A novel approach to tagging tubulin reveals microtubule assembly dynamics of the axoneme in Trypanosoma brucei.

The protozoan parasite Trypanosoma brucei assembles a new flagellum while maintaining the existing one in the same cell. Our group has previously proposed a model where the mature flagellum is locked after construction to full length. To test this hypothesis directly, we monitored flagellum assembly dynamics through inducible expression of tubulin marked with an intragenic tag. We found that addition of new tubulin occurs at the distal flagellum tip at a linear rate and is indeed restricted to the new flagellum in bi-flagellated cells. Depleting the locking protein CEP164C prior to induction resulted in simultaneous integration of new tubulin in both flagella. This is direct evidence that trypanosomes avoid competition between the two flagella by allowing tubulin incorporation only in the new organelle. However, by tracing flagella over several cell cycles we also found that flagella do not remain locked forever. An orthogonal approach with HaloTag-tagged radial spoke protein 4/6 (GeneID Tb927.11.4480) supported these findings. Given that flagellum length in trypanosomes is stable, this indicates regular events of transient disassembly, followed by assembly, at the distal tip.