Scaffold proteins are macromolecules that drive condensation, while clients are macromolecules that cannot condense on their own but become selectively enriched within condensates. A central question of our work is how client molecules become enriched in condensates and what role scaffold-client interactions play in shaping condensate composition. More than seventy scaffold proteins are known to colocalize with diverse partners, including signaling proteins such as kinases. We have previously demonstrated that focal adhesion kinase (FAK) and p130Cas are scaffold proteins that drive condensation to promote the formation of focal adhesions in cells. FAK and Cas are each capable of condensing independently but also interact with each other. Moreover, both FAK and Cas can interact with and colocalize with the client protein paxillin. Using reconstituted systems of FAK, Cas, and paxillin, we measure how FAK-Cas colocalization depends on the phosphorylation of FAK and how paxillin influences the formation of heterotypically driven condensates. To interpret these results, we use a three component Flory Huggins model. This framework captures how two scaffold proteins, which form condensates independently, can display enhanced enrichment when combined, and how clients further tune the thermodynamics of colocalized condensates. We develop theoretical predictions for the critical interaction parameters in this model and compare them directly with results from reconstituted systems. Together, our work provides a paradigmatic framework for scaffold-client interactions. It offers insight into the principles that govern client enrichment in multicomponent condensates, an organizational strategy that is both widespread and fundamental to cellular function.
Ghosal et al. (2026) studied this question.