Nucleolar dynamics are determined by the ordered assembly of the ribosome

Ribosome biogenesis occurs in the nucleolus, a nuclear biomolecular condensate that exhibits dynamic biophysical properties thought to be important for function. However, the relationship between ribosome assembly and nucleolar dynamics is incompletely understood. Here, we present a platform for high-throughput fluorescence recovery after photobleaching (HiT-FRAP), which we use to screen hundreds of genes for their impact on dynamics of the nucleolar scaffold nucleophosmin (NPM1). We find that scaffold dynamics and nucleolar morphology respond to disruptions in key stages of ribosome biogenesis. Accumulation of early ribosomal intermediates leads to nucleolar rigidification while late intermediates lead to increased fluidity. We map these biophysical changes to specific ribosomal intermediates and their affinity for NPM1. We also discover that disrupting mRNA processing impacts nucleolar dynamics and ribosome biogenesis. This work mechanistically ties ribosome assembly to the biophysical features of the nucleolus and enables study of how dynamics relate to function across other biomolecular condensates. HighlightsO_LIHigh-Throughput Fluorescence Recovery After Photobleaching (HiT-FRAP) platform discovers factors that govern macromolecular dynamics of the nucleolar scaffold NPM1 C_LIO_LINPM1 dynamics and nucleolar morphology are determined by specific ribosomal intermediates in the nucleolus C_LIO_LIMutation of interfaces in NPM1 that mediate ribosome interactions tunes nucleolar dynamics C_LIO_LIDisruption of mRNA processing pathways leads to accumulation of early rRNA precursors in the nucleolus and rigidification C_LI