Macromolecular Condensation Organizes Nucleolar Sub-Phases to Set Up a pH Gradient

Nucleoli are characterized by coexisting sub-phases of distinct macromolecular compositions. Here, we uncover biophysical principles underlying hierarchical organizations of macromolecules within nucleoli. Informatics analyses and investigations incellulo identified highly-enriched, evolutionarily-conserved intrinsically disordered regions (IDRs) in nucleolar proteins that include acidic (D/E) tracts and blocks of lysine residues interspersed by glutamic-acid-rich-regions. Sub-phase localizations are determined by molecular grammars of nucleolar proteins featuring specific IDRs tethered to nucleic acid binding domains. We find, using in vitro reconstitutions, biophysical computations, and in cellulo studies that condensation, which combines binding and phase separation driven by complex coacervation, enables hierarchal organization within nucleoli. In vitro findings reveal that D/E tracts lower the pH of condensates formed by nucleolar components. Importantly, these sequence features appear to be generative of acidic nucleoli in cellulo. Condensation, driven by specific molecular grammars, gives rise to emergent properties whereby nuclear bodies of distinct physicochemical properties coexist with one another.