Characterization of two proteins that regulate vertebrate

一个细胞为了定向迁移而极化的过程还不很清楚。所述Wnt 信号通路是胚胎发生过程中极化和细胞运动所必需的。这条路是分开的 分为两个分支，称为经典和非经典和非经典途径， 脊椎动物原肠胚形成和神经褶闭合。重要的是，非经典Wnt信号传导中的缺陷是 与包括脊柱裂在内的出生缺陷疾病以及人类癌症转移有关。尽管 令人信服的证据表明，Wnt途径可以介导细胞运动的细胞骨架变化，但目前仍不清楚 这是如何完成的沿着与分子因素和机制参与这一过程。 在我们的初步研究中，我们已经确定了形成蛋白同源蛋白Dishevelled相关 形态发生激活因子（Daam 1）将Dishevelled（Dvl）与GTvl Rho连接到非典型的GTvl Rho， Wnt信号。响应于Wnt刺激，Dvl结合Daam 1并介导Rho激活。这些步骤 导致原肠胚形成期间细胞运动和神经折叠闭合所需的细胞骨架重组， 非洲爪蟾胚胎然而，Daam 1如何修饰细胞骨架和身份还不完全清楚。 细胞骨架重组所需的Daam 1下游效应子的数量仍然未知。我们有 分离出两种蛋白质作为Daam 1的新的未表征的结合伴侣。使用免疫共沉淀（IP） 和GST-下拉分析，我们已经证实，这些蛋白质结合Daam 1，并确定了结构域内 每种蛋白质都需要结合。我们观察到在培养细胞中与Daam 1共定位， 对Wnt刺激敏感。在非洲爪蟾中，功能获得和功能丧失进一步显示了这两种蛋白质 在非经典Wnt通路中与Daam 1一起调节原肠胚形成。 在建立这两种蛋白作用机制的模型时，我们假设Wnt刺激 调节Daam/蛋白质复合物的形成，从而激活这些蛋白质，进而介导细胞骨架蛋白的形成。 细胞极性和细胞运动所需的变化。在R 03建议书中，我们会追求两个具体目标 为了验证我们的假设，即这两种蛋白质是Wnt介导的细胞骨架变化的关键因素， 利用非洲爪蟾、斑马鱼和哺乳动物细胞培养物的脊椎动物原肠胚形成和神经管闭合 基于系统。我们假设这两种蛋白质是非经典Wnt介导的细胞凋亡的重要介质。 脊椎动物原肠胚形成和神经管闭合过程中细胞运动的细胞骨架调节。我们 研究将测试这一假设，并提供深入了解Wnt信号网络。

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.