title: Imaging intraflagellar transport in trypanosomes.
abstract: 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.
authors.affiliation: Trypanosome Cell Biology Unit, Institut Pasteur & CNRS URA2581, Paris, France.
authors.forename: Julien
authors.initials: J
authors.lastname: Santi-Rocca
authors.affiliation: Bio Image Analysis Unit, Institut Pasteur & CNRS URA2582, Paris, France.
authors.forename: Nicolas
authors.initials: N
authors.lastname: Chenouard
authors.affiliation: Trypanosome Cell Biology Unit, Institut Pasteur & CNRS URA2581, Paris, France.
authors.forename: Cécile
authors.initials: C
authors.lastname: Fort
authors.affiliation: Bio Image Analysis Unit, Institut Pasteur & CNRS URA2582, Paris, France.
authors.forename: Thibault
authors.initials: T
authors.lastname: Lagache
authors.affiliation: Bio Image Analysis Unit, Institut Pasteur & CNRS URA2582, Paris, France.
authors.forename: Jean-Christophe
authors.initials: JC
authors.lastname: Olivo-Marin
authors.affiliation: Trypanosome Cell Biology Unit, Institut Pasteur & CNRS URA2581, Paris, France.
authors.forename: Philippe
authors.initials: P
authors.lastname: Bastin
mesh_terms: D002923:Cilia / Q000378:metabolism*; D005407:Flagella / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000235:genetics; D007734:Kymography / Q000379:methods; D008164:Luminescent Proteins / Q000235:genetics; D061848:Optical Imaging / Q000379:methods; D021381:Protein Transport / Q000502:physiology; D015800:Protozoan Proteins / Q000235:genetics* / Q000378:metabolism*; D034622:RNA Interference; D034741:RNA, Small Interfering; D011993:Recombinant Fusion Proteins / Q000235:genetics; D014346:Trypanosoma brucei brucei / Q000235:genetics / Q000378:metabolism*; D014404:Tubulin / Q000378:metabolism; D000097573:Red Fluorescent Protein
chemical_list: D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D015800:Protozoan Proteins; D034741:RNA, Small Interfering; D011993:Recombinant Fusion Proteins; D014404:Tubulin; D000097573:Red Fluorescent Protein
keywords: Cilia and flagella; Imaging; Intraflagellar transport; Trafficking; Trypanosome