Cell cycle-specific phase separation regulated by protein charge blockiness

1. Wippich, F. et al. Dual specificity kinase DYRK3 couples stress granule condensation/dissolution to mTORC1 signaling. Cell 152, 791–805 (2013).

2. 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).

3. Wang, A. et al. A single N-terminal phosphomimic disrupts TDP-43 polymerization, phase separation, and RNA splicing. EMBO J. 37, e97452 (2018).

4. Beutel, O., Maraspini, R., Pombo-García, K., Martin-Lemaitre, C. & Honigmann, A. Phase separation of zonula occludens proteins drives formation of tight junctions. Cell 179, 923–936 (2019).

5. Greig, J. A. et al. Arginine-enriched mixed-charge domains provide cohesion for nuclear speckle condensation. Mol. Cell 77, 1237–1250 (2020).

6. Rai, A. K., Chen, J.-X., Selbach, M. & Pelkmans, L. Kinase-controlled phase transition of membraneless organelles in mitosis. Nature 559, 211–216 (2018).

7. 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).

8. Hyman, A. A., Weber, C. A. & Jülicher, F. Liquid–liquid phase separation in biology. Annu. Rev. Cell Dev. Biol. 30, 39–58 (2014).

9. Courchaine, E. M., Lu, A. & Neugebauer, K. M. Droplet organelles? EMBO J. 35, 1603–1612 (2016).

10. Larson, A. G. et al. Liquid droplet formation by HP1α suggests a role for phase separation in heterochromatin. Nature 547, 236–240 (2017).

11. Seydoux, G. The P granules of C. elegans: a genetic model for the study of RNA–protein condensates. J. Mol. Biol. 430, 4702–4710 (2018).

12. Ubersax, J. A. & Ferrell, J. E. Jr Mechanisms of specificity in protein phosphorylation. Nat. Rev. Mol. Cell Biol. 8, 530–541 (2007).

13. Endicott, J. A., Noble, M. E. M. & Johnson, L. N. The structural basis for control of eukaryotic protein kinases. Annu. Rev. Biochem. 81, 587–613 (2012).

14. Aumiller, W. M. & Keating, C. D. Phosphorylation-mediated RNA/peptide complex coacervation as a model for intracellular liquid organelles. Nat. Chem. 8, 129–137 (2016).

15. Carlson, C. R. et al. Phosphoregulation of phase separation by the SARS-CoV-2 N protein suggests a biophysical basis for its dual functions. Mol. Cell 80, 1092–1103.e4 (2020).

16. Yamazaki, H., Kosako, H. & Yoshimura, S. H. Quantitative proteomics indicate a strong correlation of mitotic phospho-/dephosphorylation with non-structured regions of substrates. Biochim. Biophys. Acta Proteins Proteom. 1868, 140295 (2020).

17. Darling, A. L. & Uversky, V. N. Intrinsic disorder and posttranslational modifications: the darker side of the biological dark matter. Front. Genet. 9, 158 (2018).

18. Collins, M. O., Yu, L., Campuzano, I., Grant, S. G. N. & Choudhary, J. S. Phosphoproteomic analysis of the mouse brain cytosol reveals a predominance of protein phosphorylation in regions of intrinsic sequence disorder. Mol. Cell. Proteom. 7, 1331–1348 (2008).

19. Iakoucheva, L. M. The importance of intrinsic disorder for protein phosphorylation. Nucleic Acids Res. 32, 1037–1049 (2004).

20. Das, S., Eisen, A., Lin, Y.-H. & Chan, H. S. A lattice model of charge-pattern-dependent polyampholyte phase separation. J. Phys. Chem. B 122, 5418–5431 (2018).

21. Lin, Y.-H. & Chan, H. S. Phase separation and single-chain compactness of charged disordered proteins are strongly correlated. Biophys. J. 112, 2043–2046 (2017).

22. Lin, Y.-H., Song, J., Forman-Kay, J. D. & Chan, H. S. Random-phase-approximation theory for sequence-dependent, biologically functional liquid-liquid phase separation of intrinsically disordered proteins. J. Mol. Liq. 228, 176–193 (2017).

23. Castelnovo, M. & Joanny, J. F. Phase diagram of diblock polyampholyte solutions. Macromolecules 35, 4531–4538 (2002).

24. Nott, T. J. et al. Phase transition of a disordered nuage protein generates environmentally responsive membraneless organelles. Mol. Cell 57, 936–947 (2015).

25. Pak, C. W. et al. Sequence determinants of intracellular phase separation by complex coacervation of a disordered protein. Mol. Cell 63, 72–85 (2016).

26. Bishof, I. et al. RNA-binding proteins with basic-acidic dipeptide (BAD) domains self-assemble and aggregate in Alzheimer’s disease. J. Biol. Chem. 293, 11047–11066 (2018).

27. Remnant, L., Kochanova, N. Y., Reid, C., Cisneros-Soberanis, F. & Earnshaw, W. C. The intrinsically disorderly story of Ki-67. Open Biol. 11, 210120 (2021).

28. Booth, D. G. & Earnshaw, W. C. Ki-67 and the chromosome periphery compartment in mitosis. Trends Cell Biol. 27, 906–916 (2017).

29. Van Hooser, A. A., Yuh, P. & Heald, R. The perichromosomal layer. Chromosoma 114, 377–388 (2005).

30. Hernandez-Verdun, D. & Gautier, T. The chromosome periphery during mitosis. BioEssays 16, 179–185 (1994).

31. Sun, X. & Kaufman, P. D. Ki-67: more than a proliferation marker. Chromosoma 127, 175–186 (2018).

32. Cuylen-Haering, S. et al. Chromosome clustering by Ki-67 excludes cytoplasm during nuclear assembly. Nature 587, 285–290 (2020).

33. Blethrow, J. D., Glavy, J. S., Morgan, D. O. & Shokat, K. M. Covalent capture of kinase-specific phosphopeptides reveals Cdk1–cyclin B substrates. Proc. Natl Acad. Sci. USA 105, 1442–1447 (2008).

34. Mitrea, D. M. et al. Nucleophosmin integrates within the nucleolus via multi-modal interactions with proteins displaying R-rich linear motifs and rRNA. eLife 5, e13571 (2016).

35. Holt, L. J. et al. Global analysis of Cdk1 substrate phosphorylation sites provides insights into evolution. Science 325, 1682–1686 (2009).

36. Trivedi, P. et al. The inner centromere is a biomolecular condensate scaffolded by the chromosomal passenger complex. Nat. Cell Biol. 21, 1127–1137 (2019).

37. Cuylen, S. et al. Ki-67 acts as a biological surfactant to disperse mitotic chromosomes. Nature 535, 308–312 (2016).

38. Sobecki, M. et al. The cell proliferation antigen Ki-67 organises heterochromatin. eLife 5, e13722 (2016).

39. Nash, P. et al. Multisite phosphorylation of a CDK inhibitor sets a threshold for the onset of DNA replication. Nature 414, 514–521 (2001).

40. Li, H. et al. SysPTM: a systematic resource for proteomic research on post-translational modifications. Mol. Cell. Proteom. 8, 1839–1849 (2009).

41. Schweiger, R. & Linial, M. Cooperativity within proximal phosphorylation sites is revealed from large-scale proteomics data. Biol. Direct 5, 6 (2010).

42. Freschi, L., Osseni, M. & Landry, C. R. Functional divergence and evolutionary turnover in mammalian phosphoproteomes. PLoS Genet. 10, e1004062 (2014).

43. Sawle, L. & Ghosh, K. A theoretical method to compute sequence dependent configurational properties in charged polymers and proteins. J. Chem. Phys. 143, 085101 (2015).

44. Das, R. K. & Pappu, R. V. Conformations of intrinsically disordered proteins are influenced by linear sequence distributions of oppositely charged residues. Proc. Natl Acad. Sci. USA 110, 13392–13397 (2013).

45. Saiwaki, T., Kotera, I., Sasaki, M., Takagi, M. & Yoneda, Y. In vivo dynamics and kinetics of pKi-67: transition from a mobile to an immobile form at the onset of anaphase. Exp. Cell. Res. 308, 123–134 (2005).

46. Takagi, M. Generation of an antibody recognizing a set of acetylated proteins, including subunits of BAF complexes. Biochem. Biophys. Rep. 22, 100720 (2020).

47. Schindelin, J. et al. Fiji: an open-source platform for biological-image analysis. Nat. Methods 9, 676–682 (2012).

48. Milner, S. T. Polymer brushes. Science 251, 905–914 (1991).

49. Mészáros, B., Erdős, G. & Dosztányi, Z. IUPred2A: context-dependent prediction of protein disorder as a function of redox state and protein binding. Nucleic Acids Res. 46, W329–W337 (2018).

50. Erdős, G. & Dosztányi, Z. Analyzing protein disorder with IUPred2A. Curr. Protoc. Bioinformatics 70, e99 (2020).