title: Proteomic analysis of intact flagella of procyclic Trypanosoma brucei cells identifies novel flagellar proteins with unique sub-localization and dynamics.
abstract: Cilia and flagella are complex organelles made of hundreds of proteins of highly variable structures and functions. Here we report the purification of intact flagella from the procyclic stage of Trypanosoma brucei using mechanical shearing. Structural preservation was confirmed by transmission electron microscopy that showed that flagella still contained typical elements such as the membrane, the axoneme, the paraflagellar rod, and the intraflagellar transport particles. It also revealed that flagella severed below the basal body, and were not contaminated by other cytoskeletal structures such as the flagellar pocket collar or the adhesion zone filament. Mass spectrometry analysis identified a total of 751 proteins with high confidence, including 88% of known flagellar components. Comparison with the cell debris fraction revealed that more than half of the flagellum markers were enriched in flagella and this enrichment criterion was taken into account to identify 212 proteins not previously reported to be associated to flagella. Nine of these were experimentally validated including a 14-3-3 protein not yet reported to be associated to flagella and eight novel proteins termed FLAM (FLAgellar Member). Remarkably, they localized to five different subdomains of the flagellum. For example, FLAM6 is restricted to the proximal half of the axoneme, no matter its length. In contrast, FLAM8 is progressively accumulating at the distal tip of growing flagella and half of it still needs to be added after cell division. A combination of RNA interference and Fluorescence Recovery After Photobleaching approaches demonstrated very different dynamics from one protein to the other, but also according to the stage of construction and the age of the flagellum. Structural proteins are added to the distal tip of the elongating flagellum and exhibit slow turnover whereas membrane proteins such as the arginine kinase show rapid turnover without a detectible polarity.
authors.affiliation: From the ‡Trypanosome Cell Biology Unit, Institut Pasteur & CNRS URA2581.
authors.forename: Ines
authors.initials: I
authors.lastname: Subota
authors.affiliation: From the ‡Trypanosome Cell Biology Unit, Institut Pasteur & CNRS URA2581.
authors.forename: Daria
authors.initials: D
authors.lastname: Julkowska
authors.affiliation: From the ‡Trypanosome Cell Biology Unit, Institut Pasteur & CNRS URA2581.
authors.forename: Laetitia
authors.initials: L
authors.lastname: Vincensini
authors.affiliation: From the ‡Trypanosome Cell Biology Unit, Institut Pasteur & CNRS URA2581.
authors.forename: Nele
authors.initials: N
authors.lastname: Reeg
authors.affiliation: From the ‡Trypanosome Cell Biology Unit, Institut Pasteur & CNRS URA2581.
authors.forename: Johanna
authors.initials: J
authors.lastname: Buisson
authors.affiliation: From the ‡Trypanosome Cell Biology Unit, Institut Pasteur & CNRS URA2581.
authors.forename: Thierry
authors.initials: T
authors.lastname: Blisnick
authors.affiliation: From the ‡Trypanosome Cell Biology Unit, Institut Pasteur & CNRS URA2581.
authors.forename: Diego
authors.initials: D
authors.lastname: Huet
authors.affiliation: From the ‡Trypanosome Cell Biology Unit, Institut Pasteur & CNRS URA2581.
authors.forename: Sylvie
authors.initials: S
authors.lastname: Perrot
authors.affiliation: From the ‡Trypanosome Cell Biology Unit, Institut Pasteur & CNRS URA2581.
authors.forename: Julien
authors.initials: J
authors.lastname: Santi-Rocca
authors.affiliation: §Proteomics Platform, Institut Pasteur, ¶Structural Mass Spectrometry and Proteomics Unit, Institut Pasteur & CNRS UMR3528.
authors.forename: Magalie
authors.initials: M
authors.lastname: Duchateau
authors.affiliation: §Proteomics Platform, Institut Pasteur, ¶Structural Mass Spectrometry and Proteomics Unit, Institut Pasteur & CNRS UMR3528.
authors.forename: Véronique
authors.initials: V
authors.lastname: Hourdel
authors.affiliation: §Proteomics Platform, Institut Pasteur.
authors.forename: Jean-Claude
authors.initials: JC
authors.lastname: Rousselle
authors.affiliation: ‖Imagopole Platform, Institut Pasteur, Paris, France.
authors.forename: Nadège
authors.initials: N
authors.lastname: Cayet
authors.affiliation: §Proteomics Platform, Institut Pasteur.
authors.forename: Abdelkader
authors.initials: A
authors.lastname: Namane
authors.affiliation: §Proteomics Platform, Institut Pasteur, ¶Structural Mass Spectrometry and Proteomics Unit, Institut Pasteur & CNRS UMR3528.
authors.forename: Julia
authors.initials: J
authors.lastname: Chamot-Rooke
authors.affiliation: From the ‡Trypanosome Cell Biology Unit, Institut Pasteur & CNRS URA2581, pbastin@pasteur.fr.
authors.forename: Philippe
authors.initials: P
authors.lastname: Bastin
mesh_terms: D048948:14-3-3 Proteins / Q000235:genetics / Q000378:metabolism; D005407:Flagella / Q000378:metabolism*; D036681:Fluorescence Recovery After Photobleaching; D020869:Gene Expression Profiling; D008565:Membrane Proteins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D046529:Microscopy, Electron, Transmission; D040901:Proteomics; D015800:Protozoan Proteins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D034622:RNA Interference; D034741:RNA, Small Interfering; D014346:Trypanosoma brucei brucei / Q000378:metabolism*
chemical_list: D048948:14-3-3 Proteins; D008565:Membrane Proteins; D015800:Protozoan Proteins; D034741:RNA, Small Interfering
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