Proteins with proximal-distal asymmetries in axoneme localisation control flagellum beat frequency

The 9+2 microtubule-based axoneme within motile flagella is well known for its symmetry. However, examples of asymmetric structures and proteins asymmetrically positioned within the 9+2 axoneme architecture have been identified in multiple different organisms, particularly involving the inner or outer dynein arms, with a range of functions. Here, mapped, genome-wide, conserved proximal-distal asymmetries in the uniflagellate trypanosomatid eukaryotic parasites. Building on the genome-wide localisation screen in Trypanosoma brucei we identified conserved proteins with an analogous asymmetric localisation in the related parasite Leishmania mexicana . Using deletion mutants, we map which are necessary for normal cell swimming, flagellum beat parameters and axoneme ultrastructure, and using combinatorial endogenous fluorescent tagging and deletion, map co-dependencies for assembly into their normal asymmetric localisation. This revealed 15 proteins, 8 known and 7 novels, with a conserved proximal or distal axoneme-specific localisation. Most were outer dynein arm associated, and showed that there are at least two distinct classes of proximal-distal asymmetry – one dependent on the docking complex, and one independent. Many were necessary for normal frequency of the tip-to-base symmetric flagellar waveform, and our comprehensive mapping reveals unexpected contribution of proximal-specific axoneme components to frequency of distal waveform initiation.