DYT1 dystonia is a debilitating neurological movement disorder arising from mutation in the AAA+ ATPase TorsinA. The hallmark of Torsin dysfunction is nuclear envelope blebbing resulting from defects in nuclear pore complex biogenesis. Whether blebs actively contribute to disease manifestation is unknown. We report that FG-nucleoporins in the bleb lumen form aberrant condensates and contribute to DYT1 dystonia by provoking two proteotoxic insults. Short-lived ubiquitylated proteins that are normally rapidly degraded partition into the bleb lumen and become stabilized. In addition, blebs selectively sequester a specific HSP40–HSP70 chaperone network that is modulated by the bleb component MLF2. MLF2 suppresses the ectopic accumulation of FG-nucleoporins and modulates the selective properties and size of condensates in vitro. Our study identifies dual mechanisms of proteotoxicity in the context of condensate formation and establishes FG-nucleoporin-directed activities for a nuclear chaperone network.
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The LC–MS/MS datasets were uploaded to the massIVE database (accession nos MSV000090177, MSV000090186, MSV000090187 and MSV000090188 for Supplementary Tables 1–4, respectively). The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the following accession numbers: PXD036262, PXD036264, PXD036266 and PXD036267 for Supplementary Tables 1–4, respectively. 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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This work was supported by the National Institutes of Health (NIH) R01GM114401 (C.S.), DOD PR200788 (C.S.), NIH 5T32GM007223-44 (S.M.P.), NIH F31NS120528 (S.M.P.), NIH R56MH122449 (A.J.K.), NIH R01MH115939 (A.J.K.), NIH NS105640 (A.J.K.), NIH F31MH116571 (J.E.G.) and the Dystonia Medical Research Foundation (C.S. and A.J.R.). The mass spectrometers and accompanying biotechnology tools at the MS & Proteomics Services, Keck Biotechnology Resource Laboratory at Yale University were funded in part by the Yale School of Medicine and the Office of the Director, NIH (S10OD02365101A1, S10OD019967 and S10OD018034). We thank D. Görlich and the members of his laboratory for sharing reagents. We thank the Yale Keck Biophysical Resource Center, M. Graham and the Yale Center for Cellular and Molecular Imaging. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.
The authors declare no competing interests.
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a, Schematic illustration of the NE blebs that form upon Torsin deficiency. In cells with mutant TorsinA, NPC biogenesis is compromised and a subset of nascent NPCs are arrested (ref. 22). These arrested NPCs form blebs that are enriched for K48-linked ubiquitin (K48-Ub), FG-nucleoporins (FG-Nups), myeloid leukaemia factor 2 (MLF2), and a specific chaperone network. The inner nuclear membrane (INM) is depicted in grey, outer nuclear membrane in black. b, Representative IF images of TorsinKO cells expressing MLF2-FLAG and ∆133 ORF10-HA fusion constructs. ∆133 ORF10 was fused to the cytomegalovirus deubiquitinase (DUB) domain, M48 (ref. 74). M48 is a highly active DUB domain that efficiently removes ubiquitin conjugates (ref. 22,35). A C23A mutation renders the DUB domain catalytically inactive (ref. 74). Scale bar, 5 µm.
Extended Data Fig. 2 Additional abundant molecular chaperones are sequestered into NE blebs of tissue culture cells and primary mouse neurons.
a, Representative image of endogenous HSC70 in WT and TorsinKO HeLa cells. Note that HSC70 re-localizes from diffusely throughout the cell to nuclear rim foci in TorsinKO cells. b, SH-SY5Y cells expressing a dominant-negative TorsinA construct, TorsinA-EQ-HA, sequester HSPA1A and DNAJB6 into NE blebs. Yellow arrowhead, transfected cell. Blue arrow, untransfected cell. Endogenous chaperones (green) form foci around the nuclear rim upon TorsinA-EQ-HA (red) expression. c, Murine DIV4 hippocampal neurons were transfected with GFP and either MLF2-HA alone or in combination with a dominant-negative TorsinA-EQ construct. Constructs were allowed to express for 72 h before processing the DIV7 cultures for IF. Note that GFP expression was used to distinguish neurons from other cell types in the heterogeneous primary cell culture. Scale bar, 5 µm.
a, Representative IF images of TorsinKO cells treated with DMSO or VER-155008 for 24 h. VER-155008 is a small molecule inhibitor of HSP70 ATPase activity, which targets the HSP70 ATP binding pocket and approximates an ADP-bound state (ref. 40). This compound causes elevated HSPA1A expression and more K48-Ub to accumulate in blebs. Scale bar, 5 µm. b, An IP of HSPA1A from biochemically enriched ER/NE fractions from WT or TorsinKO cells treated with DMSO or VER-155008. c, A co-IP of MLF2-FLAG with HA-tagged DNAJB6 constructs lacking functional G/F-rich or S/T-rich regions in TorsinKO cells. ΔG/F indicates all phenylalanine residues have been mutated to alanine within the G/F-rich region, and ΔG/F-S/T indicates the mutation of the phenylalanine residues within the G/F- and S/T-rich regions. Note that DNAJB6-ΔG/F-S/T-HA retrieves less MLF2-FLAG, suggesting that the S/T-rich region strongly promotes its interaction with MLF2 and recruitment to blebs. d, Representative IF images of the DNAJB6 constructs described in panel (c) in TorsinKO cells. Note that interfering with the S/T-rich region, but not the G/F-rich region, prevents DNAJB6 from reaching the bleb. Scale bar, 5 µm. e, DNAJB6 peptides identified by mass spectrometry from an IP of HSPA1A from TorsinKO cells (see Fig. 4b–d). Peptides identified by mass spectrometry are highlighted in yellow and post-translationally modified residues are rendered in green. While an IP of HSPA1A from TorsinKO cells identified DNAJB6 with 24% coverage, no DNAJB6 peptides were identified in the HSPA1A IP from WT HeLa cells. See Supplementary Table 3 for complete dataset. Unprocessed blots are available in source data.
a, qPCR validation of Nup98 depletion upon 48 h of 50 nM siRNA treatment. Relative Nup98 transcript levels are normalized to RPL32. b, Nup98 and Nup96 are translated as a single precursor protein that undergoes an autocleavage event to produce the two individual proteins (ref. 45). Thus, RNAi knockdown of Nup98 results in the simultaneous depletion of Nup96. c-e, To distinguish which protein knockdown produces the cytosolic granules in TorsinKO cells, HA-tagged Nup98 or Nup96 was assessed for the ability to rescue the phenotype under knockdown conditions. c, Quantification of the rescue affect when HA-Nup96 or HA-Nup98 are expressed. The presence of cytosolic inclusions was assessed for 300 cells/condition in ≥20 ROIs. Error bar, SD. Statistical analysis was performed using a two-tailed unpaired Mann–Whitney test. ** indicates p = 0.0013. d, Representative IF images of TorsinKO cells expressing HA-Nup96 or HA-Nup98 (e) under non-targeting or siNup98-96 conditions. Results are quantified in panel (c). f, Representative IF images of endogenous Hsc70 and HSPA1A in TorsinKO cells upon Nup98 depletion. Note that these HSP70 members are not recruited to the cytosolic granules. g, Representative IF images of the Ran GTPase in TorsinKO cells under 48 h of siNT or siNup98 conditions. h, The nuclear to cytoplasmic ratio was calculated for GFP-NES in TorsinKO cells under siNT (n = 94) or siNup98 (n = 87) conditions. The ratio was calculated using CellProfiler software (ref. 64). Statistical analysis was performed using a two-tailed unpaired Mann–Whitney test. Ns, not significant. Scale bar, 5 µm for all panels. Source numerical data are available in source data.
Extended Data Fig. 5 The effects of 5% 1,6-hexanediol on NE integrity and bleb sensitivity to 2,5-hexanediol.
a, Representative IF images of TorsinKO cells expressing polyQ-97-GFP under normal IF conditions or exposed to 5% 1,6-hexanediol for five minutes prior to fixation. Note that the K48-Ub inside polyQ aggregates is not dissolved by 1,6-hexanediol but the K48-Ub inside blebs is sensitive to this alcohol. Lamin A staining demonstrates this 5% 1,6-hexanediol treatment does not break down NE membranes (see also panel (e)). b, Representative IF images of TorsinKO cells expressing polyQ-97-GFP and MLF2-HA treated with 5% 1,6-hexanediol for five minutes. A five-minute treatment with 5% 1,6-hexanediol does not dissolve polyQ aggregates but can selectively dissolve the contents of blebs. c, Treatment with the related alcohol 5% w/v 2,5-hexanediol for five minutes does not dissolve the K48-Ub or MLF2-HA sequestered inside blebs. d, Representative IF images of Ran in TorsinKO cells under normal IF conditions or after treatment with 5% 1,6-hexanediol for five minutes. Although the NE membranes are not disassembled by a 5% 1,6-hexanediol, the permeability barrier established by NPCs is lost. 1,6-hexanediol is known to disrupt the hydrophobic contacts required for the cohesion of FG-Nups within the central channel of the NPC (ref. 51). e, Representative IF images of TorsinKO cells treated with 5% w/v 1,6-hexanediol for five minutes prior to fixation. This treatment does not compromise the integrity of the NE as determined by staining for multiple inner nuclear membrane proteins. Scale bar, 5 µm for all panels.
a, Multiple sequence alignment of MLF2 homologues from Xenopus tropicalis, Danio rerio, Mus musculus, and Homo sapiens. MLF2 is a methionine- and arginine-rich protein with a high degree of conservation. Methionine residues are highlighted in yellow boxes, arginine in blue, and positively charged residues in red. Orange cylinders indicate alpha helices predicted by AlphaFold75 and blue arrows represent predicted beta sheets. b, MLF2 purified from Expi293F cells associates with HSPA1A. MLF2 was expressed as a maltose binding protein (MBP) fusion and affinity purified by virtue of FLAG- and His-tags. Lane 1, anti-FLAG resin elution composed of MLF2-Tev-MBP-His-FLAG and associating protein. Red box, gel section analysed by mass spectrometry. Lane 2, incubation with Tev-His cleaved MBP-His from MLF2. Lane 3, Ni-NTA flowthrough in which MBP-His and Tev-His are removed. Mass spectrometry of the gel segment indicated in lane 1 confirmed the identity of the associating protein as HSPA1A. c, Mass spectrometry analysis of the co-purifying protein from panel (b) revealed 55% coverage of HSPA1A. Identified peptides mapping to HSPA1A are in yellow, post-translationally modified residues in green. See Supplementary Table 4 for complete dataset. d, 10 µM TtMacNup98A condensates were formed in the presence of 5 µM 3B7C-GFP plus 10 µM HSPA1A, DNAJB6Bb, or MLF2:HSP70. 3B7C-GFP signal intensity was measured in the center of 100 condensates/condition. Bars over datapoints indicate the mean intensity value. Statistical analysis was performed using a two-tailed unpaired Mann–Whitney test comparing buffer or HSPA1A conditions to DNAJB6b or MLF2:HSP70. **** indicates p < 0.0001. Source numerical data and unprocessed blots are available in source data.
Extended Data Fig. 7 MLF2 in complex with HSP70 and DNAJB6 preserves FG-rich condensate integrity over time.
a, Phase-contrast images of 10 µM ScNup116 condensates formed in the presence of 2.5 µM DNAJB6b. Images were taken immediately after condensate formation (T = 0) or after three h of incubation at 30 °C (T = 3) in the presence of ATP. Note the buffer condition is also shown in Fig. 6e. b, Phase-contrast images of 10 µM TtMacNup98A condensates formed in the presence of 2.5 µM DNAJB6b, 5 µM HSPA1A, or 5 µM MLF2:HSP70. Images were taken at the timepoints described in panel (a). c, Images of 10 µM TtMacNup98A condensates formed in the presence of 5 µM HSPA1A plus 2.5 µM of the indicated DNAJB6b construct or 5 µM of the MLF2:HSP70 complex with 2.5 µM H31Q-DNAJB6b. Images were taken at the timepoints described in panel (a). Note the buffer condition is also shown in Fig. 6g. d, The turbidity of 10 µM ScNup116 or HsNup98 solutions in the absence or presence of 5 µM HSPA1A or the MLF2:HSP70 complex. Upon addition of 2 M urea, the condensates fully reverse and the solution loses turbidity as assessed by monitoring absorbance at 550 nm. e, 10 µM of ScNup116, TtMacNup98A, HsNup98, or 5 µM HSPA1A was incubated with 5 µM Thioflavin T (ThT) for 24 h at 30 °C. ThT fluorescence (excitation 440 nm, emission 480 nm) was monitored to detect amyloid formation. All reactions contained 2 mM ATP and an ATP regenerating system. ThT signals for all conditions were normalized to the ScNup116 maximum value. f, 10 µM of ScNup116 was monitored for amyloid formation as described above in buffer containing 5 µM HSPA1A, 2.5 µM DNAJB6b, or 5 µM of the MLF2:HSP70 complex. Where indicated, 2 mM ATP was included or omitted. g, ScNup116 amyloid formation was observed under conditions with 5 µM HSP70 or the MLF2:HSP70 complex plus 2.5 µM DNAJB6b. Where indicated, 2 mM ATP was included or omitted. ThT conditions were as described for panel (e). Scale bar 5 µM for all panels. Source numerical data are available in source data.
Schematic model for how proteotoxicity may accumulate in Torsin-deficient cells. In wild-type neurons, NPC biogenesis is unperturbed and chaperones are free to interact with clients. In DYT1 dystonia neurons, nuclear transport is perturbed due to defective NPC biogenesis. As FG-Nup containing blebs form instead of mature NPCs, they sequester proteins normally destined for degradation, chaperones, and MLF2. When essential chaperones are sequestered away from clients in Torsin-deficient cells, proteotoxic species may be allowed to form and persist to a greater extent than in cells with normal chaperone availability, sensitizing cellular proteostasis towards additional insults.
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Prophet, S.M., Rampello, A.J., Niescier, R.F. et al. Atypical nuclear envelope condensates linked to neurological disorders reveal nucleoporin-directed chaperone activities. Nat Cell Biol 24, 1630–1641 (2022). https://doi.org/10.1038/s41556-022-01001-y
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