Project Summary: Developing and adult cells are informed of their identity and given behavioral instructions through patterns of secreted morphogens. The Wnt pathway is an important morphogenic signaling pathway, directing proliferation and differentiation of stem cells, during both embryogenesis and regeneration of neural and intestinal tissues in adulthood. Wnt inputs are transduced into behavioral responses via spatiotemporal expression of the transcription factor β-catenin (β-cat), but the mechanisms for the cell’s regulation of β-cat are unclear. For this reason, perturbations to the pathway are often unpredictable and no effective therapeutics targeting abnormal Wnt pathway activation yet exist. β-cat is degraded by an oligomeric membrane-less organelle, the Destruction Complex (DC), whose function is related to its ability to form mesoscale, liquid protein droplets. Thermodynamic disruption of DC liquid-liquid phase separation (LLPS)—either dissolution or solidification of droplets—results in aberrant signaling, suggesting that the DC transduces Wnt input via a change in its material state. Due to a lack of tools for controlling DC LLPS and measuring its signaling output, the ‘thermodynamic DC’ model of Wnt signaling remains untested. The objective of this proposal is to determine if and how DC thermodynamic state transduces Wnt input into β-cat spatiotemporal expression. I will construct an in vivo phase diagram of the DC using inducible scaffold expression and optogenetic clustering to determine whether DC LLPS changes in response to Wnt input and/or gates downstream signal output. I will use a photo-switchable tag to track endogenous β-cat degradation and DC component localization as cells receive Wnt ligand, determining the timescales and dynamic range of DC output change in response to signaling input. Finally, I will use optogenetic proximity labeling and exosome secretion to determine how the interactomes of DC scaffolds change in response to Wnt input. The proposed work will yield an input/output understanding of the intracellular Wnt pathway and the first effort to directly understand the role of LLPS in live-cell signaling dynamics in general. A model of Wnt signaling that includes thermodynamic properties of the DC will both inform our understanding of how phase separation is used to make cell fate decisions and guide future therapeutic strategies leveraging LLPS.