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DYT 1肌张力障碍是一种使人衰弱的神经运动障碍，首先出现在 平均年龄在12岁左右。DYT 1肌张力障碍是由DYT 1/Tor 1a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DYT 1肌张力障碍的机制尚不清楚， TorsinA的基本细胞功能是未知的。有趣的新工作表明，torsinA可能在一个 一种新的细胞机制，通过这种机制，大的核糖核蛋白颗粒（RNP）从细胞核中输出， 在核膜中发芽当torsinA的这种功能受到干扰时，信使RNA似乎 它们无法有效地运输到蛋白质合成的突触部位，从而损害神经肌肉功能。领域 需要知道这个新模型是否正确，如果是，通过以下方法确定特定的分子机制： 其中torsinA促进核膜出芽用于RNP输出。 本申请试图使用线虫小杆线虫的卵母细胞来解决这些目标 elegans作为一个易处理的实验模型。最具特征的C.扭体线虫A同系物 （称为ooc-5，表示卵母细胞形成异常5）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的主要潜在生物化学和细胞生物学缺陷。 我们的假设是ooc-5突变通过部分干扰核输出而破坏卵子发生 组装，或我们已经定义的卵母细胞生长促进RNP的功能。鉴于其广泛的进化 保守性，OOC-5/torsinA可能在C中执行相同的基本蛋白功能。线虫卵母细胞， 哺乳动物神经元有趣的是，先前的工作表明，翻译调控的机制， 发现在C。线虫卵母细胞对哺乳动物神经元的发育和功能也很重要。 该方案充分利用了C.线虫种系系统， 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，我们的团队花了20年的时间， C.卵母细胞发育优雅最近，我们定义了RNP的蛋白质和mRNA组分， 调节C.线虫卵母细胞我们的研究 因此，该团队处于理想的位置，可以严格测试TorsinA的RNP萌芽作用的一般性。为了评估这一点 torsinA的新作用，我们将：（1）分析OOC-5控制卵母细胞生长的细胞机制 （2）定义可以恢复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.