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Kinase-controlled phase transition of membraneless organelles in mitosis

Nature volume 559, pages 211–216 (2018)Cite this article

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Abstract

Liquid–liquid phase separation has been shown to underlie the formation and disassembly of membraneless organelles in cells, but the cellular mechanisms that control this phenomenon are poorly understood. A prominent example of regulated and reversible segregation of liquid phases may occur during mitosis, when membraneless organelles disappear upon nuclear-envelope breakdown and reappear as mitosis is completed. Here we show that the dual-specificity kinase DYRK3 acts as a central dissolvase of several types of membraneless organelle during mitosis. DYRK3 kinase activity is essential to prevent the unmixing of the mitotic cytoplasm into aberrant liquid-like hybrid organelles and the over-nucleation of spindle bodies. Our work supports a mechanism in which the dilution of phase-separating proteins during nuclear-envelope breakdown and the DYRK3-dependent degree of their solubility combine to allow cells to dissolve and condense several membraneless organelles during mitosis.

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Fig. 1: DYRK3 interacts with multiple membraneless compartments.
Fig. 2: Formation of mitotic hybrid compartments upon DYRK3 inhibition.
Fig. 3: DYRK3 overexpression dissolves membraneless compartments.
Fig. 4: Increase in DYRK3-to-substrate ratio during mitosis drives dissolution of phase-separated compartments.
Fig. 5: Mitotic defects upon inhibition of DYRK3 and APC/C-dependent degradation of DYRK3.
Fig. 6: Model showing membraneless organelles transitioning through a phase diagram during the cell cycle.

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Acknowledgements

We thank members of the Pelkmans and Klemm laboratories for discussions. We further thank J. Michael Peters, A. Merdes and M. Polymenidou for reagents and acknowledge the assistance and support of the Center for Microscopy and Image Analysis, University of Zurich for performing FRAP experiments. EMBO and HFSP LTF supported A.K.R. J.-X.C was supported by an MDC-NYU PhD exchange program fellowship. L.P. is supported by the Swiss National Science Foundation and the University of Zurich.

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Nature thanks A. Gladfelter, A. Hyman and the other anonymous reviewer(s) for their contribution to the peer review of this work.

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Author notes

  1. Jia-Xuan Chen

    Present address: Cancer Research UK Cambridge Institute, University of Cambridge, Cambridge, UK

Authors and Affiliations

  1. Department of Molecular Life Sciences, University of Zurich, Zurich, Switzerland

    Arpan Kumar Rai & Lucas Pelkmans

  2. Max Delbrück Center for Molecular Medicine, Berlin, Germany

    Jia-Xuan Chen & Matthias Selbach

  3. Charité-Universitätsmedizin Berlin, Berlin, Germany

    Matthias Selbach

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  1. Arpan Kumar Rai
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Contributions

L.P. conceived the project. L.P. and A.K.R. wrote the paper. A.K.R. performed and analysed the data. J.X.C. performed SILAC pull-down experiments and bioinformatic data analysis and was supervised by M.S.

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Correspondence to Lucas Pelkmans.

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Extended data figures and tables

Extended Data Fig. 1 DYRK3 interactions are differentially regulated upon GSK-626616 treatment.

a, Experimental design of the SILAC quantitative pull-down assays to identify protein–protein interaction partners of DYRK3. b, Experimental design of triple-SILAC quantitative pull-down assays to identify change of DYRK3 interactors upon GSK-626616 inhibitor treatment. c, Scatter plot shows change in DYRK3 interactors upon GSK-626616 inhibitor treatment (normalized heavy labelled/medium labelled log2 ratios). Only proteins detected as DYRK3-specific interactors (normalized heavy labelled (H)/light labelled (L) or medium labelled (M)/light labelled log2 ratio >1) were retained on this plot. Interactors are considered differentially regulated (coloured rims) if the heavy labelled/medium labelled log2 ratio is greater than 1 or less than −1. d, Mean intensity of DYRK3 interactors upon GSK-626616 inhibitor treatment. Data are representative of two technical replicates. e, Protein abundance of DYRK3 interactors and DYRK3 upon GSK-626616 inhibitor treatment. Data are representative of two independent experiments. Box plots: centre line, median; box, interquartile range; whiskers, 1.5× interquartile range; dots, outliers.

Extended Data Fig. 2 GSK-626616-inhibited DYRK3 localizes to stress granules and forms mitotic granules.

a, Western blot shows the expression level of endogenous DYRK3 and EGFP–DYRK3 in HeLa-FlpIn-Trex cells stably expressing inducible EGFP–DYRK3(WT). EGFP–DYRK3 expression was induced with doxycycline (500 ng ml−1, 4 h). The same induction conditions were used for Fig. 1c–e and Extended Data 2b. b, Colocalization of GSK-626616-inhibited (1 μM, 2 h) EGFP–DYRK3(WT) with stress granules upon arsenite treatment (500 μM, 45 min). c, Mitotic cells show formation of small DYRK3-positive granules upon GSK-626616 treatment (1 μM, 1 h). d, Phosphoproteomic data of the human cell cycle showing changes in the regulated phospho-sites that are associated with the DYRK3-specific interactors detected in Fig. 1a. The majority (74.4%) of these phospho-sites reached peak levels at mitosis. The phospho-site occupancy level is displayed for those sites that were downregulated upon GSK-626616 inhibitor treatment as previously reported9. The phosphoproteomic data were retrieved from a previously published work37. The known localization (splicing speckle, stress granule or centrosome) of the corresponding protein is indicated for each phospho-site. Images and western blots are representative of at least three independent experiments. Scale bars, 10 μm.

Extended Data Fig. 3 DYRK3 inhibition leads to the formation of aberrant mitotic granules.

a, Dissolved staining of a splicing-speckle marker (SC35) in mitotic cells upon DMSO treatment. b, Dissolved staining of stress-granule marker (PABP) in mitotic cells upon DMSO treatment. c, Spindle-pole localization of pericentriolar material protein (PCM1) in mitotic cells upon DMSO treatment. d, Mitotic cells (HEK293T) show formation of SC35 granules upon GSK-626616 treatment (1 μM, 1 h). e, Formation of SON granules in mitotic cells upon GSK-626616 treatment (1 μM, 6 h). f, Formation of TIAR granules in mitotic cells upon GSK-626616 treatment (1 μM, 6 h). g, Left, quantification of fraction of mitotic (metaphase) cells with SC35 granules on treatment with kinase inhibitors (1 μM, 1 h). Right, quantification of fraction of mitotic (metaphase) cells with PABP granules on treatment with kinase inhibitors (1 μM, 3 h). Inhibitors and the corresponding kinase targets are mentioned. Data are mean ± s.d. from three technical replicates. Images are representative of at least three independent experiments. Scale bars, 10 μm.

Source Data

Extended Data Fig. 4 Formation of mitotic granules is DYRK3 specific.

a, Mitotic cells show no colocalization between the splicing-speckle marker (SC35) and SRPK1 upon GSK-626616 treatment (1 μM, 1 h). b, Mitotic cells show no colocalization between the splicing speckle marker (SC35) and cyclin B upon GSK-626616 treatment (1 μM, 1 h). c, Mitotic cells show no colocalization between the splicing-speckle marker (SRRM2) and CDK1 upon GSK-626616 treatment (1 μM, 1 h). d, Mitotic cells stained for pY15 CDK1. Loss of pY15 signal (CDK1 activation) in mitotic cells upon GSK-626616 (1 μM, 1 h) treatment is comparable to DMSO control. e, GSK-626616 treatment (1 μM, 1 h) does not result in a decrease in pT446 APC3 (CDK1 mitotic substrate) signal in mitotic cells compared to the DMSO control. f, Mitotic cells show staining for pT446 APC3 (CDK1 mitotic substrate). The cells were pre-permeabilized with Triton-X before fixation. pT446 APC3 signal can be observed at spindle poles for both DMSO and GSK-626616 treatment (1 μM, 1 h). g, Appearance of SC35 granules and spindle apparatus defects upon DYRK3 knockdown. Right, quantification of SC35 granule number in mitotic (metaphase) cells (four independent experiments). Box plots: centre line, population median; box, interquartile range; whiskers, 1.5× interquartile range; dots, outliers. Statistical analysis performed across cells using a Welch’s two-sided t-test. Images and data are representative of at least three independent experiments. Scale bars, 10 μm.

Source Data

Extended Data Fig. 5 Inhibition of DYRK3 does not affect all membraneless organelles in mitosis.

a, Colocalization of splicing-speckle marker and poly(A) mRNA in hybrid compartments in mitotic cells upon GSK-626616 treatment (1 μM, 3 h). b, Colocalization (arrowheads) of splicing-speckle marker and EGFP–DYRK3(WT) in hybrid compartments in mitotic cells upon GSK-626616 treatment (1 μM, 2 h). c, Dissolution of P-bodies (DDX6) in mitotic cells is unaffected upon GSK-626616 treatment (1 μM, 6 h). d, Dissolution of nucleoli (fibrillarin) in mitotic cells is unaffected upon GSK-626616 treatment (1 μM, 6 h). e, Dissolution of Cajal bodies (coilin) in mitotic cells is unaffected upon GSK-626616 treatment (1 μM, 6 hrs). f, Aberrant granules formed upon GSK-626616 treatment are not ubiquitinated aggregates. Images are representative of at least three independent experiments. Scale bars, 10 μm.

Extended Data Fig. 6 Protein abundance does not change during the formation of mitotic granules upon GSK-626616 inhibitor treatment.

a, Time-lapse images (single Z-stack) show fusion of mitotic SRRM2–mCherry granules formed in the presence of the GSK-626616 inhibitor (1 μM) in cells arrested in mitosis (thymidine–nocodazole block). b, Mean cell intensity of SRRM2–mCherry is plotted during mitotic granule formation on addition of GSK-626616 inhibitor (1 μM). Data are mean ± s.d. c, Top, mean intensity of SRRM2–mCherry in the dissolved phase is plotted during mitotic granule formation on addition of GSK-626616 inhibitor (1 μM). Bottom, time-lapse images of the cells plotted in the top panel. Images are representative of at least three independent experiments. Scale bars, 10 μm.

Source Data

Extended Data Fig. 7 Dissolution of membraneless organelles is dependent on DYRK3 localization and its kinase activity.

a, Images show cells overexpressing wild-type EGFP–DYRK3(WT) and kinase-dead EGFP–DYRK3(K218M). These images are the same as Fig. 3a showing both channels. b, Dissolution of splicing speckles upon overexpression of EGFP–DYRK3(WT) in interphase cells is reversed by GSK-626616 treatment (1 μM, 2 h). c, Top, schematic of the EGFP-tagged nuclear localization signal (NLS) mutant of DYRK3 (EGFP–NLSmut-DYRK3). Arginine and lysine residues in the NLS are mutated to alanine. Bottom, overexpressed EGFP–NLSmut-DYRK3 localizes to the cytoplasm and does not dissolve splicing speckles. d, Left, dissolution of splicing speckles upon overexpression of EGFP–NLS(SV40)-DYRK3(WT) in interphase cells. Middle, dissolution of splicing speckles upon overexpression of EGFP–NLS(SV40)-DYRK3(WT) in interphase cells is reversed upon GSK-626616 treatment (1 μM, 2 h). Right, condensed splicing speckles upon overexpression of EGFP–NLS(SV40)-DYRK3(K218M) in interphase cells. e, Images show cells overexpressing EGFP–DYRK3(WT) and EGFP–DYRK3(K218M). These images are the same as Fig. 3c showing both channels. f, Dissolution of pericentriolar satellites upon overexpression of EGFP–DYRK3(WT) in interphase cells is reversed upon GSK-626616 treatment (1 μM, 2 h). g, Images show cells overexpressing EGFP–DYRK3(WT) and EGFP–DYRK3(K218M). These images are the same as Fig. 3e showing both channels. h, Dissolution of stress granules upon overexpression of EGFP–DYRK3(WT) in interphase cells is reversed upon GSK-626616 treatment (1 μM, 2 h). i, The percentage of low complexity regions (LCR) occupying each full-length protein was computed for all DYRK3 interactors (Fig. 1a) and all the known splicing-speckle components (GO:0016607). SRRM1 shown in blue is among the proteins with the highest proportion of low complexity regions. j, Dissolution of EGFP–PCM1(1–1468) cytosolic granules upon overexpression of RFP–DYRK3(WT), and not RFP–DYRK3(K218M). k, Time-lapse images show RFP–DYRK3(WT) driven dissolution of cytosolic granules formed upon EGFP–PCM1(1–1468) overexpression. l, Dissolution of mCherry–SRRM1 nuclear granules upon overexpression of EGFP–DYRK3(WT), and not EGFP–DYRK3(K218M). m, Nucleoli are unaffected upon overexpression of EGFP–DYRK3(WT) or EGFP–DYRK3(K218M) in interphase cells. Images are representative of at least three independent experiments. Scale bars, 10 μm.

Extended Data Fig. 8 Dilution of DYRK3 substrates during G2-to-M transition.

a, Top, time-lapse images of a cell (single Z-stack) expressing EGFP–3×NLS during G2-to-M transition. Bottom, changes in mean nuclear intensity of EGFP–3×NLS during G2-to-M transition. Data are from eleven individual cells. b, Top, time-lapse images of a cell (single Z-stack) expressing pmScarlet–NES (nuclear export signal) during the G2-to-M transition. Bottom, changes in mean cytoplasmic intensity of pmScarlet–NES during the G2-to-M transition. Data are from eight individual cells. c, Top, time-lapse images of a cell (single Z-stack) expressing EGFP–DYRK3(WT) during G2-to-M transition. Bottom, changes in mean cytoplasmic intensity of EGFP–DYRK3(WT) during G2-to-M transition. Data are from nine individual cells. d, Top, Time-lapse images of a cell (single Z-stack) expressing EGFP-PCM1(1-1468) during G2-to-M transition. Bottom, changes in mean cytoplasmic intensity of EGFP-PCM1(1-1468) during G2-to-M transition. Data are from 11 individual cells. a–d, The lines (background) show mean nuclear intensity for individual cells. Data are mean ± s.d. Time point (0 min) refers to nuclear envelope breakdown. e, Overexpressed mCherry–SRRM1 forms mitotic granules which recruit endogenous splicing proteins. f, Time-lapse images (single Z-stack) show fusion of mitotic mCherry–SRRM1 granules in mitotic cells. g, Left, FRAP analysis of interphase and mitotic mCherry–SRRM1 granules in the presence and absence of the GSK-626616 inhibitor (1 μM). Data are mean ± s.d. Right, FRAP recovery of mitotic mCherry–SRRM1 granule. h, Cells arrested in mitosis show formation of splicing granules upon GSK-626616 treatment (1 μM, indicated times). Data and images are representative of at least three independent experiments. Scale bars, 10 μm.

Source Data

Extended Data Fig. 9 Spindle apparatus defects upon DYRK3 inhibition.

a, Multiple γ-tubulin foci in mitotic cells upon GSK-626616 treatment (1 μM, 6 h). b, Top, ZNF207 localization in interphase cells (no pre-permeabilization). Bottom, ZNF207 and SC35 colocalize in Triton-X pre-permeabilized interphase cells. c, ZNF207 granules in mitotic cells upon GSK-626616 treatment (1 μM, 3 h). d, EGFP–DYRK3(WT) colocalizes with ZNF207 in mitotic cells upon GSK-626616 treatment (1 μM, 3 h). EGFP–DYRK3(WT) expression was induced in HeLa-FlpIn-Trex cells by adding doxycycline (500 ng ml−1, 6 h). Images are representative of at least three independent experiments. Scale bars, 10 μm.

Extended Data Fig. 10 DYRK3 forms ubiquitin-positive aggregates on overexpression of CDC20 and CDH1 or upon proteosomal inhibition.

a, Ubiquitin does not colocalize with overexpressed EGFP inside cells. b, Ubiquitin localizes to EGFP–DYRK3(WT) aggregates, formed on HA–CDH1 and HA–CDC20 overexpression. c, Ubiquitin does not colocalize with overexpressed EGFP upon MG-132 treatment (5 μM, 4 h). d, Ubiquitin localizes to EGFP–DYRK3(WT) granules upon MG-132 treatment (5 μM, 4 h). EGFP–DYRK3(WT) expression was induced in HeLa-FlpIn-Trex cells by adding doxycycline (500 ng ml−1, 6 h). e, Top, EGFP–DYRK3(WT) overexpression prevents re-assembly of cytosolic mCherry–SRRM1 granules during late mitosis. Bottom, EGFP–DYRK3(WT) overexpression prevents re-assembly of cytosolic splicing granules during late mitosis. Images are representative of at least three independent experiments. Scale bars, 10 μm.

Supplementary information

Supplementary Figure 1

This file contains the uncropped western blots with molecular weight marker and the cropped region indicated.

Reporting Summary

Supplementary Table 1

A list of detected DYRK3 specific interaction partners.

Supplementary Table 2

A list of detected DYRK3 specific interaction partners upon GSK-626616 inhibitor treatment.

Video 1: SRRM2-mCherry granule formation upon GSK-626616 inhibitor treatment.

HeLa cell expressing SRRM2-mCherry was arrested in mitosis by thymidine-nocodazole block. Time-lapse movie shows SRRM2 granule formation upon addition of GSK-626616 (1 μM). Time point (0 min) corresponds to GSK-626616 addition. Scale bar is 10 μm. The cell shown in the video is the same as in Fig. 2f. Video is representative of at least three independent experiments.

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Rai, A.K., Chen, JX., Selbach, M. et al. Kinase-controlled phase transition of membraneless organelles in mitosis. Nature 559, 211–216 (2018). https://doi.org/10.1038/s41586-018-0279-8

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