Baron Desiree M, Ralston Katherine S, Kabututu Zakayi P, Hill Kent L
Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, CA 90095, USA.
J Cell Sci. 2007 Feb 1;120(Pt 3):478-91. doi: 10.1242/jcs.03352. Epub 2007 Jan 16.
Cilia and flagella are highly conserved, complex organelles involved in a variety of important functions. Flagella are required for motility of several human pathogens and ciliary defects lead to a variety of fatal and debilitating human diseases. Many of the major structural components of cilia and flagella are known, but little is known about regulation of flagellar beat. Trypanosoma brucei, the causative agent of African sleeping sickness, provides an excellent model for studying flagellar motility. We have used comparative genomics to identify a core group of 50 genes unique to organisms with motile flagella. These genes, referred to as T. brucei components of motile flagella (TbCMF) include 30 novel genes, and human homologues of many of the TbCMF genes map to loci associated with human ciliary diseases. To characterize TbCMF protein function we used RNA interference to target 41 TbCMF genes. Sedimentation assays and direct observation demonstrated clear motility defects in a majority of these knockdown mutants. Epitope tagging, fluorescence localization and biochemical fractionation demonstrated flagellar localization for several TbCMF proteins. Finally, ultrastructural analysis identified a family of novel TbCMF proteins that function to maintain connections between outer doublet microtubules, suggesting that they are the first identified components of nexin links. Overall, our results provide insights into the workings of the eukaryotic flagellum, identify several novel human disease gene candidates, reveal unique aspects of the trypanosome flagellum and underscore the value of T. brucei as an experimental system for studying flagellar biology.
纤毛和鞭毛是高度保守的复杂细胞器，参与多种重要功能。鞭毛是几种人类病原体运动所必需的，而纤毛缺陷会导致多种致命和使人衰弱的人类疾病。纤毛和鞭毛的许多主要结构成分已为人所知，但关于鞭毛摆动的调节却知之甚少。布氏锥虫是非洲昏睡病的病原体，为研究鞭毛运动提供了一个极好的模型。我们利用比较基因组学鉴定出了一组由50个基因组成的核心基因群，这些基因是具有活动鞭毛的生物体所特有的。这些基因被称为布氏锥虫活动鞭毛成分（TbCMF），包括30个新基因，许多TbCMF基因的人类同源基因定位于与人类纤毛疾病相关的位点。为了表征TbCMF蛋白的功能，我们使用RNA干扰靶向41个TbCMF基因。沉降分析和直接观察表明，这些敲低突变体中的大多数都存在明显的运动缺陷。表位标记、荧光定位和生化分级分离表明几种TbCMF蛋白定位于鞭毛。最后，超微结构分析鉴定出了一个新的TbCMF蛋白家族，其功能是维持外双联微管之间的连接，这表明它们是首次鉴定出的连接蛋白连接的成分。总体而言，我们的结果为真核生物鞭毛的工作原理提供了见解，鉴定出了几种新的人类疾病基因候选物，揭示了锥虫鞭毛的独特方面，并强调了布氏锥虫作为研究鞭毛生物学的实验系统的价值。
