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。多项研究现已确定 核孔素是形成核孔复合体的蛋白质成分，作为异质性的候选基因，a 左-右模式紊乱，可能导致严重的冠心病。在我们之前的工作中，我们提出了一个 核孔素如何通过建立Nup188及其 结合伴侣Nup93对于青蛙模型中的心脏发育、左右模式和纤毛是必不可少的。 重要的是，我们发现Nup188和Nup93都定位在纤毛的底部，在那里它们形成 在结构上与核孔复合体不同的组件。尽管取得了这些进展，但我们还没有 对定位于纤毛碱基的核孔蛋白有一个清晰的目录，我们也不确切地知道它是如何 它们起作用了。为了迎接这些挑战，我们打算充分利用独特和互补的专业知识 包括临床医生科学家、发育和细胞生物学家，包括超分辨率 显微镜专家。因此，我们将采取多学科的方法来：1)审问新兴的功能 在我们快速有效的非洲爪哇CHD模型中，核孔素变异与CHD有关；2)使用接近检测 像细胞培养中的BioID这样的方法来充分鉴定定位于纤毛基底部及其纤毛的核孔蛋白- 特定的结合配对；以及3)将这些数据与复杂的脉冲追逐分析结合使用 新一代多色3D超分辨率成像全面确定核孔化机制 纤毛基底部的募集及其纳米尺度分布。最后，我们将直接探索核孔素 在细胞培养和非洲爪哇模型中建立初生纤毛和复纤毛的作用。我们的结果承诺 提供一个框架来确定新出现的核孔蛋白(和纤毛碱基)基因变异是如何导致CHD的，而 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.