Using the C. elegans Oocyte to Model the Cell Biology of Early Onset Dystonia

摘要 早发性肌张力障碍或DYT1肌张力障碍是一种衰弱的神经运动障碍，首次出现在 儿童平均年龄约12岁。DYT1肌张力障碍是由DYT1/Tor1a基因突变引起的 编码进化上保守的TorsinA蛋白，导致单个谷氨酸残基的缺失 (ΔE302/303或ΔE)。ΔE突变导致肌张力障碍的机制尚不清楚，因为 TorsinA的基本细胞功能尚不清楚。耐人寻味的新研究表明，TorsinA可能在 大核糖核蛋白颗粒(RNPs)通过核外排出的新细胞机制 穿透核膜的萌芽。当TorsinA的这一功能受到干扰时，信使RNA似乎 蛋白质合成的突触部位的交通效率低下，损害了神经肌肉功能。田野 需要知道这个新模型是否正确，如果是的话，通过以下方法确定具体的分子机制 哪个TorsinA促进核膜出芽以供RNP输出。 这项申请试图使用线虫的卵母细胞来解决这些目标 雅致作为一种易于驯服的实验模型。最具特征的线虫torsinA同源物的突变 (简称OOC-5，即卵母细胞形成异常五)会导致卵母细胞生长缺陷。卵母细胞生长缺陷是 在OOC-5突变体中观察到的最早的发育异常，因此可能反映了 在缺乏TorsinA/OOC-5功能的细胞中观察到主要潜在的生化和细胞生物学缺陷。 我们的假设是，OOC-5突变通过部分干扰核输出来扰乱卵子发生 组装，或我们已经定义的促进卵母细胞生长的RNPs的功能。考虑到它广泛的进化 保守的OOC-5/TorsinA可能在线虫卵母细胞和线虫卵母细胞中执行相同的基本蛋白质功能 哺乳动物的神经元。有趣的是，先前的研究表明，翻译调控机制 在线虫卵母细胞中发现的对哺乳动物神经元的发育和功能也很重要。 这一提议利用了线虫种系系统提供的许多实验优势， 包括进行高分辨率活细胞成像的能力，以及分子遗传学和 生化操作。此外，我们团队花了20年的时间开创了控制机制的先河 线虫卵母细胞的发育。最近，我们定义了RNPs的蛋白质和mRNA成分 调节线虫卵母细胞的生长、减数分裂发育和发育潜力。我们的研究 因此，团队非常适合对TorsinA的RNP萌芽角色的一般性进行批判性测试。来评估这一点 TorsinA的新角色，我们将：(1)分析OOC-5控制卵母细胞生长的细胞机制 以及(2)定义能够恢复OOC-5/torsinA突变体功能的遗传网络。

ABSTRACT Early onset dystonia or DYT1 dystonia is a debilitating neurological movement disorder that first presents in children at a mean age of about 12. DYT1 dystonia is caused by a mutation in the DYT1/Tor1a gene that encodes the evolutionarily conserved torsinA protein resulting in the deletion of a single glutamic acid residue (ΔE302/303 or ΔE). The mechanism through which the ΔE mutation causes DYT1 dystonia is unclear because the basic cellular function of torsinA is unknown. Intriguing new work suggests that torsinA might function in a new cellular mechanism by which large ribonucleoprotein particles (RNPs) are exported from the nucleus via budding through the nuclear envelope. When this function of torsinA is perturbed, messenger RNAs appear to inefficiently traffic to their synaptic sites of protein synthesis, compromising neuromuscular function. The field needs to know whether this new model is correct, and if so, to identify the specific molecular mechanisms by which torsinA promotes nuclear envelope budding for RNP export. This application seeks to address these goals using the oocytes of the nematode Caenorhabditis elegans as a tractable experimental model. Mutations in the best characterized C. elegans torsinA homolog (called ooc-5 for oocyte formation abnormal five) cause defects in oocyte growth. The oocyte growth defect is the earliest developmental abnormality observed in ooc-5 mutants and therefore is likely reflective of the primary underlying biochemical and cell biological deficits observed in cells lacking torsinA/OOC-5 function. Our hypothesis is that ooc-5 mutations disrupt oogenesis by interfering in part with the nuclear export assembly, or function of oocyte growth-promoting RNPs that we have defined. Given its extensive evolutionary conservation, OOC-5/torsinA is likely to perform the same elemental protein function in C. elegans oocytes and mammalian neurons. Interestingly, prior work has shown that the mechanisms of translational regulation discovered in C. elegans oocytes are also important for the development and function mammalian neurons. This proposal capitalizes on the many experimental advantages afforded by the C. elegans germline system, including the ability to conduct high-resolution live-cell imaging and the ease of molecular genetic and biochemical manipulations. Further, our group has spent two decades pioneering the mechanisms controlling oocyte development in C. elegans. Recently, we defined proteins and mRNA components of RNPs that regulate the growth, meiotic development, and developmental potential of C. elegans oocytes. Our research team is thus ideally positioned to critically test the generality of an RNP budding role for torsinA. To assess this new role for torsinA, we will: (1) Analyze the cellular mechanisms by which OOC-5 controls the oocyte growth process; and (2) Define genetic networks that can restore function to ooc-5/torsinA mutants.