Non-canoical Wnt signaling and cell motility

细胞极化定向迁移的分子机制仍然不清楚 明白非经典的Wnt通路最近被证明在细胞凋亡中起重要作用。 在脊椎动物中，原肠形成和神经折叠闭合所需的极化和迁移 胚胎发生重要的是，Wnt信号传导的缺陷与神经折叠闭合障碍有关， 人类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是最好的定义不明确。 我们以前的研究已经证实，形态发生蛋白的紊乱相关激活因子， （Daaml）提供了Dishevelled（Dvl）和小GTvl Rho之间用于原肠胚形成的关键联系。 重要的是，我们的研究表明Daaml是Wnt依赖性细胞骨架变化所必需的，尽管Daaml的表达与Wnt依赖性细胞骨架的变化无关。 潜在的生物化学细节尚不清楚。为了进一步确定Daaml下游所需的因素， 细胞骨架的变化，我们进行了酵母双杂交筛选和分离蛋白质Profilin，除了 他人Profilin可以诱导肌动蛋白聚合，从而提供Daaml和肌动蛋白之间的直接连接。 细胞骨架免疫共沉淀和GST-下拉测定证实Profilin是一种真正的Daaml， 在Wnt刺激后，Daaml与Daaml共定位在细胞膜上。此外，委员会认为， 与Daaml类似，Profilin的过度表达或缺失阻断了非洲爪蟾原肠胚形成细胞的运动 并导致脊柱裂的开放神经管表型特征 在构建非经典Wnt信号如何通过Daaml调节细胞骨架变化的模型中， 我们假设Daam 1和Profilin 1是细胞运动性的肌动蛋白细胞骨架的关键调节剂。在这 我们将研究Daaml如何发挥作用，以介导细胞骨架的变化，为细胞运动使用 在两个特定的目标中，非洲爪蟾细胞和非洲爪蟾胚胎都具有各自的优势。一是 研究Daaml如何被Dvl激活，并确定其结构域在Rho激活、细胞骨架 变化和原肠胚形成。其次，我们将研究Profilin在非经典Wnt信号转导中的作用 作为Daaml细胞骨架重组的效应子。这些研究将采用功能获得和功能丧失- 利用生物化学、细胞生物学和胚胎学方法的功能方法。 这些研究将促进我们对非经典Wnt信号如何调节细胞的认识， 原肠形成细胞运动和神经折叠关闭过程中所需的极性和细胞运动。

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 includingspina 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-stimulationco-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 buildinga model for how non-canonical Wnt signaling through Daaml regulates cytoskeletal changes, we hypothesize Daaml and Profilinl 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 mammaliancells 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 Wntsignaling 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 knowledgeof how non-canonical Wnt signalingregulates cell polarity and cell motility required duringgastrulation cell movements and neural fold closure.