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.

细胞极化以进行定向迁移的过程还不是很清楚。WNT 在胚胎发育过程中，信号通路是细胞极化和运动所必需的。这条路被分成两条 分为两个分支，称为正则路径和非正则路径以及非正则路径 脊椎动物的原肠形成和神经皱折闭合。重要的是，非规范WNT信号的缺陷是 与包括脊柱裂在内的出生缺陷疾病有关，也与人类癌症转移有关。尽管 令人信服的证据表明，Wnt通路可以调节细胞骨架的变化，以促进细胞的运动，但目前尚不清楚 这是如何实现的，以及这一过程中涉及的分子因素和机制。 在我们的初步研究中，我们已经确定福尔曼同源蛋白与乱糟糟的 形态发生激活剂(Daam1)将杂乱(DVL)连接到GTP酶Rho转导非正则基因 WNT信令。作为对Wnt刺激的响应，DVL与Daam1结合并介导Rho激活。这些步骤 在原肠形成和神经折叠闭合过程中导致细胞运动所需的细胞骨架重组 非洲爪哇的胚胎。然而，目前还不完全了解Daam1是如何修改细胞骨架和身份的 细胞骨架重组所需的DAAM1下游的效应子(S)的位置尚不清楚。我们有 分离出两个蛋白作为Daam1的新的未鉴定结合伙伴。使用免疫共沉淀(IP) 和GST-Pull分析，我们已经验证了这些蛋白与Daam1结合并定义了内部结构域 结合所需的每一种蛋白质。我们观察到在培养细胞中与Daam1共定位，以及这种定位 对Wnt刺激敏感。在非洲爪哇中，功能获得和功能丧失进一步显示了这两种蛋白质 通过DAAM1调节非典范Wnt途径的原肠形成。 在建立这两种蛋白质作用机制的模型时，我们假设Wnt刺激 调节DAAM/蛋白质复合体的形成，从而激活这些蛋白质，进而调节细胞骨架 细胞极性和细胞运动所需的变化。我们将在这份R03提案中追求两个具体目标 为了验证我们的假设，即这两种蛋白质是Wnt介导的细胞骨架变化的关键因素 利用非洲爪哇、斑马鱼和哺乳动物细胞培养的脊椎动物原肠和神经管闭合 基于系统的。我们推测这两种蛋白是非规范Wnt介导的重要介质。 脊椎动物原肠发育和神经管闭合过程中细胞运动的细胞骨架调节。我们的 研究将检验这一假设，并提供对WNT信令网络的见解。