A New Phase of Wnt: Interrogating a Thermodynamic Signaling Node Using Optogenetics

项目总结： 发育中的和成年的细胞被告知它们的身份，并通过模式 分泌的形态生物质。Wnt通路是一条重要的形态发生信号通路，指导细胞增殖 干细胞在胚胎发生和神经和肠道组织再生过程中的作用 成人期。WNT的输入通过时空表达被转化为行为反应 转录因子β-Catenin(β-CAT)，但细胞对β-CAT的调控机制尚不清楚。为了这个 原因是，该途径的扰动通常是不可预测的，并且没有针对异常的有效治疗方法 WNT途径的激活仍然存在。β-CAT被低聚无膜细胞器降解，破坏 复合体(DC)，其功能与其形成中尺度液态蛋白质液滴的能力有关。热力学 直流液-液相分离(LLP)的中断--液滴的溶解或凝固--导致 异常信号，提示DC通过其物质状态的改变来传递WNT输入。由于缺乏 用于控制DC LLP和测量其信号输出的工具，WNT的热力学DC模型 信令仍未经过测试。本提案的目标是确定直流热力状态是否以及如何 将WNT输入转换为β-CAT时空表达。我将构建DC的活体相图 利用可诱导支架表达和光遗传聚类法确定DC LLP是否发生改变 对WNT输入和/或门下游信号输出的响应。我将使用一个可照片切换的标签来跟踪 当细胞接受Wnt配体时，内源性β-CAT的降解和DC成分的定位，决定了 直流输出的时标和动态范围随信号输入的变化而变化。最后，我将使用光生技术 邻近标记和外体分泌来确定DC支架的相互作用是如何变化的 转到WNT输入。拟议的工作将产生对细胞内Wnt途径的输入/输出的理解 第一次直接了解LLP在一般活细胞信号动力学中的作用。WNT的一个模型 包含DC热力学性质的信号将使我们了解相如何 分离被用来做出细胞命运的决定，并利用有限合伙人指导未来的治疗策略。

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.