Non-canonical Wnt signaling and cell motility

 描述(申请人提供)：细胞极化以进行定向迁移的分子机制(S)仍然知之甚少。最近发现，非典型的Wnt途径在细胞极化和迁移中发挥重要作用，而细胞极化和迁移是脊椎动物胚胎发育过程中原肠形成和神经折叠关闭所必需的。重要的是，非规范的Wnt信号缺陷与人类的先天缺陷疾病有关，包括神经折叠闭合障碍，如脊柱裂和癌症转移。到目前为止，非规范的Wnt信号通路如何调节肌动蛋白细胞骨架的变化仍然知之甚少。我们从上一个资助期开始的研究已经证实，Forin蛋白乱发相关的形态发生激活剂(Daam1)在DVL和小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，DAAM1是依赖Wnt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DAAM1下游细胞骨架变化所需的因子，我们进行了酵母双杂交筛选，并分离出了两个新的蛋白质以及我们已发表的其他蛋白质。免疫共沉淀和GST-Pull-Down实验证实这些蛋白是真正的Daam1相互作用因子，它们与Daam1在哺乳动物细胞中的相互作用和亚细胞共定位是受Wnt调控的。此外，这些蛋白的过度表达或耗尽，类似于Daam1，阻碍了非洲爪哇胚胎的原肠形成，并导致了脊柱裂特有的开放神经管表型。我们进一步克隆了第二个脊椎动物Daam家族成员Daam2，并开始对其进行鉴定。我们的初步研究表明，与Daam1类似，Daam2在非典范的Wnt信号转导中发挥功能，但与Daam1相反，Daam2调节脊椎动物神经管的关闭。在建立通过DAAM蛋白的非规范Wnt信号如何调节细胞骨架变化的模型中，我们假设Daam1和Daam2是原肠形成过程中肌动蛋白细胞骨架的关键调节器。在这个竞争性更新方案中，我们将研究两个新发现的与Daam1结合的蛋白质如何利用哺乳动物细胞、非洲爪哇和斑马鱼胚胎在原肠形成过程中调节细胞运动的细胞骨架变化。其次，我们将确定Daam2在脊椎动物原肠发育过程中作为非典范Wnt信号调节器的作用。我们将进一步描述Daam2和Daam1是否在原肠发育过程中具有非冗余功能，以及定义Daam1和/或Daam2共有和特有的效应蛋白。这些研究将极大地提高我们对非规范的Wnt信号如何通过DAAM福尔曼蛋白家族调节细胞极性和细胞运动所需的脊椎动物原肠形成和神经皱折关闭的理解。这些研究将进一步提供 对Wnt信号缺陷如何导致出生缺陷(如脊柱裂和癌症转移)的其他见解。

 DESCRIPTION (provided by applicant): The molecular mechanism(s) 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 non-canonical Wnt signaling are implicated human birth defects disorders including in neural fold closure disorders such as spina bifida and in cancer metastasis. To date, how the non-canonical Wnt signaling pathway regulates changes to the actin cytoskeleton remains at best poorly defined. Our studies from the prior funding period have established that the Formin protein Dishevelled- associated activator of morphogenesis (Daam1) provides a crucial link between Dishevelled (Dvl) and the small GTPase Rho for gastrulation. Importantly, our studies demonstrate Daam1 is required for Wnt-dependent cytoskeletal changes, although the underlying biochemical details are not known. To further identify factors required downstream of Daam1 for cytoskeletal changes, we performed a yeast two-hybrid screen and isolated two new proteins in addition to others we have published. Co-immunoprecipitation and GST-pulldown assays confirm that these proteins are bona-fide Daam1- interacting factors and their interaction and subcellular co-localization with Daam1 in mammalian cells is Wnt-regulated. Furthermore, over-expression or depletion of these proteins, similar to Daam1, blocks gastrulation in the Xenopus embryo and results in an open neural tube phenotype characteristic of spina bifida. We have further cloned and begun characterization of the second vertebrate Daam family member Daam2. Our preliminary studies show that similar to Daam1, Daam2 plays a functional role in non-canonical Wnt signaling but Daam2 in contrast to Daam1 regulates vertebrate neural tube closure. In building a model for how non-canonical Wnt signaling through the Daam proteins regulate cytoskeletal changes, we hypothesize Daam1 and Daam2 are key modulators of the actin cytoskeleton for cellular motility during gastrulation. In this competitive renewal proposal, we will investigate how the two new identified proteins that bind to Daam1 functions to mediate cytoskeletal changes for cell motility during gastrulation using mammalian cells, Xenopus and zebrafish embryos. Second, we will characterize the role of Daam2 as a regulator of non-canonical Wnt signaling during vertebrate gastrulation. We will further delineate whether Daam2 and Daam1 have non-redundant functions during gastrulation and well as defining effector proteins common and specific to Daam1 and/or Daam2. These studies together will significantly advance our understanding of how non-canonical Wnt signaling through the Daam family of Formin proteins regulate cell polarity and cell motility required during vertebrate gastrulation and neural fold closure. These studies will further provide additional insights into how defects in Wnt signaling contribute to birth defects such as spina bifida and cancer metastasis.