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

Wnt 的新阶段：利用光遗传学询问热力学信号节点

• 批准号: 10313754
• 负责人: Ryan Lach
• 金额: $ 4.6万
• 依托单位: UNIVERSITY OF CALIFORNIA SANTA BARBARA
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-07-07 至 2024-07-06
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10313754
• 关键词: Adult; Behavioral; Biophysics; Biotin; Cell Nucleus; Cells; Cellular Membrane; Complex; Development; Differentiation and Growth; Embryonic Development; Environment; Equation; Felis catus; Fluorescence; Fluorescence Recovery After Photobleaching; Future; Genetic Transcription; Genome; Goals; Image; Instruction; Intestines; Label; Ligands; Ligase; Light; Liquid substance; Maintenance; Maps; Mass Spectrum Analysis; Measurement; Measures; Membrane; Modeling; Nerve Regeneration; Organelles; Organism; Output; Pathway interactions; Pattern; Phase; Phosphorylation; Proliferating; Property; Proteins; Regulation; Role; Runaway; Sampling; Signal Pathway; Signal Transduction; Stimulus; Structure; System; Testing; Therapeutic; Thermodynamics; Time; Tissues; Ubiquitination; WNT Signaling Pathway; Work; behavioral response; beta catenin; biophysical properties; cell behavior; exosome; improved; in vivo; morphogens; optogenetics; photo switch; prevent; programs; response; scaffold; spatiotemporal; stem cell differentiation; stem cells; therapeutic target; therapeutically effective; tool; transcription factor; transmission process

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.

项目概要： 发育中的细胞和成体细胞被告知其身份，并通过以下模式获得行为指令： Wnt 通路是重要的形态发生信号通路，指导增殖。 和干细胞的分化，在神经和肠道组织的胚胎发生和再生过程中 成年期Wnt输入通过时空表达转变成行为反应。 转录因子 β-catenin (β-cat)，但细胞调节 β-cat 的机制尚不清楚。 原因是，对该通路的干扰通常是不可预测的，并且没有针对异常的有效治疗方法 Wnt 通路激活仍然存在，被寡聚无膜细胞器“破坏”降解。 复合物（DC），其功能与其形成介观液体蛋白质液滴的能力有关。 DC 液-液相分离 (LLPS) 的破坏（液滴溶解或凝固）会导致 信号异常，表明 DC 由于缺乏而通过其物质状态的变化来转导 Wnt 输入。 用于控制 DC LLPS 并测量其信号输出的工具，Wnt 的“热力学 DC”模型 该提案的目的是确定直流热力学状态是否以及如何状态。 将 Wnt 输入转导为 β-cat 时空表达 我将构建 DC 的体内相图。 使用诱导支架表达和光遗传学聚类来确定 DC LLPS 是否发生变化 响应 Wnt 输入和/或门下游信号输出，我将使用光切换标签来跟踪。 当细胞接受 Wnt 配体时，内源性 β-cat 降解和 DC 成分定位，从而确定 直流输出响应信号输入而变化的时间尺度和动态范围最后，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而变化 拟议的工作将产生对细胞内 Wnt 通路的输入/输出的理解和 首次直接了解 LLPS 在活细胞信号传导动力学中的作用 Wnt 模型。 包括 DC 热力学特性的信号将帮助我们理解相如何 分离用于做出细胞命运决定并指导未来利用 LLPS 的治疗策略。