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.
Highlights
Because the repartition criterion is not definitive in the case of proteins present in both the cell body and the flagellum, we investigated the arginine kinase, a protein known to exhibit such a profile [61] and that produced a relatively neutral score (1.27) but with a high number of peptides (supplemental Table S1)
The quality of the purified flagella was confirmed at the structural level by scanning and transmission electron microscopy and at the molecular level by western blotting and mass spectrometry analysis
Up to 80% of the purified flagella were surrounded by an intact membrane and the axoneme, the paraflagellar rod (PFR), and the intraflagellar transport (IFT) particles looked intact in most sections
Summary
Because the repartition criterion is not definitive in the case of proteins present in both the cell body and the flagellum, we investigated the arginine kinase, a protein known to exhibit such a profile [61] and that produced a relatively neutral score (1.27) but with a high number of peptides (supplemental Table S1). Triple staining with the anti-AK, MAb25, and a monoclonal antibody against the intraflagellar transport protein IFT172 on methanol-fixed cells showed very close co-localization between all three markers (Fig. 3D–F).
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