Absalon Sabrina, Blisnick Thierry, Kohl Linda, Toutirais Géraldine, Doré Gwénola, Julkowska Daria, Tavenet Arounie, Bastin Philippe
Trypanosome Cell Biology Unit, Pasteur Institute and Centre National de la Recherche Scientifique, 75015 Paris, France.
Mol Biol Cell. 2008 Mar;19(3):929-44. doi: 10.1091/mbc.e07-08-0749. Epub 2007 Dec 19.
Intraflagellar transport (IFT) is the bidirectional movement of protein complexes required for cilia and flagella formation. We investigated IFT by analyzing nine conventional IFT genes and five novel putative IFT genes (PIFT) in Trypanosoma brucei that maintain its existing flagellum while assembling a new flagellum. Immunostaining against IFT172 or expression of tagged IFT20 or green fluorescent protein GFP::IFT52 revealed the presence of IFT proteins along the axoneme and at the basal body and probasal body regions of both old and new flagella. IFT particles were detected by electron microscopy and exhibited a strict localization to axonemal microtubules 3-4 and 7-8, suggesting the existence of specific IFT tracks. Rapid (>3 microm/s) bidirectional intraflagellar movement of GFP::IFT52 was observed in old and new flagella. RNA interference silencing demonstrated that all individual IFT and PIFT genes are essential for new flagellum construction but the old flagellum remained present. Inhibition of IFTB proteins completely blocked axoneme construction. Absence of IFTA proteins (IFT122 and IFT140) led to formation of short flagella filled with IFT172, indicative of defects in retrograde transport. Two PIFT proteins turned out to be required for retrograde transport and three for anterograde transport. Finally, flagellum membrane elongation continues despite the absence of axonemal microtubules in all IFT/PIFT mutant.
鞭毛内运输（IFT）是纤毛和鞭毛形成所需的蛋白质复合物的双向运动。我们通过分析布氏锥虫中的9个传统IFT基因和5个新的假定IFT基因（PIFT）来研究IFT，布氏锥虫在组装新鞭毛时会保留其现有的鞭毛。针对IFT172的免疫染色或标记的IFT20或绿色荧光蛋白GFP::IFT52的表达揭示了IFT蛋白沿着轴丝以及在旧鞭毛和新鞭毛的基体和原基体区域的存在。通过电子显微镜检测到IFT颗粒，并且它们严格定位于轴丝微管3 - 4和7 - 8，这表明存在特定的IFT轨道。在旧鞭毛和新鞭毛中均观察到GFP::IFT52的快速（>3微米/秒）双向鞭毛内运动。RNA干扰沉默表明，所有单个的IFT和PIFT基因对于新鞭毛的构建都是必不可少的，但旧鞭毛仍然存在。对IFTB蛋白的抑制完全阻断了轴丝的构建。缺乏IFTA蛋白（IFT122和IFT140）导致形成充满IFT172的短鞭毛，这表明逆行运输存在缺陷。结果表明，有两种PIFT蛋白是逆行运输所必需的，三种是顺行运输所必需的。最后，尽管在所有IFT/PIFT突变体中缺乏轴丝微管，但鞭毛膜仍继续伸长。
