β-catenin Binds to Gsk-3β in Liquid-Liquid Phase Separation Compartment in HEK293 Cells

Liquid-liquid phase separation (LLPS) has emerged as a significant mechanism for cellular organization, impacting various biological processes, including Wnt/β-catenin signaling. This study investigates the role of LLPS in the regulation of β-catenin in HEK293 cells, particularly in response to Wnt3a signaling. Our findings demonstrate that β-catenin is regulated by LLPS, forming spherical droplets indicative of this phenomenon. Fluorescence recovery after photobleaching (FRAP) assays revealed that these droplets exhibit reversible dynamics, further confirming their phase-separated nature. Importantly, treatment with Wnt3a led to an increase in β-catenin levels, while simultaneously reducing the recovery of fluorescence intensity in FRAP experiments, suggesting that enhanced Wnt signaling may stimulate the release of β-catenin from LLPS. Immunoprecipitation studies indicated that β-catenin binds to glycogen synthase kinase 3β (Gsk-3β) within the LLPS state, highlighting a potential regulatory mechanism whereby LLPS facilitates the phosphorylation and subsequent degradation of β-catenin. The addition of 1,6-hexanediol disrupted the β-catenin/Gsk-3β interaction, reinforcing the idea that LLPS plays a critical role in modulating these biochemical interactions. The findings presented in this study suggest that LLPS is not only crucial for the spatial organization of β-catenin but also serves as a regulatory mechanism for its signaling functions in the Wnt pathway. Given the association of aberrant Wnt signaling with various diseases, including cancer and neurodegenerative disorders, understanding the role of LLPS in this context may provide new insights into therapeutic strategies targeting these pathological conditions.