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。使用诱导特异性受体异源二聚化同时避免不受控制的Fzd-Wnt相互作用的Wnt替代物，我们最近通过单分子成像证明Fzd与Lrp 6的异源二聚化足以启动信号体形成。我们最近的研究结果表明，受体异源二聚化和局部富集的胞质相互作用的合作伙伴启动相分离成信号体在质膜上。这种高度合作的过程可能还涉及蛋白质-脂质相互作用和脂质相分离以及由于蛋白质磷酸化的正反馈。在这个项目中，我们将使用单分子成像技术来分析信号体形成过程中质膜上受体和效应蛋白的重排。此外，我们还将通过活细胞微图案化来量化信号体形成中所涉及的相互作用，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的作用，如磷酸化和棕榈酰化的受体与细胞的决定因素，如质膜组织的皮质肌动蛋白骨架和脂质相分离。我们的目标不仅是提供一个机制的Wnt信号在细胞中的观点，但也要解开蛋白质液相分离的调节机制，在胞质溶胶中的受体在质膜上的相互作用。

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.