For virtually all cilia and eukaryotic flagella, the second messengers calcium and cyclic adenosine monophosphate are implicated in modulating dynein- driven microtubule sliding to regulate beating. Calmodulin (CaM) localizes to the axoneme and is a key calcium sensor involved in regulating motility. Using immunoprecipitation and mass spectrometry, we identify members of a CaM-containing complex that are involved in regulating dynein activity. This complex includes flagellar-associated protein 91 (FAP91), which shares considerable sequence similarity to AAT-1, a protein originally identified in testis as an A-kinase anchor protein (AKAP)– binding protein. FAP91 directly interacts with radial spoke protein 3 (an AKAP), which is located at the base of the spoke. In a microtubule sliding assay, the addition of antibodies generated against FAP91 to mutant axonemes with reduced dynein activity restores dynein activity to wild-type levels. These combined results indicate that the CaM- and spoke-associated complex mediates regulatory signals between the radial spokes and dynein arms.

Structural and functional analyses of artificial micro RNA (amiRNA) mutants reveal that the CSC plays a role not only in generating wild-type motility, but also in assembly of at least a subset of radial spokes. This study also produced the unexpected finding that, contrary to current belief, the radial spokes may not be homogeneous.

This study reveals the 3D structure of the CSC and its connections to three major axonemal complexes involved in dynein regulation, including the distal radial spoke and the nexin-DRC. The findings corroborate radial spoke heterogeneity and suggest a unique role for the distal spoke in calcium-mediated signal transduction and flagellar motility.

SummaryCalmodulin and PF6 are components of a complex that localizes to the C1 microtubule of the flagellar central apparatus