Spatiotemporal organization and signaling of the Wnt signalosome

信号体的形成已成为Wnt信号传导的一个关键特征，然而，受体聚集和下游信号激活的机制仍不清楚。利用诱导特异性受体异二聚化的Wnt替代物，同时避免不受控制的Fzd-Wnt相互作用，我们最近通过单分子成像证明，Fzd与Lrp6的异二聚化足以启动信号体的形成。我们最近的研究结果表明，受体异二聚化和细胞质相互作用伙伴的局部富集启动了质膜上信号小体的相分离。这种高度合作的过程可能还包括蛋白-脂质相互作用和脂质相分离以及由于蛋白质磷酸化而产生的正反馈。在这个项目中，我们将使用多色单分子成像来分析信号体形成过程中受体和效应蛋白在质膜上的重排。此外，我们将通过活细胞微模式量化参与信号体形成的相互作用，并通过系统地改变蛋白质密度来分析协同性的作用。此外，我们将探讨分子决定因素（如受体的磷酸化和棕榈酰化）与细胞决定因素（如皮质肌动蛋白骨架和脂质相分离的质膜组织）的作用。我们的目标不仅是提供细胞中Wnt信号传导的机制观点，而且揭示细胞质溶胶中蛋白质液相分离响应于质膜受体相互作用的调节机制。

The formation of signalosomes has emerged as a key feature of Wnt signaling, however, the mechanisms underlying receptor clustering and downstream signal activation have remained largely unclear. Using Wnt surrogates which induce specific receptor heterodimerization while avoiding uncontrolled Fzd-Wnt interactions, we have recently demonstrated by single molecule imaging that heterodimerization of Fzd with Lrp6 is sufficient to initiate signalosome formation. Our recent results suggest that receptor heterodimerization and local enrichment of the cytosolic interaction partners initiate phase separation into signalosomes at the plasma membrane. This highly cooperative process probably also involves protein-lipid interactions and lipid phase separation as well as positive feedback due to protein phosphorylation. In this project, we will use multicolor single molecule imaging to analyze the re-arrangement of receptors and effector proteins at the plasma membrane during signalosome formation. In addition, we will quantify interactions involved in signalosome formation by live cell micropatterning and analyze the role of cooperativity by systematically altering protein densities. Furthermore, we will explore the role of molecular determinants such as phosphorylation and palmitoylation of the receptors in conjunction with cellular determinants such as the plasma membrane organizations by the cortical actin skeleton and by lipid phase separation. We aim to not only provide a mechanistic view of Wnt signaling in cells, but also to unravel the regulatory mechanism of protein liquid phase separation in cytosol in response to receptor interaction at the plasma membrane.