Principles of Client Enrichment Through Scaffold–Client Interactions in Biomolecular Condensates

Scaffold proteins are macromolecules that drive condensation, whereas clients are those that cannot condense independently but become selectively enriched within condensates. A central question of our work is how client molecules become enriched within condensates and how scaffold–client interactions influence the overall condensate composition. More than seventy scaffold proteins are known to colocalize with diverse partners, including signaling proteins such as kinases. We've shown previously that Focal Adhesion Kinase (FAK) and p130Cas act as scaffolds that undergo condensation to promote focal adhesion formation in cells. Both FAK and Cas can condense independently, but they also interact with each other and with the client protein paxillin. Using reconstituted mixtures of FAK, Cas, and paxillin, we measure how FAK–Cas colocalization depends on FAK phosphorylation and how paxillin modulates the formation of heterotypically driven condensates. To interpret these results, we employ a three-component Flory–Huggins model that captures how two scaffold proteins, each capable of forming condensates independently, exhibit enhanced enrichment when combined, and how clients further modulate the thermodynamics of colocalized condensates. We then derive theoretical predictions for the critical interaction parameters and directly compare them with results from reconstituted systems. These findings establish a paradigmatic framework for scaffold and client interactions and clarify the principles that govern client recruitment in multicomponent condensates, an organizational strategy that is both widespread and fundamental to cellular function.