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

Abstract 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. These occur in multiple different organisms, particularly involving the inner or outer dynein arms. Here, we comprehensively analyse conserved proximal-distal asymmetries in the uniflagellate trypanosomatid eukaryotic parasites. Building on the genome-wide localisation screen in Trypanosoma brucei we identify conserved proteins with an analogous asymmetric localisation in the related parasite Leishmania mexicana . Using deletion mutants, we find which are necessary for normal cell swimming, flagellum beat parameters and axoneme ultrastructure. Using combinatorial endogenous fluorescent tagging and deletion, we map co-dependencies for assembly into their normal asymmetric localisation. This revealed 15 proteins, 9 known and 6 novel, with a conserved proximal or distal axoneme-specific localisation. Most are outer dynein arm associated and show that there are multiple classes of proximal-distal asymmetry – one which is dependent on the docking complex. Many of these proteins are necessary for retaining the normal frequency of the tip-to-base symmetric flagellar waveform. Our comprehensive mapping reveals unexpected contributions of proximal-specific axoneme components to the frequency of waveforms initiated distally.