Molecular dissection of the ciliary gate

睫状门的分子解剖

• 批准号: 8901154
• 负责人: Jinghua Hu
• 金额: $ 23.85万
• 依托单位: MAYO CLINIC ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-08-01 至 2018-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8901154
• 关键词: Abbreviations; Affect; Animal Model; Animals; Autosomal Dominant Polycystic Kidney; Autosomal Recessive Polycystic Kidney; Bardet-Biedl Syndrome; Binding; Biological Assay; Biology; Caenorhabditis elegans; Cell surface; Centrioles; Chickens; Cilia; Data; Defect; Development; Devices; Disease; Dissection; Distal; Dyes; Embryonic Development; Environment; Etiology; Eukaryotic Cell; Excision; Exhibits; Eye; Fiber; Functional disorder; Future; Genes; Genetic Models; Health; Hereditary Disease; Homeostasis; Homologous Gene; Human; Human Genetics; Human Pathology; Joubert syndrome; Kidney; Knock-out; Letters; Life; Light; Limb structure; Link; Liver; Location; Maintenance; Membrane; Modeling; Molecular; Mothers; Mutagenesis; Mutate; Names; Nephronophthisis; Neuraxis; Nomenclature; Nuclear Pore; Organ; Organelles; Organism; Pathology; Pathway interactions; Pattern; Personal Satisfaction; Physiological; Play; Polycystic Kidney Diseases; Pore Proteins; Process; Proteins; Research; Role; Seminal; Sensory; Sensory Receptors; Signal Pathway; Signal Transduction; Structure; Study models; Surface; Syndrome; System; TNFRSF6 gene; Testing; appendage; base; ciliopathy; cilium biogenesis; clinically relevant; design; gene function; human disease; insight; kinetosome; neuronal cell body; novel; particle; polypeptide; positional cloning; screening

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.

描述（由申请人提供）：纤毛作为大多数真核细胞表面的感觉装置，在发育中多种器官的正确形成中发挥重要作用。通过鞭毛内运输（IFT）和感觉转导能力进行的纤毛组装在所有纤毛生物中都高度保守。过去十年，随着人类疾病基因定位克隆的快速进展，多种疾病，如常染色体显性多囊肾病（ADPKD）、Joubert综合征（JBST）、Bardet-Biedl综合征（BBS）、肾结核（NPHP） ）、梅克尔-格鲁伯综合征 (MKS) 和常染色体隐性遗传性多囊肾病 (ARPKD) 的分子特征为纤毛相关疾病，现在统称为纤毛病。纤毛功能的建立和维持显然对于生物体的健康至关重要。与纤毛的普遍存在相一致，许多纤毛病作为影响多个器官的综合征性疾病发生，包括肾、肝、四肢、眼和中枢神经系统。尽管纤毛具有生理和临床相关性，但调节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学之间的联系在很大程度上仍然难以捉摸。纤毛生物学的一个核心问题是纤毛门如何在功能上将纤毛与细胞体分离并使其成为一种离散的传感细胞器。纤毛仅在母中心粒（或基体）顶部形成。在纤毛发生过程中，母体中心粒的远端附属物转变为过渡纤维（TF），形成连接基体和纤毛基膜的 9 叶片螺旋桨结构。 TF 独特的亚细胞位置使其成为睫状门的良好候选者。尽管如此，还没有关于转录因子的组成和功能的分子信息。在旨在确定秀丽隐杆线虫纤毛发生决定因素的正向诱变筛选中，我们分离并克隆了一个新基因 dyf-19，它是特征不明确的人类 fbf1 基因的唯一同源物。我们的初步数据表明，蠕虫 DYF-19 和人类 FBF1 在过渡纤维和远端附属物上表现出特定的定位模式，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 调节过渡纤维上组装的 IFT 颗粒的纤毛进入以及几种纤毛感觉受体的纤毛进入。我们的初步研究揭示了转录因子的第一个真正成分，并证明了转录因子在纤毛形成和功能中的重要作用。此外，我们还发现了另外两种真正的 TF 组件：TALPID-3 和 HYLS-1。我们的初步数据表明 DYF-19、TALPID-3 和 HYLS-1 在 TF 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纤毛病模型，而人类 hyls1 基因是纤毛病 Hydrolethalus 综合征的致病位点之一。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型生物中研究纤毛与疾病之间的联系极其困难。因此，替代实验系统是必要的。线虫能够在活体动物中探索这些问题。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秀丽隐杆线虫成为表征纤毛基因在其天然细胞环境中的生理作用的强大模型。我们的数据支持该提案的中心假设，即 DYF-19 作为功能组件，将 TF 定义为控制新生蛋白进入纤毛的“纤毛门”。这个“门”的破坏会损害纤毛的形成和功能。拟议的研究具有在不久的将来揭示纤毛研究突破的巨大潜力，并将提供关于纤毛生物发生和感觉功能如何在其原生环境中调节的开创性信息，揭示纤毛病的病因学。