Characterization of two proteins that regulate vertebrate

细胞为定向迁移而变得极化的过程尚不清楚。Wnt

The process by which a cell becomes polarized for directional migration is not well understood. The Wnt signaling pathway is required for polarization and cellular motility during embryogenesis. This pathway is divided into two branches termed canonical and non-canonical and the non-canonical pathway and is required during vertebrate gastrulation and neural fold closure. Importantly, defects in non-canonical Wnt signaling are implicated in birth defects disorders including spina bifida and also in human cancer metastasis. Despite the compelling evidence that the Wnt pathway can mediate cytoskeletal changes for cell motility, it remains unclear how this is accomplished along with the molecular factors and mechanisms involved in this process. In our preliminary studies, we have determined that the Formin homology protein Dishevelled-associated activator of morphogenesis (Daam1) links Dishevelled (Dvl) to the GTPase Rho to transduce non-canonical Wnt signaling. In response to Wnt stimulation, Dvl binds Daam1 and mediates Rho activation. These steps result in cytoskeletal reorganization required for cellular motility during gastrulation and neural fold closure in the Xenopus embryo. However, it is not fully understood how Daam1 modifies the cytoskeleton and the identity of the effector(s) downstream of Daam1 required for cytoskeletal reorganization remains unknown. We have isolated two proteins as new uncharacterized binding partners for Daam1. Using co-immunoprecipitation (IP) and GST-pulldown assays, we have verified that these proteins binds to Daam1 and defined the domains within each protein required for binding. We observed that colocalized with Daam1 in culture cells and this localization was sensitive to Wnt-stimulation. In Xenopus, gain-of-function and loss-of functions show both proteins further regulates gastrulation in the non-canonical Wnt pathway with Daam1. In building a model for the mechanism of action of these two proteins, we hypothesize that Wnt stimulation regulates a Daam/protein complex formation, which activates these proteins which in turn mediates cytoskeletal changes that are required for cell polarity and cell motility. We will pursue two specific aims in this R03 proposal to test our hypothesis that these two proteins are key factors for Wnt-mediated cytoskeletal changes for vertebrate gastrulation and neural tube closure utilizing Xenopus laevis, zebrafish and mammalian cell-culture based systems. We hypothesize that these two proteins are essential mediators for non-canonical Wnt mediated cytoskeletal regulation for cellular motility during vertebrate gastrulation and neural tube closure. Our studies will test this hypothesis and provide insights into the Wnt-signaling network.