Understanding The Role of Custos in Canonical Wnt Signaling

 描述(申请人提供)：对于生物学家来说，理解胚胎发育过程中模式形成的分子机制仍然是一个挑战。已被证明在这一过程中发挥关键作用的一个关键信号分子家族是Wnt家族。WNT蛋白是一种保守的分泌型糖蛋白，在脊椎动物发育过程中控制着主要的发育过程，包括决定细胞命运、细胞增殖、细胞运动以及原始轴和头部的形成[1-5]。除了调节胚胎发育和模式形成，Wnt信号的缺陷也与肿瘤的发生有关[2，6-8]。因此，阐明Wnt信号的分子机制可以阐明胚胎发生过程中的模式形成机制，有助于我们理解肿瘤的发生。在我们研究Wnt信号在模式形成中的作用的研究中，我们以Disheveled(DVL)为诱饵，通过酵母双杂交筛选分离了一个新的效应分子。这种名为Custos的蛋白质在非洲爪哇和斑马鱼胚胎中都是典型的Wnt信号、ç-catenin核转位和前向发育所必需的。没有Custos的胚胎在皮肤和眼睛中进一步缺乏色素沉着，这表明Custos在神经脊的发育中发挥了作用。有趣的是，在对Custos结合伙伴的酵母双杂交中试筛选中，我们分离出了核膜相关蛋白Nessprin1作为主要的Custos相互作用蛋白。在建立Custos如何发挥作用的模型时，我们提出了Custos和essprin1功能，以调节ç-catenin的细胞质到核的运输。了解Custos和Nesprint 1的功能可以为了解胚胎发育过程中规范的Wnt信号的分子机制和ç-catenin的核运输提供关键的见解，这是一个仍未解决的关键研究领域。根据我们的研究，我们推测，在脊椎动物胚胎发育过程中，Custos和nesipr1是规范的Wnt信号所必需的重要因子。这项提案的主要目的是阐明Custos和essprin1如何规范这一过程。我们将在上面描述的工作模型的基础上测试我们的假设，并描述Custos和ensprin1在Wnt信号转导中的角色和功能。我们的研究将利用非洲爪哇、斑马鱼和哺乳动物细胞培养系统的独特优势。我们在此应用程序中提出了两个主要目标。第一部分将通过单分子分辨率成像研究，描述Custos在斑马鱼和非洲爪哇动物模型中在脊椎动物胚胎发生中的作用，以及在调节ç-catenin核移位中的作用。第二部分将描述在斑马鱼和非洲爪哇动物模型中，Nesprint 1作为Custos功能调节器的作用及其在脊椎动物发育过程中的功能。综上所述，我们建议的研究将剖析？连环蛋白的细胞质到核的易位，这一研究领域到目前为止仍未得到很好的解决。此外，我们的研究将有助于更好地理解Wnt信号如何调控胚胎发育过程，以及取消调控Wnt信号如何导致包括癌症和出生缺陷疾病在内的众多毁灭性的人类病理。

 DESCRIPTION (provided by applicant): Understanding the molecular mechanisms of pattern formation during embryogenesis remains a challenge for biologists. One key family of signaling molecules that have been shown to play crucial roles in this process is the Wnt family. Wnt proteins are conserved secreted glycoproteins that govern major developmental processes including cell fate determination, cell proliferation, cell motility and establishment of the primay axis and head formation during vertebrate development [1-5]. In addition to regulating embryonic development and pattern formation, defects in Wnt signaling have also been implicated in tumorigenesis [2, 6-8]. Delineation of the molecular mechanisms of Wnt signaling can therefore illuminate the mechanisms of pattern formation during embryogenesis and contribute to our understanding of tumorigenesis. In our studies investigating the role of Wnt signaling in pattern formation, we have isolated a new effector molecule via a yeast two-hybrid screen, using Dishevelled (Dvl) as a bait. This protein termed Custos is required for canonical Wnt signaling, ß-catenin nuclear translocation and anterior development in both Xenopus and zebrafish embryos. Embryos depleted of Custos further lack pigmentation in the skin and eye suggesting a role for Custos in neural crest development. Interestingly, in a pilot yeast two-hybrid screen for binding partners for Custos, we isolated the nuclear membrane-associated protein nesprin1 as the major Custos-interacting protein. In building a model for how Custos functions, we propose Custos along with nesprin1 functions to regulate the cytoplasmic to nuclear trafficking of ß-catenin. Understanding the function of Custos and nesprin1 can provide key insights into the molecular mechanisms of canonical Wnt signaling during embryogenesis and the nuclear trafficking of ß-catenin, a key area of study that remains poorly resolved. Based on our studies, we hypothesize Custos and nesprin1 are an important factors required for canonical Wnt signaling during vertebrate embryogenesis. The primary objective of this proposal is to elucidate how Custos and nesprin1 regulates this process. We will build on our working model described above to test our hypothesis and delineate the role and function of Custos and nesprin1 in Wnt signal transduction. Our studies will utilize the distinct advantages of Xenopus laevis, zebrafish and mammalian cell culture-based systems. We have proposed two central aims in this application. The first will characterize the role of Custos in vertebrate embryogenesis in the zebrafish and Xenopus models and in role in mediating ß-catenin nuclear translocation using single molecule resolution imaging studies. The second will characterize the role of nesprin1 as a regulator of Custos function and its function during vertebrate development in both zebrafish and Xenopus models. Taken together our proposed studies will dissect the cytoplasmic to nuclear translocation of ß-catenin, an area of study that has remained exceptionally poorly resolved to date. Additionally, our studies will lead to a better understandin of how Wnt signaling regulates the process of embryogenesis as well as how deregulated Wnt signaling results in numerous and devastating human pathologies including cancers and birth defect disorders.