Understanding The Role of Custos in Canonical Wnt Signaling

 描述（由申请人提供）：了解胚胎发生过程中模式形成的分子机制仍然是生物学家面临的挑战。已被证明在这一过程中发挥关键作用的信号分子的一个关键家族是Wnt家族。Wnt蛋白是保守的分泌性糖蛋白，其在脊椎动物发育期间控制主要发育过程，包括细胞命运决定、细胞增殖、细胞运动和主轴的建立和头部形成[1-5]。除了调节胚胎发育和模式形成之外，Wnt信号传导的缺陷也与肿瘤发生有关[2，6-8]。因此，Wnt信号转导的分子机制的描绘可以阐明在胚胎发生过程中的模式形成的机制，并有助于我们理解肿瘤的发生。在我们研究Wnt信号传导在模式形成中的作用的研究中，我们使用Dishevelled（Dvl）作为诱饵，通过酵母双杂交筛选分离了一种新的效应分子。这种被称为Custos的蛋白质是非洲爪蟾和斑马鱼胚胎中典型Wnt信号传导、β-连环蛋白核转位和前部发育所必需的。缺乏Custos的胚胎在皮肤和眼睛中进一步缺乏色素沉着，表明Custos在神经嵴发育中的作用。有趣的是，在一个试点酵母双杂交筛选结合伴侣的Custos，我们分离出核膜相关蛋白nesprin 1作为主要的Custos相互作用蛋白。在建立Custos功能模型时，我们提出Custos沿着nesprin 1功能调节β-连环蛋白的细胞质到细胞核的运输。了解Custos和nesprin 1的功能可以为胚胎发生过程中经典Wnt信号传导的分子机制和β-连环蛋白的核运输提供关键的见解，这是一个关键的研究领域，仍然没有得到很好的解决。基于我们的研究，我们假设Custos和nesprin 1是脊椎动物胚胎发生过程中经典Wnt信号传导所需的重要因子。本提案的主要目的是阐明Custos和Nesprin 1如何管理这一过程。我们将建立在我们的工作模型上面描述来测试我们的假设，并描绘Custos和nesprin 1在Wnt信号转导中的作用和功能。我们的研究将利用非洲爪蟾，斑马鱼和哺乳动物细胞培养系统的独特优势。我们在本申请中提出了两个中心目标。第一个将表征Custos在斑马鱼和非洲爪蟾模型中的脊椎动物胚胎发生中的作用，以及使用单分子分辨率成像研究介导β-连环蛋白核转位中的作用。第二个将表征的作用nesprin 1作为监管机构的Custos功能和其功能在脊椎动物发育过程中的斑马鱼和非洲爪蟾模型。总之，我们提出的研究将剖析β-连环蛋白的细胞质到细胞核的易位，这是一个迄今为止解决得非常差的研究领域。此外，我们的研究将导致更好地理解Wnt信号传导如何调节胚胎发育过程，以及Wnt信号传导如何失调导致许多毁灭性的人类病理学，包括癌症和出生缺陷疾病。

 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.