Kinase-controlled phase transition of membraneless organelles in mitosis (original) (raw)

References

  1. Overbeek, J. T. G. & Voorn, M. J. Phase separation in polyelectrolyte solutions. Theory of complex coacervation. J. Cell. Comp. Physiol. 49, 7–26 (1957).
    Article CAS Google Scholar
  2. Banani, S. F., Lee, H. O., Hyman, A. A. & Rosen, M. K. Biomolecular condensates: organizers of cellular biochemistry. Nat. Rev. Mol. Cell Biol. 18, 285–298 (2017).
    Article PubMed CAS PubMed Central Google Scholar
  3. Brangwynne, C. P. et al. Germline P granules are liquid droplets that localize by controlled dissolution/condensation. Science 324, 1729–1732 (2009).
    Article ADS PubMed CAS Google Scholar
  4. Kato, M. et al. Cell-free formation of RNA granules: low complexity sequence domains form dynamic fibers within hydrogels. Cell 149, 753–767 (2012).
    Article PubMed CAS PubMed Central Google Scholar
  5. Li, P. et al. Phase transitions in the assembly of multivalent signalling proteins. Nature 483, 336–340 (2012).
    Article ADS PubMed PubMed Central CAS Google Scholar
  6. Zhang, H. et al. RNA controls polyQ protein phase transitions. Mol. Cell 60, 220–230 (2015).
    Article PubMed PubMed Central CAS Google Scholar
  7. Saha, S. et al. Polar positioning of phase-separated liquid compartments in cells regulated by an mRNA competition mechanism. Cell 166, 1572–1584.e16 (2016).
    Article PubMed PubMed Central CAS Google Scholar
  8. Jain, A. & Vale, R. D. RNA phase transitions in repeat expansion disorders. Nature 546, 243–247 (2017).
    Article ADS PubMed PubMed Central CAS Google Scholar
  9. Wippich, F. et al. Dual specificity kinase DYRK3 couples stress granule condensation/dissolution to mTORC1 signaling. Cell 152, 791–805 (2013).
    Article PubMed CAS Google Scholar
  10. Wang, J. T. et al. Regulation of RNA granule dynamics by phosphorylation of serine-rich, intrinsically disordered proteins in C. elegans. eLife 3, e04591 (2014).
    Article PubMed PubMed Central Google Scholar
  11. Saunders, T. E. et al. Noise reduction in the intracellular pom1p gradient by a dynamic clustering mechanism. Dev. Cell 22, 558–572 (2012).
    Article PubMed PubMed Central CAS Google Scholar
  12. Rincon, S. A. et al. Pom1 regulates the assembly of Cdr2–Mid1 cortical nodes for robust spatial control of cytokinesis. J. Cell Biol. 206, 61–77 (2014).
    Article PubMed PubMed Central CAS Google Scholar
  13. Spector, D. L. & Smith, H. C. Redistribution of U-snRNPs during mitosis. Exp. Cell Res. 163, 87–94 (1986).
    Article PubMed CAS Google Scholar
  14. Dammermann, A. & Merdes, A. Assembly of centrosomal proteins and microtubule organization depends on PCM-1. J. Cell Biol. 159, 255–266 (2002).
    Article PubMed PubMed Central CAS Google Scholar
  15. Sivan, G., Kedersha, N. & Elroy-Stein, O. Ribosomal slowdown mediates translational arrest during cellular division. Mol. Cell. Biol. 27, 6639–6646 (2007).
    Article PubMed PubMed Central CAS Google Scholar
  16. Ong, S.-E. et al. Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics. Mol. Cell. Proteomics 1, 376–386 (2002).
    Article PubMed CAS Google Scholar
  17. Gui, J. F., Lane, W. S. & Fu, X. D. A serine kinase regulates intracellular localization of splicing factors in the cell cycle. Nature 369, 678–682 (1994).
    Article ADS PubMed CAS Google Scholar
  18. Thiry, M. Behavior of interchromatin granules during the cell cycle. Eur. J. Cell Biol. 68, 14–24 (1995).
    PubMed CAS Google Scholar
  19. Jiang, H. et al. Phase transition of spindle-associated protein regulate spindle apparatus assembly. Cell 163, 108–122 (2015).
    Article PubMed PubMed Central CAS Google Scholar
  20. Wan, Y. et al. Splicing function of mitotic regulators links R-loop-mediated DNA damage to tumor cell killing. J. Cell Biol. 209, 235–246 (2015).
    Article PubMed PubMed Central CAS Google Scholar
  21. Sivakumar, S. & Gorbsky, G. J. Spatiotemporal regulation of the anaphase-promoting complex in mitosis. Nat. Rev. Mol. Cell Biol. 16, 82–94 (2015).
    Article PubMed PubMed Central CAS Google Scholar
  22. Merbl, Y. & Kirschner, M. W. Large-scale detection of ubiquitination substrates using cell extracts and protein microarrays. Proc. Natl Acad. Sci. USA 106, 2543–2548 (2009).
    Article ADS PubMed PubMed Central Google Scholar
  23. Aranda, S., Laguna, A. & de la Luna, S. DYRK family of protein kinases: evolutionary relationships, biochemical properties, and functional roles. FASEB J. 25, 449–462 (2011).
    Article PubMed CAS Google Scholar
  24. Kedersha, N. L., Gupta, M., Li, W., Miller, I. & Anderson, P. RNA-binding proteins TIA-1 and TIAR link the phosphorylation of eIF-2α to the assembly of mammalian stress granules. J. Cell Biol. 147, 1431–1442 (1999).
    Article PubMed PubMed Central CAS Google Scholar
  25. Cheng, K. C. C., Klancer, R., Singson, A. & Seydoux, G. Regulation of MBK-2/DYRK by CDK-1 and the pseudophosphatases EGG-4 and EGG-5 during the oocyte-to-embryo transition. Cell 139, 560–572 (2009).
    Article PubMed PubMed Central CAS Google Scholar
  26. Pellettieri, J., Reinke, V., Kim, S. K. & Seydoux, G. Coordinate activation of maternal protein degradation during the egg-to-embryo transition in C. elegans. Dev. Cell 5, 451–462 (2003).
    Article PubMed CAS Google Scholar
  27. Blower, M. D., Feric, E., Weis, K. & Heald, R. Genome-wide analysis demonstrates conserved localization of messenger RNAs to mitotic microtubules. J. Cell Biol. 179, 1365–1373 (2007).
    Article PubMed PubMed Central CAS Google Scholar
  28. Chan, F. L. et al. Active transcription and essential role of RNA polymerase II at the centromere during mitosis. Proc. Natl Acad. Sci. USA 109, 1979–1984 (2012).
    Article ADS PubMed PubMed Central Google Scholar
  29. Grenfell, A. W., Heald, R. & Strzelecka, M. Mitotic noncoding RNA processing promotes kinetochore and spindle assembly in Xenopus. J. Cell Biol. 214, 133–141 (2016).
    Article PubMed PubMed Central CAS Google Scholar
  30. Tighe, A., Staples, O. & Taylor, S. Mps1 kinase activity restrains anaphase during an unperturbed mitosis and targets Mad2 to kinetochores. J. Cell Biol. 181, 893–901 (2008).
    Article PubMed PubMed Central CAS Google Scholar
  31. Paul, F. E., Hosp, F. & Selbach, M. Analyzing protein–protein interactions by quantitative mass spectrometry. Methods 54, 387–395 (2011).
    Article PubMed CAS Google Scholar
  32. Rappsilber, J., Ishihama, Y. & Mann, M. Stop and go extraction tips for matrix-assisted laser desorption/ionization, nanoelectrospray, and LC/MS sample pretreatment in proteomics. Anal. Chem. 75, 663–670 (2003).
    Article PubMed Google Scholar
  33. Cox, J. & Mann, M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat. Biotechnol. 26, 1367–1372 (2008).
    Article PubMed CAS Google Scholar
  34. Cox, J. et al. Andromeda: a peptide search engine integrated into the MaxQuant environment. J. Proteome Res. 10, 1794–1805 (2011).
    Article ADS PubMed CAS Google Scholar
  35. Binns, D. et al. QuickGO: a web-based tool for Gene Ontology searching. Bioinformatics 25, 3045–3046 (2009).
    Article PubMed PubMed Central CAS Google Scholar
  36. Aulas, A. & Vande Velde, C. Alterations in stress granule dynamics driven by TDP-43 and FUS: a link to pathological inclusions in ALS? Front. Cell. Neurosci. 9, 423 (2015).
    Article PubMed PubMed Central CAS Google Scholar
  37. Olsen, J. V. et al. Quantitative phosphoproteomics reveals widespread full phosphorylation site occupancy during mitosis. Sci. Signal. 3, ra3 (2010).
    Article PubMed CAS Google Scholar
  38. Olsen, J. V. et al. Global, in vivo, and site-specific phosphorylation dynamics in signaling networks. Cell 127, 635–648 (2006).
    Article PubMed CAS Google Scholar
  39. Wootton, J. C. & Federhen, S. Analysis of compositionally biased regions in sequence databases. Methods Enzymol. 266, 554–571 (1996).
    Article PubMed CAS Google Scholar
  40. Cheng, C. & Sharp, P. A. Regulation of CD44 alternative splicing by SRm160 and its potential role in tumor cell invasion. Mol. Cell. Biol. 26, 362–370 (2006).
    Article PubMed PubMed Central CAS Google Scholar
  41. Belin, B. J., Cimini, B. A., Blackburn, E. H. & Mullins, R. D. Visualization of actin filaments and monomers in somatic cell nuclei. Mol. Biol. Cell 24, 982–994 (2013).
    Article PubMed PubMed Central CAS Google Scholar
  42. Bindels, D. S. et al. mScarlet: a bright monomeric red fluorescent protein for cellular imaging. Nat. Methods 14, 53–56 (2017).
    Article PubMed CAS Google Scholar
  43. Pfleger, C. M., Lee, E. & Kirschner, M. W. Substrate recognition by the Cdc20 and Cdh1 components of the anaphase-promoting complex. Genes Dev. 15, 2396–2407 (2001).
    Article PubMed PubMed Central CAS Google Scholar
  44. Carpenter, A. E. et al. CellProfiler: image analysis software for identifying and quantifying cell phenotypes. Genome Biol. 7, R100 (2006).
    Article PubMed PubMed Central CAS Google Scholar
  45. Rämö, P., Sacher, R., Snijder, B., Begemann, B. & Pelkmans, L. CellClassifier: supervised learning of cellular phenotypes. Bioinformatics 25, 3028–3030 (2009).
    Article PubMed CAS Google Scholar
  46. Jiang, H. et al. A microtubule-associated zinc finger protein, BuGZ, regulates mitotic chromosome alignment by ensuring Bub3 stability and kinetochore targeting. Dev. Cell 28, 268–281 (2014).
    Article PubMed PubMed Central CAS Google Scholar

Download references

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

Authors

  1. Arpan Kumar Rai
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  2. Jia-Xuan Chen
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  3. Matthias Selbach
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  4. Lucas Pelkmans
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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.

Corresponding author

Correspondence toLucas 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.

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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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