Non-canonical Wnt signaling and cell motility

 描述（由申请人提供）：细胞极化定向迁移的分子机制仍知之甚少。非经典的Wnt信号通路在脊椎动物胚胎发育过程中的原肠胚形成和神经折叠闭合所需的细胞极化和迁移中起着重要作用。重要的是，非经典Wnt信号传导中的缺陷涉及人类出生缺陷病症，包括神经褶皱闭合病症如脊柱裂和癌症转移。迄今为止，非经典Wnt信号通路如何调节肌动蛋白细胞骨架的变化仍然是最好的定义不清。我们在前一个资助期的研究已经确定，形成蛋白Dishevelled相关的形态发生激活因子（Daam 1）在Dishevelled（Dvl）和小GTdR Rho之间提供了一个关键的联系，用于原肠胚形成。重要的是，我们的研究表明Daam 1是Wnt依赖性细胞骨架变化所必需的，尽管其潜在的生化细节尚不清楚。为了进一步确定Daam 1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，并分离出两个新的蛋白质，除了我们已经发表的其他蛋白质。免疫共沉淀和GST-下拉试验证实，这些蛋白质是真正的Daam 1相互作用的因素和它们的相互作用和与Daam 1在哺乳动物细胞中的亚细胞共定位Wnt调节。此外，这些蛋白质的过度表达或耗尽，类似于Daam 1，阻断了非洲爪蟾胚胎中的原肠胚形成，并导致脊柱裂的开放神经管表型特征。我们已经进一步克隆并开始表征第二个脊椎动物Daam家族成员Daam 2。我们的初步研究表明，与Daam 1类似，Daam 2在非经典Wnt信号中起着功能性作用，但Daam 2与Daam 1相反，调节脊椎动物神经管闭合。在建立一个模型的非经典Wnt信号如何通过Daam蛋白调节细胞骨架的变化，我们假设Daam 1和Daam 2是关键的调节剂肌动蛋白细胞骨架的原肠胚形成过程中的细胞运动。在这个竞争性的更新建议，我们将研究如何结合到Daam 1功能的两个新确定的蛋白质介导细胞骨架的变化，细胞运动在原肠胚使用哺乳动物细胞，非洲爪蟾和斑马鱼胚胎。其次，我们将描述Daam 2在脊椎动物原肠胚形成过程中作为非经典Wnt信号转导调节剂的作用。我们将进一步描述Daam 2和Daam 1在原肠胚形成过程中是否具有非冗余功能，并定义Daam 1和/或Daam 2共同和特异的效应蛋白。这些研究一起将显着推进我们的理解，如何通过Daam家族的形成蛋白的非经典Wnt信号调节细胞极性和细胞运动需要在脊椎动物原肠胚形成和神经折叠关闭。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）的进一步了解。

 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.