Rsph4a is essential for the triplet radial spoke head assembly of the mouse motile cilia.

Motile cilia/flagella are essential for swimming and generating extracellular fluid flow in eukaryotes. Motile cilia harbor a 9+2 arrangement consisting of nine doublet microtubules with dynein arms at the periphery and a pair of singlet microtubules at the center (central pair). In the central system, the radial spoke has a T-shaped architecture and regulates the motility and motion pattern of cilia. Recent cryoelectron tomography data reveal three types of radial spokes (RS1, RS2, and RS3) in the 96 nm axoneme repeat unit; however, the molecular composition of the third radial spoke, RS3 is unknown. In human pathology, it is well known mutation of the radial spoke head-related genes causes primary ciliary dyskinesia (PCD) including respiratory defect and infertility. Here, we describe the role of the primary ciliary dyskinesia protein Rsph4a in the mouse motile cilia. Cryoelectron tomography reveals that the mouse trachea cilia harbor three types of radial spoke as with the other vertebrates and that all triplet spoke heads are lacking in the trachea cilia of Rsph4a-deficient mice. Furthermore, observation of ciliary movement and immunofluorescence analysis indicates that Rsph4a contributes to the generation of the planar beating of motile cilia by building the distal architecture of radial spokes in the trachea, the ependymal tissues, and the oviduct. Although detailed mechanism of RSs assembly remains unknown, our results suggest Rsph4a is a generic component of radial spoke heads, and could explain the severe phenotype of human PCD patients with RSPH4A mutation.

Effects of external strain on the regulation of microtubule sliding induced by outer arm dynein of sea urchin sperm flagella.

Oscillatory bending movement of eukaryotic flagella is powered by orchestrated activity of dynein motor proteins that hydrolyse ATP and produce microtubule sliding. Although the ATP concentration within a flagellum is kept uniform at a few millimoles per litre level, sliding activities of dyneins are dynamically coordinated along the flagellum in accordance with the phase of bending waves. Thus at the organellar level the dynein not only generates force for bending but also modulates its motile activity by responding to bending of the flagellum. Single molecule analyses have suggested that dynein at the molecular level, even if isolated from the axoneme, could alter the modes of motility in response to mechanical strain. However, it still remains unknown whether the coordinated activities of multiple dyneins can be modulated directly by mechanical signals. Here, we studied the effects of externally applied strain on the sliding movement of microtubules interacted with an ensemble of dynein molecules adsorbed on a glass surface. We found that by bending the microtubules with a glass microneedle, three modes of motility that have not been previously characterized without bending can be induced: stoppage, backward sliding and dissociation. Modification in sliding velocities was also induced by imposed bending. These results suggest that the activities of dyneins interacted with a microtubule can be modified and coordinated through external strain in a quite flexible manner, and that such a regulatory mechanism may be the basis of flagellar oscillation.