Nucleolar integrity in isolated nucleoli is governed by electrostatic interactions and altered by disease-associated proteins.

The nucleolus is a biomolecular condensate maintained by multivalent intermolecular interactions. However, the physicochemical interactions that preserve the nucleolar integrity remain poorly understood. We established an isolated nucleolus assay to investigate the mechanisms that maintain nucleolar integrity. Increasing the ionic strength or adding basic amino acids disrupted the nucleolar organization, whereas 1,6-hexanediol and RNase treatment had little effect, highlighting the major role of electrostatic interactions. Arginine-rich dipeptide repeat proteins associated with C9orf72-linked neurodegeneration caused greater disruption than free arginine, depending on the repeat length and concentration. The assay further revealed that unlike the wild-type HMGB1, a disease-associated HMGB1 mutant resisted arginine-induced release from the nucleoli. These findings identify electrostatic interactions as major determinants of nucleolar integrity and provide a platform for investigating disease-associated alterations in nucleolar organization.