Wnt signal transduction is controlled by the destruction complex (DC), a condensate comprising scaffold proteins and kinases that regulate β-catenin stability. Overexpressed DC scaffolds undergo liquid–liquid phase separation (LLPS), but DC mesoscale organization at endogenous expression levels and its role in β-catenin processing were previously unknown. Here, we find that DC LLPS is nucleated by the centrosome. Through a combination of CRISPR-engineered custom fluorescent tags, finite element simulations, and optogenetic tools that allow for manipulation of DC concentration and multivalency, we find that centrosomal nucleation drives processing of β-catenin by colocalizing DC components to a single reaction crucible. Enriching GSK3β partitioning on the centrosome controls β-catenin processing and prevents Wnt-driven embryonic stem cell differentiation to mesoderm. Our findings demonstrate the role of nucleators in controlling biomolecular condensates and suggest tight integration between Wnt signal transduction and the cell cycle.

Wnt signal transduction is mediated by a protein assembly called the Destruction Complex (DC) made from scaffold proteins and kinases that are essential for transducing extracellular Wnt ligand concentrations to changes in nuclear β-catenin, the pathway’s transcriptional effector. Recently, DC scaffold proteins have been shown to undergo liquid-liquid phase separation in vivo and in vitro providing evidence for a mesoscale organization of the DC. However, the mesoscale organization of DC at endogenous expression levels and how that organization could play a role in β-catenin processing is unknown. Here we find that the native mesoscale structure is a dynamic biomolecular condensate nucleated by the centrosome. Through a combination of advanced microscopy, CRISPR-engineered custom fluorescent tags, finite element simulations, and optogenetic tools, that allow for independent manipulation of the biophysical parameters that drive condensate formation, we find that a function of DC nucleation by the centrosome is to drive efficient processing of β-catenin by co-localizing DC components to a single reaction hub. We demonstrate that simply increasing the concentration of a single DC kinase onto the centrosome controls β-catenin processing. This simple change in localization completely alters the fate of the Wnt-driven human embryonic stem cell differentiation to mesoderm. Our findings demonstrate the role of nucleators in dynamically controlling the activities of biomolecular condensates and suggest a tight integration between cell cycle progression and Wnt signal transduction.