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The chaperone DNAJB6 surveils FG-nucleoporins and is required for interphase nuclear pore complex biogenesis

Nature Cell Biology volume 24, pages 1584–1594 (2022)Cite this article

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Abstract

Biogenesis of nuclear pore complexes (NPCs) includes the formation of the permeability barrier composed of phenylalanine-glycine-rich nucleoporins (FG-Nups) that regulate the selective passage of biomolecules across the nuclear envelope. The FG-Nups are intrinsically disordered and prone to liquid–liquid phase separation and aggregation when isolated. How FG-Nups are protected from making inappropriate interactions during NPC biogenesis is not fully understood. Here we find that DNAJB6, a molecular chaperone of the heat shock protein network, forms foci in close proximity to NPCs. The number of these foci decreases upon removal of proteins involved in the early steps of interphase NPC biogenesis. Conversely, when this process is stalled in the last steps, the number of DNAJB6-containing foci increases and these foci are identified as herniations at the nuclear envelope. Immunoelectron tomography shows that DNAJB6 localizes inside the lumen of the herniations arising at NPC biogenesis intermediates. Loss of DNAJB6 results in the accumulation of cytosolic annulate lamellae, which are structures containing partly assembled NPCs, a feature associated with disturbances in NPC biogenesis. We find that DNAJB6 binds to FG-Nups and can prevent the aggregation of the FG region of several FG-Nups in cells and in vitro. Together, our data show that the molecular chaperone DNAJB6 provides quality control during NPC biogenesis and is involved in the surveillance of native intrinsically disordered FG-Nups.

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Fig. 1: DNAJB6 localizes in foci to the NE, and disruption of DNAJB6 expression leads to accumulation of annulate lamellae.
Fig. 2: DNAJB6 foci formation is cell cycle dependent and is related to interphase NPC assembly.
Fig. 3: DNAJB6 localizes to herniations at the NE.
Fig. 4: DNAJB6 interacts with FG-Nup components of the NPC.
Fig. 5: DNAJB6b modulates the LLPS and aggregation of FG-Nup fragments in vitro.
Fig. 6: The S/T domain of DNAJB6 mediates the interaction with FG-Nups.

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Data availability

Large-scale EM data are available via nanotomy.org (http://www.nanotomy.org/OA/Kuiper2022NCB/). All other data supporting the findings of this study are available from the corresponding author on reasonable request. Source data are provided with this paper.

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Acknowledgements

E.F.E.K. is supported by a topmaster fellowship from the Groningen University Institute for Drug Exploration (GUIDE). H.H.K. and S.B. are supported by a grant from the CTH (grant no. 686728). L.M.V., H.H.K., S.B., M.K.M., P.G., T.B. and A.S. are supported by an NWO Groot (grant no. 685709). L.M.V., P.G., T.B. and A.S. are supported by a Vici grant (VI.C.192.031). M.M. is supported by an ALW Open Programme (ALWOP.355). We thank J. Brunsting and A. Blaauwbroek for practical assistance. We thank E. Lemke for providing us with the GFP-Nup153AG construct, D. Görlich for the yNup116FG and yNup145NFG constructs, and C. Schlieker for the HeLa 4TorKO cell line. We thank S. Cascarina for assistance with the LCD-Composer. We thank W. Browne for his help with the Circular dichroism measurements. Fluorescence and live cell imaging was performed in the UMCG Microscopy and Imaging Center (UMIC), sponsored by ZonMW grant 91111.006 and NWO 175-010-2009-023. EM 2D imaging was performed at the UMIC. Tomography imaging was performed at the EM facility of the Faculty of Science and Engineering of Groningen, the Netherlands.

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Author notes

  1. E. F. Elsiena Kuiper

    Present address: Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, USA

  2. Muriel Mari

    Present address: Department of Biomedicine, Aarhus University, Aarhus, Denmark

  3. Steven Bergink

    Present address: University College, University of Groningen, Groningen, the Netherlands

  4. These authors contributed equally: Paola Gallardo, Tessa Bergsma, Muriel Mari.

Authors and Affiliations

  1. Department of Biomedical Sciences of Cell & Systems, University of Groningen, University Medical Center Groningen, Groningen, the Netherlands

    E. F. Elsiena Kuiper, Muriel Mari, Maiara Kolbe Musskopf, Jeroen Kuipers, Ben N. G. Giepmans, Harm H. Kampinga & Steven Bergink

  2. European Research Institute for the Biology of Ageing, University of Groningen, University Medical Center Groningen, Groningen, the Netherlands

    Paola Gallardo, Tessa Bergsma, Anton Steen & Liesbeth M. Veenhoff

  3. UMCG Microscopy and Imaging Center (UMIC), University of Groningen, University Medical Center Groningen, Groningen, the Netherlands

    Jeroen Kuipers

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Contributions

E.F.E.K., H.H.K., L.M.V. and S.B. conceived the project. The experiments in Figs. 1, 2 and 4 were designed, performed and analysed by E.F.E.K., the TEM in Fig. 1f by J.K., the EM and tomography in Fig. 3 by M.M., the in vitro data in Figs. 5 and 6 by P.G., T.B. and A.S., and the PLA and DNAJB6b mutant experiments in Figs. 4b and 6c by M.K.M. Supervision was done by H.H.K., L.M.V., A.S., B.N.G.G. and S.B. The manuscript was written by E.F.E.K., S.B. and L.M.V., with input from all authors.

Corresponding authors

Correspondence to E. F. Elsiena Kuiper, Harm H. Kampinga, Liesbeth M. Veenhoff or Steven Bergink.

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Extended data

Extended Data Fig. 1 Disruption of DNAJB6 expression leads to accumulation of annulate lamellae.

a, DNAJB6 in HEK293T cells localizes to the cytoplasm and nucleus, and in foci at the nuclear rim. A Z-projection is shown. Graph depicts a quantification of the distribution of the number of DNAJB6 foci per cell. n = 3 independent experiments with 1272 cells analyzed; Log2 graph box and whiskers minimum to maximum with all data points; median indicated with a line and mean with a+. b, Different cell lines (BeWo, U2OS and HeLA) stained for DNAJB6 (green) and FG-Nups (mAb414, magenta). c, HEK293T DNAJB6 KO cells stained for DNAJB6 (green) and Lamin B1 (magenta). d, Images of mAb414 or Nup153-stained nuclear pores (focused on the top of the nucleus) in HEK293T and HEK293T DNAJB6 KO cells. Quantification of average nearest neighbour distance (in pixels) shows larger distances between NPCs in DNAJB6 KO cells. Every point represents one cell; WT n = 32 individual cells, DNAJB6 KO n = 33 individual cells; mean ± SEM. e, siRNA-mediated knockdown of DNAJB6 (green) (72 h) induces cytoplasmic accumulations of NPCs stained by mAb414 (magenta). f, HEK293T cells transfected with siRNA against the 5’UTR of DNAJB6 show accumulations of NPCs and by simultaneous expression of GFP-DNAJB6bWT formation of these can be prevented. With a quantification of mAb414-positive accumulations after knockdown of DNAJB6. n = 3 independent experiments; unpaired t-test, 2-tailed p-value; *p = 0.0139, **p = 0.0097; mean ± SEM. g, In DNAJB6 KO cells, NPCs in the cytoplasm (annulate lamellae) visualized by transmission electron microscopy and pseudo-colored in bright pink, can be distinguished in cytoplasmic strings. Arrows point at individually identifiable NPCs. Nucleus indicated with N, mitochondrion indicated with M. Scale bar represents 500 nm. h, As in g, but now the white arrow head points at individually identifiable NPCs in the NE and black arrow heads at NPCs in the annulate lamellae. i, Individual channels of Fig. 1h. RanGap1, and different nucleoporins of the NPC Nup358, Nup133, Nup205, Nup98, TPR, Nup50, and Nup153 are stained (magenta) together with DNAJB6 (green) in WT and DNAJB6 KO cells. Source numerical data are available in source data.

Source data

Extended Data Fig. 2 Closely related JDPs of DNAJB6 also accumulate in foci and deletion can induce annulate lamellae.

a, Representative images of HEK293T and HEK293T DNAJB6 KO cells cultured in medium containing the uridine analog 5-ethynyluridine (5-EU). 5-EU incorporation into newly transcribed RNA was detected by Cy3-azide (green). The ratio of nuclear to cytoplasmic signal intensities of RNA relative to HEK293T WT cells. Each individual point represents a single cell; WT n = 3 independent experiments with 327 cells examined, DNAJB6 KO n = 3 independent experiments with 323 cells examined; unpaired t-test, 2-tailed p-value; ***p < 0.0001; mean ± SEM. b, Colocalization of GFP-tagged DNAJB6b, DNAJB8, and DNAJB2 with endogenous DNAJB6. DNAJB1 and DNAJA1 do not colocalize with DNAJB6 foci at the NE. c, annulate lamellae formation under siRNA-mediated knockdown of DNAJB6, DNAJB2, DNAJB1, and DNAJA1. Knockdown of DNAJB6 and DNAJB2 leads to significant increase in annulate lamellae formation. All experiments n = 3 independent experiments with cells examined: control 4392, DNAJB6b 5073, DNAJB2 4118, DNAJB1 4059, DNAJA1 4184; unpaired t-test, 2-tailed p-value; **p = 0.0048 mock vs DNAJB6b, **p = 0.003 mock vs DNAJB2; mean ± SEM. Scale bars on all fluorescent images represent 10 μm, and on magnifications 2 μm. Source numerical data are available in source data.

Source data

Extended Data Fig. 3 DNAJB6 foci formation is cell cycle dependent and is related to interphase NPC assembly.

a, Cell cycle progression profiles as analyzed by fluorescence-activated cell sorting (FACS). Cells were stained with propidium iodide to detect DNA content. b, DNAJB6 foci in HEK293T cells in asynchronized cells, and cells 1.5 h and 12 h after release from a double thymidine cell cycle synchronization. c, siRNA-mediated knockdown of ELYS and Nup153 in HEK293T cells visualized on SDS-PAGE with specific antibodies. d, e, Bar graphs of the data in Fig. 2b depicting a distribution of the amount of DNAJB6 foci per cell for the indicated siRNA treatment. NB: these conditions share the same control. d, *p = 0.0129, e, *p = 0.0304, **p = 0.0080, ***p = 0.0006; Control n = 5 independent experiments with 8298 cells examined, ELYS n = 4 independent experiments with 6839 cells examined, Nup153 n = 4 independent experiments with 7690 cells examined, automated with ICY; mean ± SEM. f, Quantification of percentage of cells with cytoplasmic NPC accumulations (AL) stained with mAb414 under siRNA knockdown of ELYS, Nup153, or Nup358. Control n = 5 independent experiments with 7317 cells examined, ELYS n = 4 independent experiments with 6323 cells examined, Nup153 n = 3 independent experiments with 4798 cells examined, Nup358 n = 2 independent experiments with 2264 cells examined; unpaired t-test, 2-tailed p-value; **p = 0.0037, ***p < 0.0001; mean ± SEM. g, qPCR on mRNA levels of Torsin A and B under control or knockdown conditions, normalized to GAPDH. Tor1A control n = 3 independent experiments, Tor1A, Tor1B control, and Tor1B n = 2 independent experiments. h, Bar graph of the data in Fig. 2c, depicting a distribution of the amount of DNAJB6 foci per cell. Control n = 3 independent experiments with 1272 cells examined, gRNA TOR1A + TOR1B n = 3 independent experiments with 952 cells examined, automated with ICY; mean is indicated with a cross; unpaired t-test, 2-tailed p-value; only significant differences are indicated; ***p = 0.0005; mean ± SEM. i, HeLa WT and HeLa 4TorKO cells stained for DNAJB6 (green) and FG-Nups with mAb414 (magenta). j, Overexpression of either TorsinA-GFP wildtype or ΔE302 (green) leads to an increase in foci at the nuclear envelope, containing DNAJB6 (magenta). k, Ubiquitin (magenta) colocalizes with DNAJB6 (green) at foci at the nuclear envelope and are increased upon Torsin knockdown (gRNA vector containing mTurquoise2 in the blue channel). Scale bars on all fluorescent images represent 10 μm, and on magnifications 2 μm. Source numerical data are available in source data.

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Extended Data Fig. 4 DNAJB6 localizes to herniations at the NE and is dependent on intact NPCs.

a, For immunoelectron tomography, surface labelling was used to select areas of interest prior to recording tilt series. Tomographic slice (Z slice 4) revealing the specific immunogold (15 nm) labelling on the surface of the cryosection. Asterisk indicating herniations. Indicated tomographic slices extracted from the tomogram showing herniations at the NE. In the bottom images, nuclear membranes are traced (pink). More electron dense areas can be observed at the neck of the herniations where the NE bends into the herniation, indicated by black arrow heads. NPCs are indicated with white arrow heads. Nucleus is indicated with N and nuclear envelope with NE. Scale bars represent 232 nm. Lower images are from the three-dimensional model (see Supplementary Video 2) of DNAJB6-positive herniations, obtained by electron tomography of 400 to 450 nm thick cryosections. The NE is represented in pink. Left is a view from the nuclear side showing the openings of the NPCs and of the herniations. Right shows the herniations with the localization of the DNAJB6 gold labelling in red. b, HSP70 (magenta) colocalizes with DNAJB6 (green) in foci at the NE. c, MLF2-GFP colocalizes with DNAJB6 (magenta) in foci at the NE. d, WT HEK293T cells treated with 5% 1,6-hexanediol for 2 minutes lose almost all DNAJB6 foci (green), while cells treated with 2,5-hexanediol have only a slight reduction in foci. Scale bars on all fluorescent images represent 5 μm, and on magnifications 2 μm.

Extended Data Fig. 5 The interaction of DNAJB6 with hNup153FG depends on the FG-motifs in hNup153FG.

a, Schematic overview of the BioID2-DNAJB6b fusion protein and how samples were obtained. Fluorescent image shows HEK293T cells expressing BioID2-DNAJB6b, treated for 24 h with 50 μM biotin and stained with Alexa Fluor 488-conjugated streptavidin, biotinylated substrates are visualized in foci at the NE. b, Streptavidin blot belonging Fig. 4a where HEK293T cells expressing V5-DNAJB6b or BioID2-DNAJB6b are treated for 24 h with 50 μM biotin. c, Proximity ligation assay (PLA) performed with antibodies against FG-Nups (mAb414) or DNAJB6 alone, or only both secondary antibodies as negative technical controls for PLA. d, SDS-PAGE showing increase in HSPA6 levels after heat shock (43 °C for 30 minutes) but not in DNAJB6 KO nor after overexpression of hNup153FG. e, GFP-Nup153 full length localizes predominantly to the NE in HEK293T cells. Due to overexpression, there is also small clustering at the NE, but no aggregates can be observed. f, GFP-Nup153AG overexpression in HEK293T cells is homogeneously distributed in the cytoplasm. g, HEK293T cells overexpressing GFP-Nup153FG and mCherry-DNAJB6b show colocalization in spherical accumulations (arrowheads). h, Filter-trap assay of HEK293T cells with GFP-Nup153AG or GFP-Nup153FG overexpression. Three serial 5-fold dilutions were loaded onto cellulose-acetate membranes and probed with anti-GFP antibodies to detect aggregation of the GFP-Nup153FG or GFP-Nup153AG fragment without or with co-overexpression of V5-DNAJB6b. i, HEK293T cells overexpressing V5-DNAJB6b or BioID2-DNAJB6b, either with GFP-Nup153FG or GFP-Nup153AG, are treated for 24 h with 50 μM biotin. Biotinylated close proximity partners of DNAJB6b are pulled down by biotin affinity immunoprecipitation with streptavidin beads, run on SDS-PAGE. GFP and DNAJB6 are detected with specific antibodies. Scale bars on all fluorescent images represent 10 μm. Source numerical data and unprocessed blots are available in source data.

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Extended Data Fig. 6 The effect of crowding agents and DNAJB6b on the aggregation of human and yeast FG-Nup fragments in vitro.

a, 6 μM of purified FG-Nup fragments were incubated in assay buffer (150 mM NaCl, 50 mM TrisHCl, pH 8) containing the indicated concentration (w/v) of the crowding agents Serine, PEG3350, or Ficoll, at 25 °C for 1 hour, and loaded on a filter trap. Graphs show quantification of Nup band intensity in the described conditions. n = 3 independent experiments; mean ± SEM. hNup153FG did not form any quantifiable bands on filter trap with the addition of Serine or Ficoll. b-e, Individual repetitions of the data in Fig. 5a. The effect of DNAJB6b on the aggregation of human and yeast FG-Nup fragments in vitro. Filter trap assays to detect aggregation of purified FG-Nup fragments, incubated with the indicated molar ratios of DNAJB6b, probed with anti-His antibodies to detect FG-Nup fragments. hNup153FG (b), yNup100FG (c), and yNup116FG (d) were incubated in the assay buffer (150 mM NaCl, 50 mM TrisHCl, pH 8) containing 10% PEG3350 (w/v), at 25 °C for either 1 hour (hNup153FG, yNup100FG) or 3 hours (yNup116FG), and yeast Nup145NFG (e) was incubated in a buffer comprising 50 mM NaCl, 100 mM sodium phosphate, pH6, 10% PEG3350 (w/v), at 25 °C during 1 hour. Graphs show band intensities on filter trap quantified relative to the average intensity in the absence of DNAJB6b. Source numerical data and unprocessed blots are available in source data.

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Extended Data Fig. 7 Individual repetitions of the data in Fig. 5b.

a, b, The effect of control proteins relative to DNAJB6b on the aggregation of human and yeast FG-Nup fragments in vitro. 6 μM of purified hNup153FG (a) or yNup100FG (b) were incubated in the absence (control) or presence of BSA, MBP-mCherry, DNAJA1, or DNAJB6b at a 6 μM concentration (molar ratio 1:1). BSA and MBP-mCherry served as non-chaperone controls and DNAJA1 served as chaperone control. Graphs represent band intensities relative to the average intensity of the FG-Nup fragments alone (control) of each replicate. n = 3 independent experiments. c, d, 6 μM of purified hNup153FG (c) or yNup100FG (d) were incubated in the absence (control) or presence of the GFP-tagged intrinsically disordered proteins (IDPs) 1 and 2 (IDP1 is the disordered region of yHeh2; IDP2 is a synthetic IDP; for details see methods) at a 6 μM concentration (molar ratio 1:1). Band intensities are quantified relative to the average intensity of the FG-Nup fragments alone. Source numerical data and unprocessed blots are available in source data.

Extended Data Fig. 8 Individual repetitions of the data in Fig. 5c.

a, b, The effect of DNAJB6 on the time-dependent formation of hNup153FG and yNup100FG aggregates in vitro. Formation of hNup153FG (a) and yNup100FG (b) aggregates captured on filter trap over time in the absence and presence of DNAJB6b at a molar ratio of 1:1. Time is indicated in minutes. Graphs depict quantification of Nup band intensity (in arbitrary units, a.u.) over time in the absence (green) or presence (grey) of DNAJB6b. Source numerical data and unprocessed blots are available in source data.

Extended Data Fig. 9 Condensation of DNAJB6b and FG-Nup fragments, and individual repetitions of the data in Fig. 6b.

a, DNAJB6b condensates by itself and is SDS soluble. Purified DNAJB6b was incubated with or without 10% PEG3350 (w/v). After 1 hour incubation, either 5% 2.5-hexanediol, 5% 1.6-hexanediol, or 0.5% SDS was added for 10 minutes and bright field images were taken. b, Fluorescence and bright field microscopy images showing co-localisation of Fluorescein-5-Maleimide labelled hNup153FG and yNup100FG in the absence or the presence of unlabelled DNAJB6b at a 3 μM concentration (molar ratio 1:1). Deconvolved fluorescence images are depicted as maximum Z-Projection of 30 consecutive slices of 0.2 micron. Scale bar represents 2 μm. c, Coomassie-stained SDS-PAGE showing one replicate of the sedimentation assay (quantified in Fig. 5e-f) to assess the soluble fraction of yNup100FG (3 μM) in the presence or absence of DNAJB6b (1.5 μM) for the indicated times. To determine the fraction 1,6-hexanediol soluble condensates, 10% 1,6-hexanediol was added for 10 minutes prior to centrifugation. Total yNup100FG protein is slightly shifted on the blot due to effects of PEG on mobility. d, Individual repetitions of the data in Fig. 6b. Filter-trap assays showing the effect of wildtype or indicated DNAJB6 mutants (18x S/T > A, 12x F > A, Δ79-115) on the aggregation of yNup100FG (3 μM; molar ratio 1:1). Graphs represent band intensities relative to the average intensity of the yNup100FG fragment alone (control). Source numerical data and unprocessed blots are available in source data.

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Extended Data Fig. 10 Characterization of purified FG-Nup fragments, control proteins and DNAJB6 mutants.

a, Coomassie Brilliant Blue stained SDS-PAGE of indicated purified protein samples. b, Circular dichroism spectroscopy of DNAJB6b and DNAJB6b mutants (18x S/T > A, 12x F > A, and Δ79-115). Purified proteins were diluted to a concentration of either 0.2 μM (DNAJB6b WT), 0.67 μM (18xS/T > A) or 15 μM (12xF > A and Δ79-115) in the assay buffer (50 mM TrisHCl, 150 mM NaCl, pH 8.0). Ellipticity was recorded between 500 and 200 nm in a quartz (QS) cuvette. Data was converted to Molar ellipticity ([q]) to correct for protein concentration, cuvette pathlength and number of backbone peptide units. Source numerical data are available in source data.

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Supplementary information

Reporting Summary

Peer Review File

Supplementary Table 1

Amino acid composition comparison with the S+T, G and F content of the S/T domain of DNAJB6.

Supplementary Video 1

HEK293T cells stained for DNAJB6 (green) showing foci at the NE, Z-stacks. Hoechst in blue.

Supplementary Video 2

Tomography 3D model of NE herniation; DNAJB6 stained with anti-DNAJB6.

Supplementary Video 3

Tomography 3D model of NE herniation; GFP-DNAJB6b stained with anti-GFP.

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Kuiper, E.F.E., Gallardo, P., Bergsma, T. et al. The chaperone DNAJB6 surveils FG-nucleoporins and is required for interphase nuclear pore complex biogenesis. Nat Cell Biol 24, 1584–1594 (2022). https://doi.org/10.1038/s41556-022-01010-x

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