Abstract Radial spokes (RS), the T-shaped, multiprotein complexes of motile cilia, transmit regulatory signals from the central apparatus to the outer doublet complexes, including inner dynein arms. In the vast majority of ciliated species, RSs assemble as repeats of triplets (RS1-RS2-RS3), and each spoke is associated with a different subset of inner dynein arms. Studies in Chlamydomonas and mice sperm flagella led to the identification of RS proteins (RSPs) and revealed that some structural components are either RS1- or RS2-specific. In contrast, the protein composition of RS3 remains largely unknown. We used the ciliate Tetrahymena thermophila to investigate the protein composition of individual RSs, including the poorly characterized RS3. The Tetrahymena genome encodes three RSP3 paralogs. Using engineered RSP3 knock-out mutants and previously studied RS mutants with CFAP61 , CFAP91 , or CFAP206 deletion and complementary approaches, including bioinformatics, total ciliome comparisons, and cryo-electron tomography with subtomogram averaging, we identified Tetrahymena RSP orthologs and solved the composition of individual RSs, showing their subunit heterogeneity. We found that RSP3 proteins are components of RS1 and RS2 but not RS3. Based on the presence of the RSP3 paralog, we distinguished sub-types of RS1 (RSP3A- or RSP3B-containing) and RS2 spokes (RSP3B- or RSP3C-containing). We identified novel RS-associated proteins, including several enzymes that may locally regulate ADP/ATP levels, GMP-recycling-related enzymes, and enzymes regulating phosphorylation levels. These discoveries will help to better understand the molecular mechanism(s) that regulate cilia beating and overall cilia metabolism. Impact Statement Identification of the subtypes of RS1 and RS2 spokes and RS1-3-specific RSPs. Discovery of the novel radial spoke structural components and RS-associated enzymes regulating ADP/ATP ratio and protein phosphorylation.

Heterogeneity of radial spokes structural components and associated enzymes inTetrahymenacilia