The role of SRGAP2 in vertebrate gastrulation and neural tube closure

 描述(由申请人提供)：在原肠形成和神经管关闭过程中，控制细胞极化和迁移事件的分子机制的完整模型仍然不完整。虽然已知非典型的Wnt信号通路在这些事件发生所需的细胞骨架重排和细胞反应中起着关键作用，但该通路的关键元件仍然缺失。最令人担忧的是，Wnt信号的异常与脊柱裂等出生缺陷有关，并与癌症转移有关。因此，对参与这一途径的分子进行更彻底的研究仍然是一个重要的目标。我们过去的工作表明，在原肠胚形成过程中，Wnt介导的细胞骨架变化的一个重要组成部分是乱发相关的形态发生蛋白激活因子(Daam1)。Daam1架起了杂乱(DVL)和小GTP酶Rho之间的桥梁，是Wnt依赖的Rho激活所必需的，但这一过程的生化细节仍未确定。为了确定可能参与这一过程的其他因素，我们使用Daam1的C-末端区域进行了酵母双杂交筛选。在这个筛选过程中，我们鉴定了SRGAP2，一种RhoGAP蛋白。RhoGAP蛋白催化GTP结合的Rho向GDP结合的Rho的转变，使其失活。因此，SRGAP2可能在调控Daam1介导的Rho激活中起关键作用。通过免疫共沉淀和GST-Pull-Down实验确定SRGAP2为真正的DAAM1结合伙伴。这两个蛋白还显示出亚细胞共定位，就在细胞膜下方，也沿着肌动蛋白应激纤维。此外，在非洲爪哇胚胎中，与Daam1类似的方式，SRGAP2的过度表达或缺失会导致原肠发育缺陷和神经管外露，这是脊柱裂的典型特征。为了继续描述非规范的Wnt信号通路，需要更好地了解Daam1介导的Rho激活的机制。我们推测，Daam1和SRGAP2是Wnt依赖的Rho激活和脊椎动物原肠形成和神经管关闭所必需的细胞骨架重排的关键调节器。这项资助申请旨在研究SRGAP2在非典范Wnt信号中的作用，以及它如何调节脊椎动物原肠发育和神经管关闭所需的细胞骨架重排和细胞反应。我们将以三个具体目标来处理我们的调查。首先，我们将研究SRGAP2和Daam1相互作用所需的功能结构域，并检查这种相互作用是否依赖于Wnt。其次，我们将利用非洲爪哇胚胎来探索SRGAP2在原肠发育和神经管关闭过程中的作用。在第三个目标中，我们将研究SRGAP2和DAAM1的亚细胞定位模式，这种模式是如何被WNT处理改变的，以及这种模式的变化可能如何影响肌动蛋白细胞骨架。这些实验将使我们更深入地了解SRGAP2如何调节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形成和神经管关闭的细胞运动事件。重要的是，他们将提供更多的见解，了解这一过程中的缺陷是如何在脊柱裂等出生缺陷疾病以及癌症转移中表现出来的。

 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.