Discussion

• Dynamic morphological changes of intracellular organelles are regulated by

  Protein phosphorylation or dephosphorylation
• Modulates stereospecific interactions among unstructured proteins

  Phosphorylation
• How does phosphorylation control molecular interactions and regulate their macroscopic behaviors

  Still unknown
• Localizes to nucleoli during interphase and relocates to the chromosome periphery during mitosis


  Ki-67
• Generates alternating charge blocks and increases propensity for liquid-liquid phase separation

  Mitotic hyperphosphorylation of disordered repeat domains of Ki-67
• Underwent strong LLPS in vitro and induced chromosome periphery formation in vivo


  Phosphomimetic sequence and sequences with enhanced charge blockiness
• Diminished a charge block and suppressed LLPS, resulting in nucleolar dissolution

  Mitotic hyperphosphorylation of NPM1
• Modulated via phosphorylation by enhancing or reducing the charge blockiness of disordered regions, rather than by attaching phosphate groups to specific sites


  Cell cycle-specific phase separation
• How are intracellular organelles formed

  Interactions among proteins and nucleic acids by LLPS
• How does phosphorylation regulate formation and dissolution of organelles

  Changes the structure, interactions and intracellular localization of substrate proteins, and regulates intracellular signaling pathways
• Regulates protein-based phase separation and protein-nucleic acid coacervation

  Phosphorylation
• Viral replication is regulated by

  LLPS of viral nucleocapsid protein
• Chain with segregated charge blocks has stronger phase separation than


  Chain with randomly distributed charge blocks
• Shuffling of charged residues along a polypeptide


  Dispersion of liquid-like organelles in the cell
• Addition of multiple negatively charged groups 


  May enhance or reduce charge blockiness of IDR and affect propensity for LLPS in the cell
• Separates chromosomes from each other and prevents their coalescence during mitosis

  Ki-67
• Multiple domains, N-terminal PP1-binding domain, RD, and LR domain 


  Ki-67
• Hyperphosphorylates Ki-67

  CDK1
• Significantly phosphorylated upon entry into mitosis

  70 residues in the RD
• Converts individual repeats into strong diblock ampholytes, in which a positive block is followed by a negative block

  Mitotic phosphorylation
• Enhances alternating charge blocks throughout RD

  Mitotic phosphorylation
• Diminishes alternating charge blocks that otherwise exist in the non-phosphorylated form

  Mitotic hyperphosphorylation of NPM1
• Introduce negative charges to enhance or reduce the alternating charge blocks in the IDR and modulate propensity for LLPS


  Mitotic hyperphosphorylation
• Propensity of LLPS decreased as


  Repeat number increased
• In vitro phosphorylation of R12 by CDK1

  Enhanced LLPS
• Phosphomimetic mutations


  Enhanced LLPS
• Alternating charge blocks


  Necessary for LLPS
• Basis on the extent of charge blockiness along the polypeptide

  Blockiness of like charges or degree of segregation
• Existence of alternating charge blocks


  Governs LLPS in vitro, neither the exact position of charged residues nor negative shift of net charge is a determinant
• Test whether the Ki-67 RD could form a liquid phase on an artificial chromosome surface in vitro

  Phosphomimetic mutant and the charge-block mutant assembled stronger than the wild type
• Necessary and sufficient for localization at the mitotic chromosome periphery

  Block-polyampholyte repeat
• Liquid like behavior of Ki-67 at chromosome periphery was confirmed by


  Treating the cells with ammonium acetate and FRAP analysis
• Replacement of all mitotic phosphosites with non-phosphorylatable residues abolished mitotic phosphorylation


  Diminished peripheral localization
• Phosphomimetic mutant and charge-block mutant showing similar LLPS in the in vitro droplet assay


  Localized at chromosome periphery
• Examination of the ability to form a functional chromosome periphery

  Ki-67 knockout cells
• Mitotic chromosomes coalesced, forming a large single mass of chromatin


  Ki-67 knockout cells
• Number of repeats rather than specific amino-acid sequence of R12

  Formation of chromosome periphery
• Charge blockiness, not negative shift of the net charge

  Formation of chromosome periphery
• Efficient LLPS in vitro and forming functional mitotic chromosome periphery in vivo


  Alternating charge blocks of Ki-67 RD
• Dephosphorylated form (interphase)


  Strong block-polyampholytic charge distribution