Dynamic morphological changes of intracellular organelles are often regulated by protein phosphorylation or dephosphorylation^1,2,3,4,5,6. Phosphorylation modulates stereospecific interactions among structured proteins, but how it controls molecular interactions among unstructured proteins and regulates their macroscopic behaviours remains unknown. Here we determined the cell cycle-specific behaviour of Ki-67, which localizes to the nucleoli during interphase and relocates to the chromosome periphery during mitosis. Mitotic hyperphosphorylation of disordered repeat domains of Ki-67 generates alternating charge blocks in these domains and 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. Conversely, 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.

细胞内细胞器的动态形态变化通常受蛋白质磷酸化或去磷酸化的调节^1,2,3,4,5,6. 磷酸化调节结构化蛋白质之间的立体特异性相互作用，但它如何控制非结构化蛋白质之间的分子相互作用并调节它们的宏观行为仍然未知。在这里，我们确定了 Ki-67 的细胞周期特异性行为，它在间期定位于核仁，并在有丝分裂期间重新定位到染色体外围。Ki-67 无序重复结构域的有丝分裂过度磷酸化在这些结构域中产生交替的电荷块，并增加它们的液-液相分离 (LLPS) 倾向。拟磷序列和具有增强电荷阻断性的序列在体外经历强 LLPS 并在体内诱导染色体外围形成。相反，NPM1 的有丝分裂过度磷酸化减少了电荷块并抑制了 LLPS，导致核仁溶解。细胞周期特异性相分离可以通过磷酸化来调节，方法是增强或减少无序区域的电荷阻塞，而不是通过将磷酸基团连接到特定位点。