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。但是，我们对这种疾病的遗传原因一无所知。现在已经确定了多个研究核孔蛋白是建立核孔复合物的蛋白质成分，作为杂质的候选基因，A 左右模式的障碍，可能导致严重的CHD形式。在我们以前的工作中，我们提出了一个核孔蛋白如何通过确定NUP188及其ITS的模型可能导致左右模式结合伴侣NUP93对于青蛙模型中的心脏发展，左右模式和纤毛至关重要。重要的是，我们发现NUP188和NUP93都位于Cilia的基础上，它们形成了在结构上与核孔复合物不同的组件。尽管取得了这种进步，我们还没有具有将核孔蛋白定位到纤毛碱的清晰目录，我们也不确切地理解如何它们起作用。为了应对这些挑战，我们打算充分利用独特和互补的专业知识我们的团队包括临床科学家，发育和细胞生物学家，包括超级分辨率显微镜家。因此，我们将采用多学科的方法：1）询问新兴的功能在我们的快速有效的爪蟾模型中，与CHD相关的核孔蛋白变体； 2）使用接近检测细胞培养中的生物类药物等方法充分鉴定核孔蛋白定位于纤毛碱及其纤毛 - 特定的约束伙伴； 3）与复杂的脉冲练习分析一起使用这些数据耦合下一代多色3D超级分辨率成像，以充分定义核孔蛋白的机制招募及其在纤毛基地的纳米级分布。最后，我们将直接探索核孔素在细胞培养和爪蟾模型中构建原发性和多核的功能。我们的结果有望提供一个框架来确定新兴核孔蛋白（和纤毛基碱）基因变体如何下降还告知在细胞和组织水平上起作用的基本机制。