The Role of Torsins at the Nuclear Envelope Using a Novel C. elegans Model

使用新型线虫模型研究 Torsins 在核膜中的作用

基本信息

批准号：
8733065
负责人：
Michael James White VanGompel
金额：
$ 5.51万
依托单位：
UNIVERSITY OF CALIFORNIA AT DAVIS
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-09-30 至 2015-09-29
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8733065
关键词：
ATP phosphohydrolase Address Animal Model Biological Biological Assay Biological Models Biology Caenorhabditis elegans Cell Nucleus Cell Polarity Cell physiology Cellular biology Cytoplasm DYT1 gene Data Defect Development Developmental Cell Biology Developmental Process Dextrans Discipline Disease Dyes Dystonia Early Onset Dystonia Embryo Embryonic Development Endoplasmic Reticulum Event Exclusion Family Family member Functional disorder Future Genes Goals Homologous Gene Human Image Injection of therapeutic agent Label Laboratories Lead Life Link Lipodystrophy Mammalian Cell Mediating Meiosis Membrane Methods Modeling Movement Muscle Contraction Muscular Dystrophies Mutation Nematoda Neuromuscular Diseases Neurons Neuropathy Nuclear Nuclear Envelope Nuclear Outer Membrane Nuclear Pore Nuclear Pore Complex Nuclear Pore Complex Proteins Oocytes Oogenesis Pathway interactions Patients Phenotype Positioning Attribute Premature aging syndrome Protein Binding Proteins RNA Interference Regulation Reporter Research Personnel Role Rosa Staging Tertiary Protein Structure Testing TorsinA Training career dextran disease phenotype env Gene Products insight mutant novel nucleocytoplasmic transport protein complex public health relevance research study

项目摘要

DESCRIPTION (provided by applicant): Proteins in the nuclear envelope (NE) have been shown to be important regulators of cell function and development, and disruption of these proteins leads to a range of diseases termed laminopathies. The Torsin family of AAA+ ATPases have recently been proposed to regulate NE proteins. Mutation in the human gene that encodes TorsinA, DYT1, leads to the neuromuscular disease early onset dystonia. Mammalian cells with out TorsinA function have NE structural defects, and the outer nuclear membrane KASH domain protein Nesprin-3 can interact with TorsinA. However, how TorsinA functions at the NE, and how this function relates to the disease phenotype is not known. The long term goal of this project is to understand the cell biology of Torsin function at the NE, and how Torsins regulate later development. In this proposal, I will investigate Torsin function at the NE using C. elegans as a model. Our laboratory previously identified ooc-5, a C. elegans homolog of TorsinA. Mutations in ooc-5 lead to smaller oocytes and polarity and spindle positioning defects in early embryos. Interestingly, it has been shown that depletion of nucleoporins, components of nuclear pore complexes, in C. elegans leads to an ooc-5 like phenotype, suggesting a link between Torsins and nuclear pores in the worm. Furthermore, preliminary studies have revealed defects in the localization of multiple nuclear envelope proteins in ooc-5 mutants. Using GFP reporter strains and RNAi-mediated gene depletion, I propose to determine which NE protein defects are primary, and which are secondary downstream defects. I will also determine if nuclear envelope barrier and transport functions are disrupted in ooc-5 mutants by using nuclear-localized GFP markers and dextran dye injections. This will be the first test of the functional consequences of nuclear envelope defects in a Torsin mutant model. Finally, I will test which defects at the NE cause later polarity phenotypes, providing a link between the cell biology of Torsin function and the regulation of development. Results from this study will be applicable to both human Torsin function and mechanisms underlying early onset dystonia, and also to the study of embryonic polarity establishment in C. elegans.

描述（由申请人提供）：核膜（NE）中的蛋白质已被证明是细胞功能和发育的重要调节因子，这些蛋白质的破坏会导致一系列称为核纤层蛋白病的疾病。最近提出 AAA+ ATP 酶的 Torsin 家族可调节 NE 蛋白。编码 TorsinA、DYT1 的人类基因突变会导致神经肌肉疾病早发性肌张力障碍。不具有TorsinA功能的哺乳动物细胞具有NE结构缺陷，外核膜KASH结构域蛋白Nesprin-3可以与TorsinA相互作用。然而，TorsinA 如何在 NE 发挥作用以及该功能与疾病表型有何关系尚不清楚。该项目的长期目标是了解 Torsin 在 NE 功能的细胞生物学，以及 Torsins 如何调节后期发育。在这个提案中，我将研究 Torsin 函数 NE 使用秀丽隐杆线虫作为模型。我们的实验室之前鉴定出 ooc-5，它是 TorsinA 的线虫同源物。 ooc-5 突变导致早期胚胎中卵母细胞变小以及极性和纺锤体定位缺陷。有趣的是，研究表明，线虫中核孔蛋白（核孔复合物的组成部分）的消耗会导致类似 ooc-5 的表型，这表明 Torsins 和线虫核孔之间存在联系。此外，初步研究揭示了 ooc-5 突变体中多个核膜蛋白的定位缺陷。我建议使用 GFP 报告菌株和 RNAi 介导的基因缺失来确定哪些 NE 蛋白缺陷是主要缺陷，哪些是次要下游缺陷。我还将通过使用核定位 GFP 标记和葡聚糖染料注射来确定 ooc-5 突变体中的核膜屏障和运输功能是否被破坏。这将是对 Torsin 突变模型中核膜缺陷的功能后果的首次测试。最后，我将测试 NE 的哪些缺陷会导致后来的极性表型，从而提供 Torsin 功能的细胞生物学与发育调节之间的联系。这项研究的结果将适用于人类 Torsin 功能和早发性肌张力障碍的机制，也适用于秀丽隐杆线虫胚胎极性建立的研究。