The role of SRGAP2 in vertebrate gastrulation and neural tube closure

SRGAP2在脊椎动物原肠胚形成和神经管闭合中的作用

• 批准号: 8970386
• 负责人: Raymond Habas
• 金额: $ 7.8万
• 依托单位: TEMPLE UNIV OF THE COMMONWEALTH
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-01 至 2017-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8970386
• 关键词: Actins; Affect; Applications Grants; Binding; Biochemical; Biological Assay; C-terminal; Cell Culture Techniques; Cell Polarity; Cell membrane; Cells; Co-Immunoprecipitations; Complex; Congenital Abnormality; Cytoskeleton; Data; Defect; Disclosure; Disease; Elements; Embryo; Embryonic Development; Event; Family; Fiber; GTP Binding; GTPase-Activating Proteins; Glycoproteins; Goals; Guanine Nucleotide Exchange Factors; Guanosine Triphosphate; Guanosine Triphosphate Phosphohydrolases; Human; Human Development; Human Pathology; Hydrolysis; Individual; Investigation; Laboratories; Link; Malignant Neoplasms; Mammalian Cell; Mediating; Molecular; Molecular Models; Monomeric GTP-Binding Proteins; Morphogenesis; Neoplasm Metastasis; Neural Fold; Neural Tube Closure; Neural Tube Defects; Pathology; Pathway interactions; Pattern; Play; Process; Proteins; Regulation; Role; Signal Pathway; Signal Transduction; Signal Transduction Pathway; Signaling Molecule; Spinal Dysraphism; Stress Fibers; System; Tertiary Protein Structure; Testing; Tissues; Wnt proteins; Work; Xenopus; Xenopus laevis; Yeasts; base; beta catenin; cell behavior; cell motility; convergent extension; design; gain of function; gastrulation; in vivo; insight; loss of function; member; migration; molecular modeling; novel; overexpression; polymerization; public health relevance; research study; response; rho; xenopus development; yeast two hybrid system

 DESCRIPTION (provided by applicant): A complete model of the molecular mechanisms governing cell polarization and the migration events during gastrulation and neural tube closure remains incomplete. While it is known that the non-canonical Wnt signaling pathway plays a critical role in the cytoskeletal rearrangements and cellular responses required for these events to occur, vital elements of this pathway remain missing. Of principal concern, is that aberrant Wnt signaling has been linked to birth defects such as spina bifida and implicated in cancer metastasis. Therefore, a more thorough investigation of the molecules involved in this pathway remains an essential goal. Our past work has shown that one essential component of Wnt-mediated cytoskeletal changes during gastrulation is Dishevelled-associated activator of morphogenesis protein (Daam1). Daam1 bridges the gap between Dishevelled (Dvl) and the small GTPase Rho and is required for Wnt-dependent Rho activation, but the biochemical details of this process are still undefined. To identify additional factors that might be involved n this process, we performed a yeast two-hybrid screen using a C-terminal region of Daam1. During this screen we identified SRGAP2, a RhoGAP protein. RhoGAP proteins catalyze the transition of GTP-bound Rho to GDP-bound Rho, inactivating it. Thus SRGAP2 may play a key role in modulating Daam1 mediated Rho activation. Confirmation of SRGAP2 as a genuine Daam1- binding partner was determined by co-immunoprecipitation and GST-pulldown assays. The two proteins also show subcellular co-localization just below the cellular plasma membrane and also along the actin stress fibers. Additionally, in a manner similar to Daam1, overexpression or depletion of SRGAP2, in Xenopus laevis embryos, produces gastrulation defects and neural tube disclosures typical of spina bifida. The continued delineation of the non-canonical Wnt signaling pathway, requires a better understanding of the mechanisms involved in Daam1 mediated Rho activation. We hypothesize that Daam1 and SRGAP2 are key modulators of Wnt-dependent Rho activation and the cytoskeletal rearrangements necessary for vertebrate gastrulation and neural tube closure. This grant application is designed to investigate the role of SRGAP2 in non-canonical Wnt signaling and how it functions to mediate the cytoskeletal rearrangements and cellular responses required for vertebrate gastrulation and neural tube closure. We will approach our investigation using three specific aims. First, we will examine the functional domains of SRGAP2 and Daam1 required for their interaction, and examine if this interaction is Wnt-dependent. Second, we will use Xenopus laevis embryos to explore the role of SRGAP2 during gastrulation and neural tube closure. In the third aim, we will investigate the subcellular localization patterns of SRGAP2 and Daam1, how this pattern is altered by Wnt treatment and how changes in this pattern may affect the actin cytoskeletal. These experiments together will provide a more thorough understanding of how SRGAP2 functions to regulate Wnt-induced Rho activity levels and changes to the cytoskeleton and how this regulation affects the cellular motility events of gastrulation and neural tube closure. Importantly, they will provide additional insights into how defects in this process are manifested in birth defects disorders such as spina bifida and also in cancer metastasis.

 描述（由申请人提供）：虽然已知非典型Wnt信号通路在细胞骨架重排中发挥着关键作用，但控制细胞极化和原肠胚形成和神经管闭合期间迁移事件的分子机制的完整模型仍然不完整。以及发生这些事件所需的细胞反应，但该通路的重要元件仍然缺失。主要关注的是，异常的 Wnt 信号传导与脊柱裂等出生缺陷有关，并与癌症有关。因此，对参与该途径的分子进行更彻底的研究仍然是一个重要目标，表明原肠胚形成过程中 Wnt 介导的细胞骨架变化的一个重要组成部分是形态发生蛋白 (Daam1) 的混乱相关激活剂。它弥合了 Disheveled (Dvl) 和小 GTPase Rho 之间的差距，并且是 Wnt 依赖性 Rho 激活所必需的，但该过程的生化细节仍不确定。为了了解这一过程中可能涉及的其他因素，我们使用 Daam1 的 C 末端区域进行了酵母双杂交筛选。在此筛选过程中，我们鉴定了 SRGAP2，一种 RhoGAP 蛋白，可催化 GTP 结合的 Rho 向 GDP 的转变。结合 Rho，使其失活，因此 SRGAP2 可能在调节 Daam1 介导的 Rho 激活中发挥关键作用，证实 SRGAP2 是真正的 Daam1 结合伴侣。通过免疫共沉淀和 GST 下拉测定确定，这两种蛋白还以类似于 Daam1、SRGAP2 过度表达或缺失的方式在细胞质膜下方和肌动蛋白应激纤维上共定位。在非洲爪蟾胚胎中，会产生典型的脊柱裂原肠胚形成缺陷和神经管暴露。继续描述非经典 Wnt 信号通路需要更好的方法。了解 Daam1 介导的 Rho 激活机制。我们研究 Daam1 和 SRGAP2 是 Wnt 依赖性 Rho 激活以及脊椎动物原肠胚形成和神经管闭合所需的细胞骨架重排的关键调节剂。我们了解非经典 Wnt 信号传导及其如何介导脊椎动物原肠胚形成和神经管闭合所需的细胞骨架重排和细胞反应。首先，我们将检查 SRGAP2 和 Daam1 相互作用所需的功能域，并检查这种相互作用是否依赖于 Wnt；其次，我们将使用非洲爪蟾胚胎来探索 SRGAP2 的作用。在第三个目标中，我们将研究 SRGAP2 和 Daam1 的亚细胞定位模式、Wnt 处理如何改变这种模式以及这种模式的变化如何影响肌动蛋白。这些实验将帮助我们更全面地了解 SRGAP2 如何发挥作用来调节 Wnt 诱导的 Rho 活性水平和细胞骨架的变化，以及这种调节如何影响原肠胚形成和神经管闭合的细胞运动事件。深入了解这一过程中的缺陷如何在脊柱裂等出生缺陷疾病以及癌症转移中表现出来。