Spatiotemporal organization and signaling of the Wnt signalosome

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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.