FAP106 is an interaction hub required for assembly of conserved and lineage-specific microtubule inner proteins at the cilium inner junction

ABSTRACT Motility of pathogenic protozoa depends on flagella (synonymous with cilia) with axonemes containing nine doublet microtubules (DMTs) and two singlet microtubules. Microtubule inner proteins (MIPs) within DMTs influence axoneme stability and motility and provide lineage-specific adaptations, but individual MIP functions and assembly mechanisms are mostly unknown. Here, we show in the sleeping sickness parasite Trypanosoma brucei , that FAP106, a conserved MIP at the DMT inner junction, is required for trypanosome motility and functions as a critical interaction hub, directing assembly of several conserved and lineage-specific MIPs. We further use comparative cryogenic electron tomography (cryoET) and quantitative proteomics to identify novel MIP candidates, and RNAi knockdown plus fitting of AlphaFold models into cryoET maps to demonstrate one of these, MC8, is a trypanosome-specific MIP required for parasite motility. Our work advances understanding of MIP assembly mechanisms and identifies lineage-specific motility proteins that are attractive targets to consider for therapeutic intervention.