Characterization of two proteins that regulate vertebrate

调节脊椎动物的两种蛋白质的表征

• 批准号: 10531664
• 负责人: Raymond Habas
• 金额: $ 7.74万
• 依托单位: TEMPLE UNIV OF THE COMMONWEALTH
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-01 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10531664
• 关键词: Actins; Binding; Binding Proteins; Biological Assay; Cell Culture Techniques; Cell Polarity; Cells; Co-Immunoprecipitations; Complex; Congenital Abnormality; Cytoskeletal Modeling; Cytoskeleton; Data; Defect; Development; Disease; Dsh protein; Embryo; Embryonic Development; Focal Adhesions; Genetic; Human; Human Pathology; Individual; Link; Malignant Neoplasms; Mammalian Cell; Mediating; Mediator of activation protein; Modeling; Molecular; Morphogenesis; Movement; Nature; Neoplasm Metastasis; Neural Fold; Neural Tube Closure; Pathology; Pathway interactions; Pattern; Process; Proteins; Role; Signal Transduction Pathway; Signaling Molecule; Spinal Dysraphism; Structure; System; Testing; Tubular formation; WNT Signaling Pathway; Xenopus; Xenopus laevis; Zebrafish; base; cell behavior; cell growth regulation; cell motility; convergent extension; gain of function; gastrulation; in vivo; insight; loss of function; migration; overexpression; polymerization; protein complex; response; rho; rho GTP-Binding Proteins; xenopus development

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.

细胞变得两极化以进行定向迁移的过程尚不清楚。 wnt 胚胎发生过程中极化和细胞运动性需要信号通路。这条路分裂 分为两个分支，称为规范和非规范和非规范途径，在 脊椎动物的胃肠道和神经褶皱闭合。重要的是，非典型Wnt信号的缺陷是 与包括脊柱裂有关的先天缺陷疾病以及人类癌转移的疾病。尽管有 令人信服的证据表明，Wnt途径可以介导细胞骨骼变化以使细胞运动性变化，这仍然不清楚 如何与此过程中涉及的分子因子和机制一起完成。 在我们的初步研究中，我们确定了formin同源性蛋白质菜肴相关 形态发生的激活剂（DAAM1）将衣服（DVL）与GTPase Rho联系起 wnt信号传导。为了响应Wnt刺激，DVL结合DAAM1并介导RHO激活。这些步骤 导致胃静脉运动在胃部运动和神经折叠过程中所需的细胞骨架重组 Xenopus胚胎。但是，尚未完全了解DAAM1如何修饰细胞骨架和身份 细胞骨架重组所需的DAAM1下游的效应子仍然未知。我们有 将两种蛋白质分离为DAAM1的新的未表征结合伙伴。使用共免疫沉淀（IP） 和GST-Pulldown分析 结合所需的每种蛋白质。我们观察到在培养细胞中与DAAM1共定位，并且这种定位 对WNT刺激很敏感。在Xenopus中，功能障碍和功能丧失进一步显示两种蛋白质 用DAAM1调节非典型WNT途径中的胃结构。 在建立这两种蛋白质作用机理的模型时，我们假设Wnt刺激 调节DAAM/蛋白质复合物的形成，该形成激活这些蛋白质，进而介导细胞骨架 细胞极性和细胞运动所需的变化。我们将在此R03提案中实现两个具体目标 为了测试我们的假设，即这两种蛋白是Wnt介导的细胞骨架变化的关键因素 脊椎动物胃胃和神经管闭合，利用爪蟾，斑马鱼和哺乳动物细胞培养 基于系统。我们假设这两种蛋白质是非典型Wnt介导的必不可少的介体 脊椎动物胃肠道和神经管闭合期间细胞运动的细胞骨架调节。我们的 研究将检验这一假设，并为WNT信号网络提供见解。