Functional studies of an evolutionarily conserved, cytochrome b5 domain protein reveal a specific role in axonemal organisation and the general phenomenon of post‐division axonemal growth in trypanosomes

Abstract

Eukaryotic cilia and flagella are highly conserved structures composed of a canonical 9+2 microtubule axoneme. Several recent proteomic studies of cilia and flagella have been published, including a proteome of the flagellum of the protozoan parasite Trypanosoma brucei. Comparing proteomes reveals many novel proteins that appear to be widely conserved in evolution. Amongst these, we found a previously uncharacterised protein which localised to the axoneme in T. brucei, and therefore named it Trypanosome Axonemal protein (TAX)-2. Ablation of the protein using RNA interference in the procyclic form of the parasite has no effect on growth but causes a reduction in motility. Using transmission electron microscopy, various structural defects were seen in some axonemes, most frequently with microtubule doublets missing from the 9+2 arrangement. RNAi knockdown of TAX-2 expression in the bloodstream form of the parasite caused defects in growth and cytokinesis, a further example of the effects caused by loss of flagellar function in bloodstream form T. brucei. In procyclic cells we used a new set of vectors to ablate protein expression in cells expressing a GFP:TAX-2 fusion protein, which enabled us to easily quantify protein reduction and visualise axonemes made before and after RNAi induction. This establishes a useful generic technique but also revealed a specific observation that the new flagellum on the daughter trypanosome continues growth after cytokinesis. Our results provide evidence for TAX-2 function within the axoneme, where we suggest that it is involved in processes linking the outer doublet microtubules and the central pair.