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（由适用提供）：控制细胞极化的分子机制的完整模型以及在过度和神经管闭合期间的迁移事件仍然不完整。虽然众所周知，非典型的Wnt信号通路在这些事件发生的细胞骨架重排和细胞反应中起着关键作用，但该途径的重要元素仍然缺失。主要关注的是，异常的Wnt信号传导与诸如脊柱裂片之类的先天缺陷有关，并在癌症转移中隐含。因此，对该途径涉及的分子进行了更彻底的研究仍然是一个基本目标。我们过去的工作表明，在混血过程中，Wnt介导的细胞骨架变化的一个必不可少的成分是形态发生蛋白（DAAM1）的DISEVELLED相关激活剂。 DAAM1桥接了Diseveled（DVL）和小的GTPase Rho之间的差距，并且是Wnt依赖性RHO激活所必需的，但是此过程的生化细节仍然不确定。为了确定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两杂交筛选。在此屏幕上，我们确定了rhogap蛋白的SRGAP2。 Rhogap蛋白会催化GTP结合的Rho向GDP结合的Rho的过渡，使其失活。该SRGAP2可能在调节DAAM1介导的RHO激活中起关键作用。通过共免疫沉淀和GST-Pulldown分析确定SRGAP2作为真正的DAAM1-结合伙伴的确认。两种蛋白还显示在细胞质膜下方以及肌动蛋白应激纤维的下细胞共定位。另外，以类似于DAAM1的方式，在Xenopus laevis胚胎中，SRGAP2的过表达或部署会产生过失缺陷，而神经管披露则是脊柱裂的典型的。持续描述非典型的Wnt信号通路，需要更好地了解DAAM1介导的RHO激活中涉及的机制。我们假设DAAM1和SRGAP2是Wnt依赖性RHO激活的关键调节剂，并且是脊椎动物胃和神经元管闭合所需的细胞骨架重排。该赠款应用旨在研究SRGAP2在非典型Wnt信号传导中的作用，以及它如何介导脊椎动物过度的细胞骨架重排和细胞响应的功能，并进行了脊椎动物过性和神经元管闭合所需的细胞反应。我们将使用三个具体目标对我们进行调查。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xenopus laevis胚胎来探索SRGAP2在胃管和神经管闭合过程中的作用。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该模式的变化如何影响肌动蛋白细胞骨架。这些实验将共同了解SRGAP2如何调节Wnt诱导的RHO活性水平以及细胞骨架的变化以及该调节如何影响过失和神经管闭合的细胞运动事件。重要的是，他们将提供更多有关此过程中缺陷的见解。