Dissecting the Architecture of the Leishmania Paraflagellar Rod

Motile cilia are hair-like appendages on cells, whose beating movement facilitates locomotion of single-cell organisms, sperm cells, and the flow of extracellular fluid across tissues of the airways, brain and female reproductive tract. Although the core architecture of the cilium is conserved from protists to humans, molecular and structural differences exist both between species and, in mammals, between various tissues within a species, perhaps to accommodate motility in different media. The radial spoke (RS) is an example for a large ciliary complex that its presence is conserved, but its composition and structure diverged in cilia of various cell types. The RS is crucial for generation and maintenance of rhythmic ciliary beating, but its roles in facilitating cilia motility are poorly understood. We produced recombinant complexes of 9-12 proteins that recapitulate the “head” region of the RS of the single-cell algae Chlamydomonas, human sperm, and human ciliated epithelia. We obtained high-resolution structures of our three synthetic complexes using cryo-EM and compared them, identifying structural features in RS heads that are conserved between species, such as an architecture of intertwined proteins and a negatively-charged surface, as well as features that changed between single-cell organisms and mammals, such as the shape of the surface. Supported by motility analyses of Chlamydomonas mutants that were perturbed in the RS head, the structural characteristics of RS heads reveal important principles that underly the mechanism of ciliary beating and explain the necessity for modifications in RS composition and form among cilia that beat in different environments.