The stability of NPM1 oligomers regulated by acidic disordered regions controls the quality of liquid droplets

The nucleolus is a membrane-less nuclear body that typically forms through the process of liquidliquid phase separation (LLPS) involving its components. NPM1 drives LLPS within the nucleolus and its oligomer formation and inter-oligomer interactions play a cooperative role in inducing LLPS. However, the molecular mechanism underlaying the regulation of liquid droplet quality formed by NPM1 remains poorly understood. In this study, we demonstrate that the N- terminal and central acidic residues within the intrin- sically disordered regions (IDR) of NPM1 contribute to attenuating oligomer stability, although differences in the oligomer stability were observed only under strin- gent conditions. Furthermore, the impact of the IDRS is augmented by an increase in net negative charges resulting from phosphorylation within the IDRS. Sig- nificantly, we observed an increase in fluidity of liquid droplets formed by NPM1 with decreased oligomer stability. These results indicate that the difference in oligomer stability only observed biochemically under stringent conditions has a significant impact on liquid droplet quality formed by NPM1. Our findings provide new mechanistic insights into the regulation of nucleolar dynamics during the cell cycle.