In situ cryo-ET reveals restricted docking of intraflagellar transport at the base of the trypanosome flagellum

Abstract

Cilia and flagella are conserved organelles essential for sensory and motility functions and are assembled and maintained by bidirectional intraflagellar transport (IFT) along the axoneme. In Trypanosoma brucei, IFT is concentrated at the flagellar base near the transition zone and is restricted to four microtubule doublets (MTDs) as it traverses from the transition zone to the axoneme before reaching the extra-axonemal structure, the paraflagellar rod (PFR), associated region. To gain an insight on its initial working model before IFT restriction occurs, we revealed the in situ architecture of the IFT complex during the initial and complete landing to the transition zone using cryo-focused ion beam (cryo-FIB) milling and cryo-electron tomography (cryo-ET). IFT proteins assemble into polymeric trains upon landing. However, in the proximal portion of the flagellum, subtomogram averaging revealed that each single train is associated with individual MTDs at variable A- or B-tubule positions. By using miniaturized and enucleated T. brucei zoids that preserve the full circular arrangement of MTDs without milling, we revealed an alternating but spatially restricted IFT train pattern during the landing process, resembling the anterograde train scaffold known in Chlamydomonas. Magnify expansion microscopy further confirmed this restricted distribution of IFT proteins at the basal pool. Depletion of the IFT component IFT46 protein reduced the number of simultaneously docked trains without altering the restricted landing spatial pattern. These findings reveal a previously unrecognized spatial regulation governing IFT train initiation at the flagellar base, which could operate independently of IFT-B structural integrity, bridging the gap between the IFT architecture and doublet-spatial docking prior to anterograde transport.