Multiphase Coalescence Mediates Hippo Pathway Activation

The formation of biomolecular condensates facilitates spatiotemporal control of biochemical reactions inside cells, and this process is often reversed by the dissolution of condensates. Whether the function of condensates can be suppressed without condensate dissolution remains unknown. Here we show that upstream regulators of the Hippo signaling pathway form functionally antagonizing condensates, and the coalescence of these distinct condensates into a common phase provides a novel mode of counteracting the function of biomolecular condensates without condensate dissolution. Specifically, the negative regulator SLMAP forms Hippo-inactivating condensates to facilitate pathway inhibition by the STRIPAK phosphatase complex. In response to cell-cell contact or osmotic stress, the positive regulators AMOT and KIBRA form Hippo-activating condensates to facilitate pathway activation. The functionally antagonizing SLMAP and AMOT/KIBRA condensates further coalesce into a common phase to inactivate STRIPAK function by excluding SIKE, an essential STRIPAK subunit that is normally enriched in the Hippo-inactivating SLMAP condensates. These findings provide a new paradigm for restricting the activity of biomolecular condensates without condensate dissolution, shed light on the molecular principles of multiphase organization, and offer a new conceptual framework for understanding upstream regulation of the Hippo signaling pathway.