Molecular dissection of the ciliary gate

描述(由申请人提供)：纤毛是大多数真核细胞表面的感觉装置，在发育过程中各种器官的正确形成中发挥着重要作用。纤毛组装通过鞭毛内运输(IFT)和感觉转导能力是高度保守的所有纤毛生物。随着人类疾病基因定位克隆的快速发展，许多疾病，如常染色体显性遗传性多囊肾病(ADPKD)、Joubert综合征(JBST)、Bardet-Biedl综合征(BBS)、肾病(NPHP)、Meckel-Gruber综合征(MKS)和常染色体隐性遗传性多囊肾病(ARPKD)在分子上被定性为纤毛相关疾病，现在统称为纤毛疾病。纤毛功能的建立和维持显然对生物体的健康至关重要。与无处不在的纤毛的存在相一致，许多纤毛疾病以综合征的形式出现，影响到多个器官，包括肾脏、肝脏、四肢、眼睛和中枢神经系统。尽管纤毛具有生理和临床相关性，但调控纤毛生物发生和功能的核心机制以及疾病基因功能和病理之间的联系在很大程度上仍然难以捉摸。纤毛生物学中的一个中心问题是纤毛门如何在功能上将纤毛从细胞体中分离出来，并使其成为一个离散的传感细胞器。纤毛只在母中心粒(或母体)上形成。在纤毛发生过程中，母中心粒的远端附属物转化为过渡纤维(TF)，形成一个9叶螺旋状结构，连接基底体和纤毛基底膜。TF独特的亚细胞位置使其成为睫状门的良好候选者。然而，目前还没有关于转录因子的组成和功能的分子信息。在一项旨在确定线虫纤毛发生决定因素的正向诱变筛选中，我们分离和克隆了一个新的基因dyf-19，它是特征不佳的人fbf1基因的唯一同源基因。我们的初步数据显示，蠕虫DYF-19和人FBF1在移行纤维和远端附件上显示了特定的定位模式，表明DYF-19/FBF1具有高度保守的Tf相关功能。进一步的分析表明，DYF-19调节聚集的IFT颗粒在过渡纤维上的纤毛进入，以及几种睫状体感受器的纤毛进入。我们的初步研究揭示了TFS的第一个Bona FDE组分，并证明了TFS在纤毛形成和功能中的重要作用。此外，我们还鉴定了另外两个真正的转铁蛋白成分，TALPID-3和HYLS-1。我们的初步数据表明，DYF-19、TALPID-3和HYLS-1在转录因子的背景下功能上相互作用。最有趣的是，TALPID-3基因敲除鸡被证实是一种睫毛病模型，而人类hyls1基因是纤毛病脑积水综合征的一个致病基因。由于纤毛在哺乳动物早期胚胎发育中的重要作用，在人类和其他哺乳动物模式生物中，纤毛与疾病之间的联系的研究非常困难。因此，替代的实验系统是必要的。线虫使得在活的动物身上探索这些问题成为可能。线虫高度保守的纤毛发生蛋白、纤毛发生途径和纤毛感觉功能使其成为研究纤毛基因在其天然细胞环境中的生理作用的有力模型。我们的数据支持这一建议的中心假设，即DYF-19作为一个功能组件，将TFS定义为控制新生蛋白质进入纤毛的“纤毛门”。这种“门”的破坏会影响纤毛的形成和功能。这些拟议的研究具有在不久的将来揭示纤毛研究的突破的巨大潜力，并将提供关于纤毛的生物发生和感觉功能在其自然环境中如何调节的开创性信息，揭示纤毛疾病的病因。

DESCRIPTION (provided by applicant): Cilia serve as sensory devices on most eukaryotic cells surface and play an essential role in the proper formation of a diversity of organs in development. Ciliary assembly via intraflagellar transport (IFT) and sensory transduction capabilities are highly conserved in all ciliated organisms. With rapid advancements in the positional cloning of human disease genes in the past decade, a wide variety of disorders, such as autosomal dominant polycystic kidney disease (ADPKD), Joubert syndrome (JBST), Bardet-Biedl syndrome (BBS), nephronophthisis (NPHP), Meckel-Gruber syndrome (MKS), and autosomal recessive polycystic kidney disease (ARPKD), have been characterized molecularly as cilia-related diseases, now known collectively as ciliopathies. The establishment and maintenance of ciliary function are clearly essential for the well-being of an organism. Consistent with the ubiquitous presence of cilia, many ciliopathies occur as syndromic disorders that affect multiple organs, including the kidney, liver, limb, eye, and central nervous system. Despite the physiological and clinical relevance of cilia, the core machinery that regulates cilia biogenesis and function as well as the connection between the disease gene function and pathology remain largely elusive. One central question in cilia biology is that how the ciliary gat functionally separates the cilium from the cell body and makes it a discrete sensing organelle. Cilia only form atop mother centrioles (or basal bodies). During ciliogenesis, the distal appendages of the mother centriole transform to transition fibers (TFs), which form a 9-bladed propeller structure connecting the basal body to the ciliary base membrane. The distinct subcellular location of TFs makes it a good candidate for the ciliary gate. Nonetheless, no molecular information is available regarding the composition as well as the function of TFs. In a forward mutagenesis screening that aimed to identify the determinants of ciliogenesis in C. elegans, we isolated and cloned a novel gene dyf-19, which is the sole homolog of poorly characterized human fbf1 gene. Our preliminary data showed that worm DYF-19 and human FBF1 exhibit specific localization pattern on transition fibers and distal appendages, suggesting a highly conserved TF-related function for DYF- 19/FBF1. Further analyses suggested that DYF-19 regulates the ciliary entry of assembled IFT particles on transition fibers as well as the ciliary entry of several ciliary sensory receptors. Our preliminary studies reveal the first bona fde component of TFs and demonstrate the essential roles of the TFs in cilia formation and function. Additionally, we identified two more genuine TF components, TALPID-3 and HYLS-1. Our preliminary data indicate that DYF-19, TALPID-3, and HYLS-1 functionally interact in the context of TFs. Most interestingly, talpid-3 knockout chicken is confirmed to be a ciliopathy model and human hyls1 gene is one causal locus for the ciliopathy Hydrolethalus syndrome. Due to the essential roles of cilia in mammalian early embryonic development, the study of the connections between cilia and disease are extremely difficult in humans and other mammalian model organisms. Thus, alternative experimental systems are necessary. C. elegans enables the exploration of these questions in living animals. The highly conserved ciliogenic proteins, ciliogenesis pathway, and cilia sensory function make Caenorhabditis elegans a powerful model for characterizing the physiological roles of ciliary genes in their native cellular environments. Our data support the central hypothesis of this proposal that DYF-19 acts as a functional component to define TFs as a "ciliary gate" that governs access of nascent proteins into the cilia. and that disruption of this "gate" compromises cilia formation and function. The proposed studies have great potential to unveil breakthroughs in cilia research in the near future, and would provide seminal information about how cilia biogenesis and sensory function are regulated in their native environment, shed light on the etiologies of ciliopathies.