TRIP6 condensates constrain Hippo pathway hyperactivation under osmotic stress

Abstract

The Hippo pathway integrates mechanical and chemical cues to regulate cell proliferation and tissue homeostasis, whereas its dysregulation contributes to impaired regeneration and tumorigenesis. Liquid-liquid phase separation has emerged as an organizing principle for Hippo signal transduction, but how cells constrain excessive pathway activation under stress remain poorly defined. Here we demonstrate that TRIP6, a LIM-domain scaffold and tension-sensitive Hippo inhibitor, undergoes intrinsic phase separation both in vitro and in living cells. Osmotic stress triggers rapid redistribution of TRIP6 condensates from large cortical assemblies to dispersed cytoplasmic puncta. Domain mapping shows that the N-terminal IDR2 drives TRIP6 phase separation, whereas the C-terminal LIM domains negatively regulate condensate fluidity. Mechanistically, TRIP6 condensates selectively recruit the LATS1 inhibitor AJUBA and the MST1 scaffold SAV1 while excluding MST1 itself. The phase-separation-deficient {Delta}IDR2 mutant fails to undergo stress-induced condensate redistribution, exhibits weakened LATS1 binding, and shows increased YAP phosphorylation and cytoplasmic retention under osmotic stress, with reduced cell proliferation and stress resilience. Finally, TRIP6 condensates disassemble in a mouse model of elevated epidermal tissue tension, identifying them as mechanosensitive platforms that link phase transitions to Hippo-dependent growth control. Together, these findings establish TRIP6 condensates as a negative regulatory node that buffers excessive Hippo activation to preserve cellular homeostasis under hyperosmotic conditions.