New Mechanisms of Heterotaxy and Congenital Heart Disease: Nucleoporins at Cilia

项目摘要 先天性心脏病（CHD）是美国婴儿死亡率和发病率的主要原因之一。 然而，我们对这种疾病的遗传原因知之甚少。多项研究已经发现 核孔蛋白是构建核孔复合物的蛋白质组分，作为异位症的候选基因， 左-右模式紊乱可能导致严重的CHD。在我们以前的工作中，我们提出了一个 通过建立Nup 188和它的结构， 结合伴侣Nup 93对于青蛙模型中的心脏发育、左右图案化和纤毛是必需的。 重要的是，我们发现Nup 188和Nup 93都定位于它们形成的纤毛基部 组装体在结构上不同于核孔复合物。尽管取得了这些进展，但我们还没有 我们有一个明确的目录，核孔蛋白，本地化纤毛基地，我们也不知道确切如何 他们的功能。为了应对这些挑战，我们打算充分利用 包括临床科学家、发育和细胞生物学家， 显微镜专家因此，我们将采取多学科的方法：1）询问新兴的功能 在我们快速有效的非洲爪蟾CHD模型中与CHD相关的核孔蛋白变体; 2）使用邻近检测 细胞培养中的BioID等方法，以充分鉴定定位于纤毛基底及其纤毛的核孔蛋白， 特异性结合伴侣;和3）将这些数据与复杂的脉冲追踪分析相结合， 下一代多色3D超分辨率成像，以充分定义核孔蛋白的机制 补充和它们在纤毛基部的纳米级分布。最后，我们将直接探索核孔蛋白 在细胞培养和非洲爪蟾模型中构建初级和多纤毛的功能。我们的成果承诺 提供了一个框架，以确定新出现的核孔蛋白（和纤毛基地）基因变异如何成为冠心病的基础， 也告知在细胞和组织水平上起作用的基本机制。

Project Summary Congenital heart disease (CHD) is one of the major causes of infant mortality and morbidity in the US. However, we know little about the genetic causes of this disease. Multiple studies have now identified nucleoporins, the protein components that build nuclear pore complexes, as candidate genes for Heterotaxy, a disorder of left-right patterning that can lead to a severe form of CHD. In our previous work, we proposed a likely model for how nucleoporins could contribute to left-right patterning by establishing that Nup188 and its binding partner Nup93 are essential for cardiac development, left-right patterning and cilia in a frog model. Importantly, we discovered that both Nup188 and Nup93 are localized to the bases of cilia where they form assemblies that are structurally distinct from nuclear pore complexes. Despite this progress, we do not yet have a clear catalogue of the nucleoporins that localize to cilia bases, nor do we understand precisely how they function. To meet these challenges, we intend to fully leverage the unique and complementary expertise of our team that includes clinician scientists, developmental and cell biologists, including super-resolution microscopists. We will thus take a multidisciplinary approach to: 1) Interrogate the function of emerging nucleoporin variants linked to CHD in our rapid and efficient Xenopus CHD model; 2) Use proximity-detection methods like BioID in cell culture to fully identify the nucleoporins that localize to cilium bases and their cilium- specific binding partners; and 3) Use these data in concert with sophisticated pulse-chase analyses coupled to next generation multi-color 3D super resolution imaging to fully define the mechanisms of nucleoporin recruitment and their nanoscale distribution at the cilium base. Lastly, we will directly explore nucleoporin function in building both primary and multi-cilia in cell culture and Xenopus models. Our results promise to provide a framework to identify how emerging nucleoporin (and cilium base) gene variants underlie CHD while also informing fundamental mechanisms that function at the cell and tissue-level.