inter- Sciences, Shanghai, SCSP-504) were used throughout. PolyJet reagent domain interactions between the G/F-rich region and the J (SignaGen, SL100688) was used for the transfection of plasmids into HeLa domain create an autoinhibitory state for the binding of Hsp70 cells. Cells were transfected for 24 h before proceeding with subsequent (49). Thus, we hypothesize that phase separation mediated by experiments.

Gu et al. PNAS Latest Articles | 9of11 Downloaded by guest on September 24, 2021 Analysis of Hdj1 Assembly in SGs. For tracking Hdj1 WT and its variants as- spanning 185 × 185 μm2. Each pixel was 180 nm in length. Finally, the ag- sembly in SGs, HeLa cells were transfected with 0.3 μg of the plasmid of myc- gregation area per transfected cell and mean integrated fluorescent inten- Hdj1 WT or its variants. Transfected HeLa cells were fixed and immunostained sity of CFP or pFTAA were calculated at each image. At least, nine images with anti-myc and anti-G3BP1 after stress. The percentage of myc-Hdj1-posi- were taken for each slide, n ≥ 4. tive SGs containing cells was then calculated: (number of cells with myc-Hdj1- positive SGs/number of transfected cells with SGs) × 100. Every transfected cell in the selected area was counted. In each experiment, 140–200 cells per slide Quantification and Statistical Analysis. Statistical parameters including the were counted, n = 3. As for quantification of the Hdj1-positive SG area per definitions and exact values of n (e.g., number of biological repeats, number cell, a low threshold for the Hdj1 signal was used to generate a mask to of flies, etc.), distributions and deviations are reported in the figures and remove the G3BP1 signal. Over 100 cells from three repeats were counted. figure legends. The statistical significance in this study is determined by Mann–Whitney U test or the unpaired two-tailed Student’s t test at *P < < < Analysis of Hdj1 and FUS Assembly in Cells. As for quantification of colocali- 0.05, **P 0.01, and ***P 0.001. Statistical analysis was performed in zation of aggregated CFP-FUS with the Hdj1 variant, HeLa cells were GraphPad Prism. transfected with 0.7 μg of the CFP-FUS-P525L plasmid and 0.2 or 0.02 μgof Detailed methods and materials are provided in the SI Appendix. the myc-Hdj1 plasmid per well. At 24 h posttransfection, cells were fixed and immunostained with anti-myc. The percentage of myc-Hdj1-positive CFP-FUS Data Availability. All data and procedures are provided in the article and aggregation containing cells was then calculated: (number of cells with myc- SI Appendix. Hdj1-positive aggregation/number of transfected cells) × 100. Every transfected cell in the selected area was counted. In each experiment, 70–100 cells per slide were counted, n = 3. ACKNOWLEDGMENTS. We are grateful for the assistance on NMR data collection from the National Center for Protein Science, Shanghai. This work was supported by the Major State Basic Research Development Program Measurement of FUS Cytoplasmic Aggregation. HeLa cells were transfected (2016YFA0501902), the National Natural Science Foundation (NSF) of China with CFP-FUS-P525L plus empty vector, Hdj1 WT, or F/R-A. At 24 h post- (Grants 91853113 and 31872716), the Science and Technology Commission of transfection, cells were fixed, permeabilized, and stained by pFTAA which Shanghai Municipality (Grant 18JC1420500), the “Eastern Scholar” Project can preferentially recognize the β-sheet-rich cellular aggregates (38). The supported by Shanghai Municipal Education Commission, Shanghai Munici- pFTAA molecule was prepared as reported procedures (55). Each image was pal Science and Technology Major Project (2019SHZDZX02), and Innovation taken by the Leica TCS SP8 confocal microscopy system equipped with a Program of Shanghai Municipal Education Commission (Grant 2019-01-07- 63 ×objective (water immersion) with Z stacks over 6 μm and an area 00-02-E00037).

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