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

Abstract

Eukaryotic motile cilia/flagella are conserved organelles important for cell propulsion and fluid flow, typically built around a "9+2" axoneme of nine doublet microtubules (DMTs) encircling a central pair of singlet microtubules. The DMT lumen is lined with an interconnected network of microtubule inner proteins (MIPs), some conserved and others lineage-specific. MIPs augment the tubulin lattice of the DMT, directly impacting stability, fine structure, and motility, thus providing an important source of lineage-specific adaptations.Trypanosoma bruceiis a flagellated eukaryotic pathogen with distinctive motility that is critical for pathogen transmission and pathogenesis. Prior studies revealed lineage-specificT. bruceiMIPs, but their identities are unknown. To identifyT. bruceiMIPs, we examined flagellum structure and composition following knockdown of FAP106, a conserved MIP at the inner junction (IJ) connecting A- and B-microtubules of the DMT. FAP106 knockdown resulted in short flagella and defective parasite motility, supporting a role for MIPs inT. bruceiflagellum stability and motility. Cryogenic electron tomography (cryoET) and quantitative proteomics identified several conserved MIPs and lineage-specific MIP structures and MIP candidate proteins (MCs) that depend on FAP106 for stable assembly. We further demonstrate by knockdown and fitting AlphaFold models to cryoET maps that one of these, MC8, is a newly identified lineage-specific MIP required for normal parasite motility. This work provides an important advance toward elucidating the order of assembly of MIPs at the cilium inner junction and identifies trypanosome proteins specific to these deadly pathogens that represent targets to consider for therapeutic intervention.

FAP20 is required for flagellum assembly inTrypanosoma brucei

“…Prior studies showed that simultaneous knockdown of both PACRG-A and -B did slow cell growth, although the defect was not evident until approximately 72hpi and cells remained viable (Dawe et al, 2005). The rapid, lethal phenotype of FAP20 KD also differs from knockdown of T. brucei MIPs examined to date, which have minimal impact on parasite growth or viability (Shimogawa et al, 2023).…”

“…1A). Recent work in trypanosomes and other organisms has provided insight into proteins that mediate DMT assembly and function (Stoddard et al, 2018, Ichikawa et al, 2019, Imhof et al, 2019, Ma et al, 2019, Owa et al, 2019, Gui et al, 2021, Gui et al, 2022, Li et al, 2022, Kubo et al, 2023, Shimogawa et al, 2023). Of particular interest is the inner junction (IJ) that connects the B-tubule to the A-tubule at the inner face of each DMT (Yanagisawa et al, 2014, Dymek et al, 2019, Khalifa et al, 2020, Shimogawa et al, 2023).…”

“…Recent work in trypanosomes and other organisms has provided insight into proteins that mediate DMT assembly and function (Stoddard et al, 2018, Ichikawa et al, 2019, Imhof et al, 2019, Ma et al, 2019, Owa et al, 2019, Gui et al, 2021, Gui et al, 2022, Li et al, 2022, Kubo et al, 2023, Shimogawa et al, 2023). Of particular interest is the inner junction (IJ) that connects the B-tubule to the A-tubule at the inner face of each DMT (Yanagisawa et al, 2014, Dymek et al, 2019, Khalifa et al, 2020, Shimogawa et al, 2023). The IJ contains a non-tubulin filament together with microtubule inner proteins (MIPs) that interconnect with the IJ filament and each other, facilitating attachment of protofilament 10 in the B-tubule to protofilament 1 of the A-tubule (Yanagisawa et al, 2014, Dymek et al, 2019, Khalifa et al, 2020, Shimogawa et al, 2023) (Fig.…”

“…Of particular interest is the inner junction (IJ) that connects the B-tubule to the A-tubule at the inner face of each DMT (Yanagisawa et al, 2014, Dymek et al, 2019, Khalifa et al, 2020, Shimogawa et al, 2023). The IJ contains a non-tubulin filament together with microtubule inner proteins (MIPs) that interconnect with the IJ filament and each other, facilitating attachment of protofilament 10 in the B-tubule to protofilament 1 of the A-tubule (Yanagisawa et al, 2014, Dymek et al, 2019, Khalifa et al, 2020, Shimogawa et al, 2023) (Fig. 1A).…”

Microtubule inner proteins ofPlasmodiumare essential for transmission of malaria parasites

“…Like other eukaryotes, human infecting protozoan parasites can contain intriguing microtubule arrays ( 21, 22 ) and feature MIPs within axonemes ( 13, 23 ) and cytoplasmic microtubules ( 1, 21, 24 ). In Plasmodium spp., the causative agents of malaria in humans and many vertebrates (Figure 1A), cytoplasmic microtubules are closely associated to the membranes of the pellicle, the outer multi-membrane layer of the parasite that surrounds most of the parasite cytoplasm and consists of the parasite plasma membrane and the inner membrane complex (IMC) (Figure 1B).…”