Design of de novo nucleolar surface proteins

Biomolecular condensates are membraneless intracellular structures formed from the phase separation of proteins and nucleic acids. Although biomolecular condensates do not have a phospholipid membrane at their surface, recent studies reveal conspicuous assemblies of proteins at the interface between the dense and dilute phases of nucleoli, P granules, and other condensates. The molecular and biophysical rules that govern the surfactant-like localization of these proteins are only beginning to be understood. Here, we created de novo nucleolar surface proteins (NoLSurfers) by fusing diverse synthetic nucleoli-philic and nucleoli-phobic protein segments. By quantitatively analyzing the spatial distribution of different combinations of nucleoli-philic and nucleoli-phobic protein segments, we find that the nucleolar surface localization of NoLSurfers is largely determined by the oligomerization state of nucleoli-philic proteins and their immiscibility with the underlying condensate structure. Although nucleoli-philic proteins with low oligomerization are miscible in the dense phase of nucleoli, nucleoli-philic proteins with high oligomerization become immiscible and show surface localization, and in some cases, they form de novo condensates in the nucleoplasm. These engineered NoLSurfers are useful as a tool to both elucidate central aspects of the biophysics of condensate interfaces and to potentially modulate condensate properties and function.