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Motile cilia and flagella are built from stable populations of doublet microtubules that comprise their axonemes. Their unique stability is brought about, at least in part, by a network ofMicrotubuleInnerProteins (MIPs) found in the lumen of their doublet microtubules. Rib72A and Rib72B were identified as microtubule inner proteins (MIPs) in the motile cilia ofTetrahymena thermophila. Loss of these proteins leads to ciliary defects and loss of multiple MIPs. We performed mass spectrometry coupled with proteomic analysis and bioinformatics to identify the MIPs lost inRIB72A/Bknockout (KO)Tetrahymenacells. From this analysis we identified a number of candidate MIPs and pursued one, Fap115, for functional characterization. We find that loss of Fap115 results in disrupted cell swimming and aberrant ciliary beating. Cryo-electron tomography reveals that Fap115 localizes to MIP6a in the A-tubule of the doublet microtubules. Overall, our results highlight the complex relationship between MIPs, ciliary structure, and ciliary function.

Basal bodies (BBs) are microtubule-based organelles that template and stabilize cilia at the cell surface. Centrins ubiquitously associate with BBs and function in BB assembly, maturation, and stability. Human POC5 (hPOC5) is a highly conserved centrin-binding protein that binds centrins through Sfi1p-like repeats and is required for building full-length, mature centrioles. Here, we use the BB-rich cytoskeleton ofTetrahymena thermophilato characterize Poc5 BB functions.TetrahymenaPoc5 (TtPoc5) uniquely incorporates into assembling BBs and is then removed from mature BBs prior to ciliogenesis. Complete genomic knockout ofTtPOC5leads to a significantly increased production of BBs yet a markedly reduced ciliary density, both of which are rescued by reintroduction of TtPoc5. A secondTetrahymena POC5-like gene,SFR1, is similarly implicated in modulating BB production. WhenTtPOC5andSFR1are co-deleted, cell viability is compromised, and levels of BB overproduction are exacerbated. Overproduced BBs display defective transition zone formation and a diminished capacity for ciliogenesis. This study uncovers a requirement for Poc5 in building mature BBs, providing a possible functional link betweenhPOC5mutations and impaired cilia.SUMMARY STATEMENTLoss ofTetrahymena thermophilaPoc5 reveals an important role for this centrin-binding protein in basal body maturation, which also impacts basal body production and ciliogenesis.