Cell cycle-specific phase separation

• Dynamic morphological changes of intracellular organelles are often regulated by protein phosphorylation or dephosphorylation

  
• Phosphorylation modulates stereospecific interactions among structured proteins, but how it controls molecular interactions among unstructured proteins and regulates their macroscopic behaviours remains unknown
• Ki-67


  Localizes to the nucleoli during interphase and relocates to the chromosome periphery during mitosis

  
• Mitotic hyperphosphorylation of Ki-67


  1. Generates alternating charge blocks in the disordered repeat domains

  2. Increases their propensity for liquid–liquid phase separation (LLPS)

  
• A phosphomimetic sequence and the sequences with enhanced charge blockiness

  Underwent strong LLPS in vitro and induced chromosome periphery formation in vivo

  
• Mitotic hyperphosphorylation of NPM1


  Diminished a charge block and suppressed LLPS, resulting in nucleolar dissolution

  
• Cell cycle-specific phase separation

  Can be modulated via phosphorylation by enhancing or reducing the charge blockiness of disordered regions, rather than by attaching phosphate groups to specific sites

  
• Numerous recent studies have reported the liquid-like behaviour of intracellular membraneless organelles, such as nucleoli, stress granules and processing bodies

  
• Liquid–liquid phase separation (LLPS), coacervation or condensation


  Reversible formation and dissolution of these organelles during the cell cycle and intracellular signalling plays critical roles in cellular responses and homeostasis and is regulated by various post-translational modifications
• Phosphorylation regulates not only protein-based phase separation in positive or negative manners but also protein–nucleic acid coacervation

  
• A recent study showed that viral replication is regulated by phosphorylation-dependent LLPS of viral nucleocapsid protein
• Intrinsically disordered regions (IDRs)


  How phosphorylation of these regions regulates LLPS mechanistically remains elusive

  
• Recent studies using charged polymers and theoretical modelling demonstrated that a polyampholyte chain with segregated charged residues (charge blocks) exhibits stronger phase separation than the chain with the same number of charged residues randomly distributed
• Charge blocks also play important roles in phase separation in vivo, and shuffling of the charged residues along a polypeptide results in the dispersion of liquid-like organelles in the cell

  
• Many IDRs of cellular proteins are hyperphosphorylated during mitosis, for example, Ki-67, RIF1, INCENP and NPM1

  
• The addition of multiple negatively charged groups may enhance or reduce such 'charge blockiness' of IDRs and affect the propensity for LLPS in the cell
• Ki-67


  A nucleolar phosphoprotein that plays a critical role in organizing the periphery of mitotic chromosomes, which are thought to have a liquid-like property

  
• Human Ki-67


  • Composed of multiple domains, including an N-terminal PP1-binding domain, a central repeat domain (RD) composed of 16 repeats of an ~110-amino-acid unit, and a C-terminal chromatin-targeting domain (LR domain) for chromosome binding

  
• Phosphoproteomic analyses demonstrated that Ki-67 is hyperphosphorylated by CDK1 and other mitotic kinases

  
• Quantitative mass spectrometric analysis of mitotic phosphorylation identified more than 70 residues in the RD that are significantly phosphorylated upon entry into mitosis

  
• Comparison of the charge distributions between the mitotic (hyperphosphorylated) form and interphase (dephosphorylated) form revealed that mitotic phosphorylation converts the individual repeats into strong diblock ampholytes, in which a positive charge block is followed by a negative block

  
• Mitotic hyperphosphorylation of nucleophosmin (NPM1), an IDR-rich nucleolar protein that interacts with Ki-67 and plays a critical role in assembling nucleolar components in interphase cells, diminishes the alternating charge blocks that otherwise exist in the non-phosphorylated form

  
• Mitotic hyperphosphorylation may introduce negative charges to enhance or reduce the alternating charge blocks in the IDRs and modulate the propensity for LLPS

  
• Recombinant proteins of human Ki-67 RD formed liquid-like droplets in vitro in the presence of 100 mM NaCl and 15% polyethylene glycol

  
• A clear inverse correlation between the number of repeats and the propensity of LLPS (quantified by the saturation concentration, Csat) was observed; Csat sharply decreased as the repeat number increased
• In vitro phosphorylation of recombinant R12 by CDK1 increased droplet formation

  
• Phosphomimetic mutations in nine mitotic phosphosites (Pm9) in R12 enhanced droplet formation

  
• Phosphomimetic mutations in another repeat (R7) also enhanced LLPS
• The phosphomimetic mutations did not induce the formation of any secondary structures

  
• Charge-block mimetic mutant (CBm) construction

  1. Neutralization of the positive charge block at the amino-terminal region reduced the formation of liquid droplets

  2. Replacement of neutral residues in the middle region with E residues, which mimics phosphorylated charge blocks, substantially promoted LLPS

  3. Replacement of neutral residues in the carboxyl-terminal region with E residues also promoted LLPS

  
• Extent of charge blockiness along the polypeptide

  • Evaluated based on either the blockiness of like charges (BLC) or degree of segregation (Dseg)
  • Inverse correlation with Csat

  
• A series of mutants (CBm-5–16) carrying different net charges and/or charge blockiness were constructed and subjected to LLPS assay
• Csat more closely correlated with charge blockiness (BLC and Dseg) than with the net charge
• The existence of alternating charge blocks governs LLPS in vitro, and neither the exact position of the charged residues nor a specific net charge is critical
• Tic mutant (Pm9)


  A similar effect was observed when E residues were introduced in the carboxyl-terminal region (CBm-4)
• The relationship between charge blockiness and LLPS was investigated quantitatively

  The extent of charge blockiness along the polypeptide was evaluated on the basis of either the blockiness of like charges (BLC) or degree of segregation (Dseg)
• Plotting Csat against BLC and Dseg

  Revealed a clear inverse correlation
• A series of mutants (CBm-5–16) carrying different net charges (between −4 and +5) and/or charge blockiness


  • Were constructed and subjected to LLPS assay
• Csat more closely correlated with charge blockiness (BLC and Dseg)


  Than with the net charge
• These results demonstrate that the existence of alternating charge blocks governs LLPS in vitro and indicate that neither the exact position of the charged residues nor a negative shift of the net charge is a critical determinant