Exploration of the functions of the ciliopathy Arls in cilia

 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，在发育过程中的器官发生和组织模式形成中发挥重要作用。通过鞭毛内转运（IFT）和感觉转导能力的纤毛组装在所有纤毛生物中是高度保守的。在过去的十年中，随着人类疾病基因定位克隆的快速发展，各种各样的疾病，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），肾单位结核（NPHP），Meckel-Gruber综合征（MKS）和常染色体隐性遗传性多囊肾病（ARPKD），已被分子表征为纤毛疾病。纤毛功能的建立和维持对于生物体的健康显然是必不可少的。与纤毛的普遍存在一致，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、肝肢、眼、中枢神经系统（CNS）和脂肪储存组织。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仍然存在， 以及疾病基因功能和病理学之间的联系仍然知之甚少。酶促小GTP酶作为分子开关，其控制基本细胞过程并且通常与各种人类病理状况相关。来自其他实验室和我们实验室的研究表明，三种保守且特征不佳的ADP-核糖基化因子样（ARL）小GTP酶ARL 3、ARL 6和ARL 13 B作为突出的纤毛开关，其功能被破坏使人或小鼠易患纤毛病。最大的障碍是，鸟嘌呤核苷酸交换因子（GEF）和GTP酶激活蛋白（GAP），开关睫状体ARLs，分别和ARLs的效应器尚未确定。在其他疾病中，如结节性硬化症，确定由TSC 2 GAP抑制的GT3在理解疾病方面具有变革性意义，我们提出，这里的相应知识将对理解纤毛病产生类似的戏剧性影响。由于高度保守的纤毛途径和纤毛病基因，秀丽隐杆线虫已被建立为一个简单而有效的模型，用于表征纤毛病蛋白在其天然细胞环境中的生理作用。在上一个基金会期间，我们成功地建立了C。elegans作为研究纤毛病ARLs作用的模型。我们最近的数据表明，睫状ARLs可能组织成两个不同的功能模块在神秘的反转（InV）室的纤毛。一个功能模块包含ARL-13-ARL-3-NPHP-2-NPHP-119，其中NPHP-119和肾单位萎缩蛋白NPHP-2与ARL-13协同作用，但与ARL-3拮抗，调节纤毛发生。第二个含有ARL-6-ARL-13-BBSome，其可能通过调节纤毛感觉受体的适当定位来调节纤毛信号传导。我们的初步研究结果也支持了从蠕虫到哺乳动物细胞中纤毛病ARLs的作用是高度保守的。基于这些，我们的中心假设是，三个纤毛病ARLs和它们的调节器被组织成不同的复合物，以协调纤毛生物发生和信号，分别。我们将使用C。elegans以鉴定体内调节剂和功能，以及哺乳动物系统以确定对人类纤毛病的适用性。具体目标一是确定ARL蛋白模块中各组分的调控因子类型，并希望鉴定出纤毛病变ARL的GEFs、GAP或效应因子;具体目标2是确定ARL-13、NPHP-2和ARL-119是否以及如何在InV隔室中协调IFT完整性和/或轴丝稳定性，ARL-3是否通过依赖去乙酰化酶HDAC-6的方式拮抗ARL-13-NPHP-22-NPHP-119的作用;具体目的3是确定ARL-6-ARL-13-BBSomeTM模块是否通过ARL-13促进ARL-6活化、BBSomeTM-货物组装以及随后感觉受体在InV隔室中的适当纤毛定位。这些研究将为揭示纤毛生物学的突破提供巨大的潜力，并将提供有关纤毛生物发生和感觉功能如何在其天然环境中调节的开创性信息，阐明纤毛病的病因，并可能为疾病诊断和治疗提供新的靶点。

 DESCRIPTION (provided by applicant): Cilia serve as sensory devices on most eukaryotic cell surfaces and play essential roles in organogenesis and tissue pattern formation during 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 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 kidneys, liver limbs, eyes, central nervous system (CNS), and fat storage tissue. 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 poorly understood. Enzymatic small GTPases act as molecular switches, which control fundamental cellular processes and are often correlated with various human pathological conditions. Studies from other and our laboratories demonstrated that three conserved and poorly characterized ADP-ribosylation factor-like (ARL) small GTPases, ARL3, ARL6, and ARL13B, act as prominent ciliary switches, with disrupted function predisposing human or mice to ciliopathies. The paramount obstacle being that the guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs) that switch ciliary ARLs on and off, respectively, and the effectors of ARLs have not been identified. In other disorders, such as tuberous sclerosis, identifying the GTPase inhibited by the TSC2 GAP was transformative in understanding the disease, and we propose that the corresponding knowledge here would have a similar dramatic effect on understanding ciliopathies. Due to highly conserved cilia pathways and ciliopathy genes, Caenorhabditis elegans has been established as a simple and effective model for characterizing the physiological roles of ciliopathy proteins in their native cellular environments. In last fundig period, we have successfully established C. elegans as a model to investigate the roles of ciliopathy ARLs. Our recent data suggested that ciliary ARLs are likely organized into two distinct functional modules in the enigmatic inversin (InV) compartment of cilia. One function module contains ARL-13-ARL-3-NPHP-2-UNC-119, in which UNC-119 and nephronophthisis protein NPHP-2 act synergistically with ARL-13, but antagonistically with ARL-3, in regulating ciliogenesis. The second one contains ARL-6-ARL-13-BBSome, which may regulate cilia signaling through regulating the proper localization of ciliary sensory receptors. Our preliminary results also supported that the roles of ciliopathy ARLs are highly conserved from worm to mammalian cells. Based on these, our central hypothesis is that the three ciliopathy ARLs and their regulators are organized into distinct complexes to coordinate cilia biogenesis and signaling, respectively. We will employ C. elegans to identify in vivo regulators and functions, and mammalian systems to determine the applicability to human ciliopathies. Specific Aim 1 is to characterized the type of regulators for each component in ARL-containing protein module, and we hope to identify GEFs, GAPs, or effectors for ciliopathy ARLs; Specific Aim 2 is to ascertain whether and how ARL-13, NPHP-2, and UNC-119 coordinate IFT integrity and/or axonemal stability in the InV compartment, and whether ARL-3 antagonizes the roles of ARL-13-NPHP-22-UNC-119 through deacetylase HDAC-6-dependent manner; Specific Aim 3 is to determine whether the ARL-6-ARL-13-BBSome module coordinates cilia signaling through the mechanism that ARL-13 promotes ARL-6 activation, BBSome-cargo assembly, and subsequent proper ciliary localization of sensory receptors in the InV compartment. The proposed studies have great potentials for unveiling breakthroughs in cilia biology, 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, and potentially provide novel targets fo disease diagnosis and treatment.