Intraflagellar transport selectivity occurs within the proximal portion of the trypanosome flagellum

Intraflagellar transport (IFT) trains move bidirectionally along the doublet microtubules (DMTs) of the axoneme within the flagellum. In Trypanosoma brucei, IFT trains predominantly associate with four of the nine DMTs. Using high-resolution microscopy, we reveal how this selective association is put in place. IFT proteins form a ring surrounding the 9 DMTs above the transition fibers. Volume electron microscopy revealed densities along all DMTs in the proximal portion of the flagellum, exhibiting thinner, shorter profiles with branches absent in mature IFT trains. As the axoneme extends within the flagellar pocket, IFT trains are detected but are often positioned outside DMTs 3–4/7–8. After the axoneme exits the flagellar pocket, IFT trains localize exclusively to DMTs 3–4 and 7–8. Super-resolution and expansion microscopy demonstrated that IFT proteins follow the same distribution as the IFT-like densities. This suggests they represent IFT trains undergoing assembly and/or disassembly and reveals their unexpected ability to shift from one DMT to another.