Santi-Rocca Julien, Chenouard Nicolas, Fort Cécile, Lagache Thibault, Olivo-Marin Jean-Christophe, Bastin Philippe
Trypanosome Cell Biology Unit, Institut Pasteur & CNRS URA2581, Paris, France.
Bio Image Analysis Unit, Institut Pasteur & CNRS URA2582, Paris, France.
Methods Cell Biol. 2015;127:487-508. doi: 10.1016/bs.mcb.2015.01.005. Epub 2015 Mar 7.
Trypanosoma brucei is a flagellated eukaryotic pathogen responsible for sleeping sickness in central Africa. Because of the presence of a long motile flagellum (>20 μm) and its amenity to genetic manipulation, it is becoming an attractive model to study the assembly and the functions of cilia and flagella. In recent years, several aspects have been investigated, especially intraflagellar transport (IFT) that has been exhaustively characterized at the light microscopy level. In this manuscript, we review various methods to express fluorescent fusion proteins and to record IFT in living trypanosomes in normal or mutant contexts. We present an approach for separating anterograde and retrograde IFT, hence facilitating quantification of train speed, frequency, and size. A statistical analysis to discriminate different subpopulations of IFT trains is also summarized. These methods have proven their efficiency for the study of IFT in trypanosomes and could be applied to any other organism.
布氏锥虫是一种有鞭毛的真核病原体，在中非导致昏睡病。由于其存在长的运动鞭毛（>20μm）且易于进行基因操作，它正成为研究纤毛和鞭毛组装及功能的有吸引力的模型。近年来，已经对几个方面进行了研究，特别是在光学显微镜水平上对鞭毛内运输（IFT）进行了详尽的表征。在本手稿中，我们综述了在正常或突变背景下在活的锥虫中表达荧光融合蛋白和记录IFT的各种方法。我们提出了一种分离顺行和逆行IFT的方法，从而便于对运输速度、频率和大小进行量化。还总结了用于区分不同IFT运输亚群的统计分析方法。这些方法已证明在锥虫中研究IFT的有效性，并且可应用于任何其他生物体。
