Non-canoical Wnt signaling and cell motility

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DESCRIPTION (provided by applicant): The molecular mechanisms by which a cell becomes polarized for directional migration remains poorly understood. The non-canonical Wnt pathway has recently been shown to play important roles in cell polarization and migration which are required for gastrulation and neural fold closure during vertebrate embryogenesis. Importantly defects in Wnt signaling are implicated in neural fold closure disorders in humans including spina bifida and also in cancer metastasis. To date, how the Wnt signaling pathway mediates changes to the actin cytoskeleton remains at best poorly defined. Our previous studies have established that the Dishevelled-associated activator of morphogenesis protein (Daaml) provides a crucial link between Dishevelled (Dvl) and the small GTPase Rho for gastrulation. Importantly, our studies demonstrate Daaml is required for Wnt-dependent cytoskeletal changes, although the underlying biochemical details are not known. To further identify factors required downstream of Daaml for cytoskeletal changes, we performed a yeast two-hybrid screen and isolated the protein Profilin, in addition to others. Profilin can induce actin polymerization and thus provides a direct link between Daaml and the actin cytoskeleton. Co-immunoprecipitation and GST-pulldown assays confirm that Profilin is a bona-fide Daaml-interacting factor and upon Wnt-stimulation co-localizes with Daaml at the cellular membrane. Furthermore, over-expression or depletion of Profilin, similar to Daaml, blocks gastrulation cell movements in Xenopus and results in an open neural tube phenotype characteristic of spina bifida. In building a model for how non-canonical Wnt signaling through Daaml regulates cytoskeletal changes, we hypothesize Daaml and Profilin are key modulators of the actin cytoskeleton for cellular motility. In this proposal, we will investigate how Daaml functions to mediate cytoskeletal changes for cell motility using the individual advantages of both mammalian cells and Xenopus embryos in two specific aims. First, we will examine how Daaml is activated by Dvl and define the role of its domains in Rho activation, cytoskeletal changes and gastrulation. Second, we will investigate how Profilin functions in non-canonical Wnt signaling as an effector for Daaml for cytoskeletal reorganization. These studies will employ gain-of-function and loss-of- function approaches utilizing biochemical, cell biological and embryological approaches. These studies together will advance our knowledge of how non-canonical Wnt signaling regulates cell polarity and cell motility required during gastrulation cell movements and neural fold closure.