Non-canoical Wnt signaling and cell motility

描述(由申请人提供)：细胞为定向迁移而极化的分子机制仍然知之甚少。最近研究表明，非典型的Wnt途径在脊椎动物胚胎发育过程中原肠形成和神经折叠关闭所需的细胞极化和迁移中发挥重要作用。重要的是，Wnt信号的缺陷与人类的神经折叠闭合障碍有关，包括脊柱裂和癌症转移。到目前为止，Wnt信号通路如何介导肌动蛋白细胞骨架的变化仍然知之甚少。我们以前的研究已经证实，乱糟糟的相关形态发生蛋白激活因子(Daam1)在DVL和小的GTP酶Rho之间提供了原肠形成的关键联系。重要的是，我们的研究表明，DAAML是Wnt依赖的细胞骨架变化所必需的，尽管潜在的生化细节尚不清楚。为了进一步确定Daaml下游细胞骨架变化所需的因素，我们进行了酵母双杂交筛选，并分离了Profilin等蛋白。Profilin可以诱导肌动蛋白聚合，从而在Daam1和肌动蛋白细胞骨架之间提供直接联系。免疫共沉淀和GST-Pull-Down分析证实Profilin是一个真正的Daaml相互作用因子，并且在Wnt刺激时与Daaml共定位在细胞膜上。此外，Profilin的过度表达或耗尽，类似于Daaml，阻止了非洲爪哇的原肠细胞运动，并导致脊柱裂特有的开放神经管表型。在建立通过Daaml的非规范Wnt信号如何调节细胞骨架变化的模型中，我们假设Daaml和Profilin是肌动蛋白细胞骨架的关键调节器。在这项提案中，我们将研究Daaml如何利用哺乳动物细胞和非洲爪哇胚胎在两个特定目标上的个体优势来调节细胞运动的细胞骨架变化。首先，我们将研究DVL是如何激活Daaml的，并确定其结构域在Rho激活、细胞骨架变化和原肠形成中的作用。其次，我们将研究Profilin如何在非规范的Wnt信号中作为Daaml的细胞骨架重组的效应器发挥作用。这些研究将采用功能获得和功能丧失的方法，利用生化、细胞生物学和胚胎学方法。这些研究将促进我们对非规范的Wnt信号如何调节原肠形成、细胞运动和神经折叠关闭所需的细胞极性和细胞运动性的了解。

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.