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

项目概要： 发育中的和成年的细胞被告知它们的身份，并通过以下模式给予行为指令： 分泌的形态发生素Wnt信号通路是一种重要的形态发生信号通路， 在胚胎发生以及神经和肠组织再生过程中，干细胞的分化 成年Wnt输入通过Wnt的时空表达被转换为行为反应 转录因子β-catenin（β-cat），但β-cat的细胞调节机制尚不清楚。为此 因此，对通路的干扰通常是不可预测的，并且没有针对异常的 Wnt通路激活仍然存在。β-cat被低聚无膜细胞器破坏 复合物（DC），其功能与其形成中尺度液体蛋白液滴的能力有关。热力学 DC液-液相分离（LLPS）的破坏-液滴的溶解或固化-导致 异常信号传导，表明DC通过其材料状态的变化转导Wnt输入。由于缺乏 用于控制DC LLPS并测量其信号输出的工具中，Wnt的“热力学DC”模型 信号仍然未经测试。这个建议的目的是确定直流热力学状态是否以及如何 将Wnt输入转导为β-cat时空表达。我将构建DC的体内相图 使用可诱导的支架表达和光遗传学聚类来确定DC LLPS是否改变， 响应于Wnt输入和/或选通下游信号输出。我会用一个可转换的标签来追踪 当细胞接受Wnt配体时，内源性β-cat降解和DC成分定位，确定了 DC输出的时间尺度和动态范围响应于信令输入而改变。最后，我将使用光遗传学 邻近标记和外泌体分泌，以确定DC支架的相互作用组如何响应 Wnt输入。拟议的工作将产生细胞内Wnt通路的输入/输出的理解， 这是直接了解LLPS在一般活细胞信号动力学中的作用的第一次努力。Wnt模型 包括DC的热力学性质的信号既将告知我们如何理解相位 分离用于做出细胞命运决定并指导利用LLPS的未来治疗策略。

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.