Acute versus chronic loss of mammalian Azi1/Cep131 results in distinct ciliary phenotypes.
Acute versus chronic loss of mammalian Azi1/Cep131 results in distinct ciliary phenotypes.
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DOI：
10.1371/journal.pgen.1003928
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发表时间：
2013
期刊：
影响因子：
4.5
通讯作者：
Mill P
中科院分区：
文献类型：
--
作者：
Hall EA;Keighren M;Ford MJ;Davey T;Jarman AP;Smith LB;Jackson IJ;Mill P
纤毛和中心体功能缺陷会导致一系列与临床相关的疾病，称为纤毛病。然而，这些结构的复杂分子组成混淆了对任何单个基因产物在正常和疾病条件下所做的功能剖析。作为哺乳动物纤毛发生相关基因siRNA筛选的一部分，我们和其他人已经确定保守的中心体蛋白Azi1/Cep131是纤毛形成所需的，支持了之前的Danio rerio和果蝇突变研究。在小鼠成纤维细胞中，通过敲击导致Azi1的急性丢失导致纤毛发生的强劲减少，我们通过表达具有siRNA抗性的Azi1-GFP来挽救这一点。定位研究表明，Azi1定位于中心粒卫星，沿微管的交通在基底周围变得丰富。Azi1也定位于过渡区，这是调节进入睫状室的交通的重要结构。为了研究Azi1在发育过程中的需求和组织动态平衡，我们建立了Azi1基因缺失小鼠(Azi1Gt/Gt)。令人惊讶的是，Azi1Gt/GT MEF没有可识别的纤毛表型，而且对Azi1 siRNA击倒具有抵抗力，这表明存在一种补偿机制，即使缺少Azi1，纤毛发生也可以进行。Azi1基因缺失小鼠的纤毛在功能上是正常的，因为胚胎模式和成年动态平衡完全不受影响。然而，在高度专业化的精子鞭毛中，Azi1的丢失没有得到补偿，导致套囊和鞭毛中基于微管的显著运输缺陷，导致男性不育。我们对Azi1基因敲除(急性缺失)和基因缺失(慢性缺失)的分析表明，Azi1在纤毛发生中起着保守但非必要的运输作用。重要的是，我们的体内分析揭示了Azi1介导了鞭毛发生所必需的新的运输功能。我们的研究强调了在确定人类疾病候选基因的功能特征时，除了小鼠基因敲除研究外，蛋白质的急性去除的重要性。纤毛是大多数哺乳动物细胞表面的细长突起，具有感觉功能，有时还具有运动功能。它们对哺乳动物的发育是必不可少的，纤毛缺陷会导致一组人类疾病，称为纤毛病，具有不同的症状，包括胚胎死亡、肺和肾脏缺陷、失明和不育。纤毛是由数百种成分组成的复杂结构，其单个功能尚不清楚。我们筛选了在纤毛形成中重要的哺乳动物基因，并确定了Azi1/Cep131，这是一个以前被证明是鱼类和苍蝇纤毛形成和功能所必需的基因。我们发现，如果我们大幅降低小鼠细胞中Azi1的水平，形成纤毛的细胞会更少，但如果我们产生的细胞长期缺乏Azi1，纤毛就会正常形成。此外，没有任何Azi1的小鼠是健康和存活的，证实了正常的纤毛功能。然而，在这些小鼠中，精子尾巴高度专业化的纤毛结构没有形成，导致男性不育。我们认为Azi1在初级纤毛和特殊的精子鞭毛中都具有保守的运输作用。突然移除azi1会导致不稳定，导致现有纤毛网络崩溃，而长期缺失azi1会使系统重新平衡，纤毛正常形成。
Defects in cilium and centrosome function result in a spectrum of clinically-related disorders, known as ciliopathies. However, the complex molecular composition of these structures confounds functional dissection of what any individual gene product is doing under normal and disease conditions. As part of an siRNA screen for genes involved in mammalian ciliogenesis, we and others have identified the conserved centrosomal protein Azi1/Cep131 as required for cilia formation, supporting previous Danio rerio and Drosophila melanogaster mutant studies. Acute loss of Azi1 by knock-down in mouse fibroblasts leads to a robust reduction in ciliogenesis, which we rescue by expressing siRNA-resistant Azi1-GFP. Localisation studies show Azi1 localises to centriolar satellites, and traffics along microtubules becoming enriched around the basal body. Azi1 also localises to the transition zone, a structure important for regulating traffic into the ciliary compartment. To study the requirement of Azi1 during development and tissue homeostasis, Azi1 null mice were generated (Azi1Gt/Gt). Surprisingly, Azi1Gt/Gt MEFs have no discernible ciliary phenotype and moreover are resistant to Azi1 siRNA knock-down, demonstrating that a compensation mechanism exists to allow ciliogenesis to proceed despite the lack of Azi1. Cilia throughout Azi1 null mice are functionally normal, as embryonic patterning and adult homeostasis are grossly unaffected. However, in the highly specialised sperm flagella, the loss of Azi1 is not compensated, leading to striking microtubule-based trafficking defects in both the manchette and the flagella, resulting in male infertility. Our analysis of Azi1 knock-down (acute loss) versus gene deletion (chronic loss) suggests that Azi1 plays a conserved, but non-essential trafficking role in ciliogenesis. Importantly, our in vivo analysis reveals Azi1 mediates novel trafficking functions necessary for flagellogenesis. Our study highlights the importance of both acute removal of a protein, in addition to mouse knock-out studies, when functionally characterising candidates for human disease. Cilia are slender projections from the surface of most mammalian cells and have sensory and sometimes motile functions. They are essential for mammalian development and defects in cilia lead to a group of human diseases, termed ciliopathies, with variable symptoms including embryonic lethality, lung and kidney defects, blindness and infertility. Cilia are complex structures composed of hundreds of components, whose individual functions are poorly understood. We screened for mammalian genes important in building cilia, and identified Azi1/Cep131, a gene previously shown to be required for cilia formation and function in fish and flies. We show that if we acutely reduce levels of Azi1 in mouse cells, fewer cells form cilia, but if we generate cells chronically lacking all Azi1, cilia form normally. In addition, mice without any Azi1 are healthy and viable, confirming normal cilia function. However, in these mice, the highly specialised ciliary structure of the sperm tail does not form, resulting in male infertility. We suggest Azi1 has conserved trafficking roles in both primary cilia and the specialised sperm flagella. Abruptly removing Azi1 results in instability causing the existing cilia network to collapse, whereas chronic deletion of Azi1 allows the system to re-equilibrate, and cilia to form normally.
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影响因子：
56.9
作者：
Celeste, A;Petersen, S;Nussenzweig, A
通讯作者：
Nussenzweig, A
影响因子：
64.5
作者：
Chaki M;Airik R;Ghosh AK;Giles RH;Chen R;Slaats GG;Wang H;Hurd TW;Zhou W;Cluckey A;Gee HY;Ramaswami G;Hong CJ;Hamilton BA;Cervenka I;Ganji RS;Bryja V;Arts HH;van Reeuwijk J;Oud MM;Letteboer SJ;Roepman R;Husson H;Ibraghimov-Beskrovnaya O;Yasunaga T;Walz G;Eley L;Sayer JA;Schermer B;Liebau MC;Benzing T;Le Corre S;Drummond I;Janssen S;Allen SJ;Natarajan S;O'Toole JF;Attanasio M;Saunier S;Antignac C;Koenekoop RK;Ren H;Lopez I;Nayir A;Stoetzel C;Dollfus H;Massoudi R;Gleeson JG;Andreoli SP;Doherty DG;Lindstrad A;Golzio C;Katsanis N;Pape L;Abboud EB;Al-Rajhi AA;Lewis RA;Omran H;Lee EY;Wang S;Sekiguchi JM;Saunders R;Johnson CA;Garner E;Vanselow K;Andersen JS;Shlomai J;Nurnberg G;Nurnberg P;Levy S;Smogorzewska A;Otto EA;Hildebrandt F
通讯作者：
Hildebrandt F
影响因子：
--
作者：
Akimov, Vyacheslav;Rigbolt, Kristoffer T. G.;Blagoev, Blagoy
通讯作者：
Blagoev, Blagoy
DOI：
10.1002/ajmg.c.30231
发表时间：
2009-11-15
影响因子：
3.1
作者：
Baker, Kate;Beales, Philip L.
通讯作者：
Beales, Philip L.
影响因子：
21.3
作者：
Chih, Ben;Liu, Peter;Peterson, Andrew S.
通讯作者：
Peterson, Andrew S.