Nucleolar dynamics are determined by the ordered assembly of the ribosome

Ribosome biogenesis occurs in the nucleolus, a biomolecular condensate whose material properties are thought to be important for function. However, the molecular basis of nucleolar dynamics and their relationship to ribosome assembly remain incompletely understood. We present a platform for high-throughput FRAP (HiT-FRAP) and use it to screen hundreds of genes for their impact on dynamics of the nucleolar scaffold nucleophosmin (NPM1). We find that NPM1 dynamics and nucleolar morphology are sensitive to ribosome assembly state: accumulation of early pre-ribosomal intermediates slows NPM1 dynamics and compacts the condensate, while accumulation of abortive late precursors accelerates dynamics and disrupts condensate integrity. These opposing biophysical states correlate with the strength of NPM1–pre-ribosome interactions. Importantly, mutations in the NPM1 intrinsically disordered region that alter pre-ribosome binding directly tune nucleolar dynamics. These results establish that ribosomal precursor assembly state determines nucleolar material properties through the strength of scaffold–pre-ribosome interactions and introduce HiT-FRAP as a platform for interrogating condensate dynamics broadly.