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

异向性与先天性心脏病的新机制：纤毛核孔蛋白

基本信息

批准号：
10443780
负责人：
Mustafa K Khokha
金额：
$ 59.21万
依托单位：
YALE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-04-01 至 2023-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10443780
关键词：
3-Dimensional Alleles Animal Disease Models Binding Binding Proteins Biochemical Biotin Biotinylation Candidate Disease Gene Cardiac Cardiac development Catalogs Cause of Death Cell Culture Techniques Cell Fractionation Cell Nucleus Cells Child Health Chimeric Proteins Cilia Clustered Regularly Interspaced Short Palindromic Repeats Color Congenital Abnormality Copy Number Polymorphism Coupled Cytoplasm Data Defect Development Disease Disease model Embryo Engineering Etiology Funding Genes Genetic Genetic Counseling Genomics Goals Grant Growth Heart Image Impairment Infant Infant Mortality Knowledge Label Lead Left Ligase Light Link Microscopy Modeling Molecular Morbidity - disease rate Mucociliary Clearance Nanoscopy Nephrotic Syndrome Nuclear Pore Nuclear Pore Complex Nuclear Pore Complex Proteins Parents Pathogenesis Pathway interactions Patient-Focused Outcomes Patients Pattern Phenotype Physicians Physiologic pulse Play Proteins Quantitative Microscopy Radial Rana Resolution Role S-nitro-N-acetylpenicillamine Scientist Situs Inversus Streptavidin Structure Talents Technology Testing Tissues Translating Transmission Electron Microscopy Variant Work Xenopus base cardiogenesis cohort congenital heart disorder detection method experimental study genetic variant human genomics improved infant morbidity infant morbidity/mortality insight interdisciplinary approach kinetosome knock-down nanoscale next generation nucleocytoplasmic transport recruit single molecule spatiotemporal

项目摘要

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.

项目概要先天性心脏病（CHD）是美国婴儿死亡和发病的主要原因之一。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蛋白，构建核孔复合物的蛋白质成分，作为异序的候选基因，左右模式紊乱可导致严重的冠心病。在我们之前的工作中，我们提出了一个通过建立 Nup188 及其它的核孔蛋白如何促进左右模式的可能模型结合伴侣 Nup93 对于青蛙模型中的心脏发育、左右模式和纤毛至关重要。重要的是，我们发现 Nup188 和 Nup93 都定位于它们形成的纤毛基部结构上不同于核孔复合体的组装体。尽管取得了这些进展，但我们还没有我们对定位于纤毛碱基的核孔蛋白有一个清晰的目录，但我们也不知道到底是如何定位的他们发挥作用。为了应对这些挑战，我们打算充分利用独特且互补的专业知识我们团队的成员包括临床科学家、发育和细胞生物学家，包括超分辨率显微镜学家。因此，我们将采取多学科方法来： 1）询问新兴技术的功能在我们快速高效的非洲爪蟾 CHD 模型中，核孔蛋白变体与 CHD 相关； 2) 使用接近检测细胞培养中的 BioID 等方法可完全识别定位于纤毛碱基及其纤毛的核孔蛋白特定的结合伙伴； 3) 将这些数据与复杂的脉冲追踪分析结合使用下一代多色 3D 超分辨率成像，全面定义核孔蛋白的机制纤毛基部的招募及其纳米级分布。最后，我们将直接探索核孔蛋白在细胞培养和爪蟾模型中构建初级纤毛和多纤毛的功能。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）基因变异如何导致冠心病还告知在细胞和组织水平发挥作用的基本机制。