Takagishi, M. | bioRxiv Science

bioRxiv2025

Molecular organization of the distal tip of vertebrate motile cilia

The beating of cilia on multi-ciliated cells (MCCs) is essential for normal development and homeostasis in animals. But while the structure and function of basal bodies and axonemes have received significant attention recently, the distal tips of MCC cilia remain relatively poorly defined. Here, we characterize the molecular organization of the distal tip of vertebrate MCC cilia, characterizing two distinct domains occupied by distinct protein constituents. Using frog, mouse, and human MCCs, we find that two largely uncharacterized proteins, Ccdc78 and Ccdc33 occupy a previously undefined region at the extreme distal tip, and these are required for the normal organization of all other known tip proteins. Ccdc78 and Ccdc33 each display robust microtubule-bundling activity both in vivo and in vitro, yet each is independently required for normal length regulation of MCC cilia. Moreover, loss of each protein elicits a distinct pattern of defective cilia beating and resultant fluid flow. Thus, two previously undefined proteins form a key module essential for organizing and stabilizing the distal tip of motile cilia in vertebrate MCCs. We propose that these ill-defined proteins represent potential disease loci for motile ciliopathies.

cell biology↗

bioRxiv2023

Kif6 regulates cilia motility and polarity in brain ependymal cells

Ependymal cells, lining brain ventricular walls, display tufts of cilia that beat in concert promoting laminar Cerebrospinal fluid (CSF) flow within brain ventricles. The ciliary axonemes of multiciliated ependymal cells display a 9+2 microtubule array common to motile cilia. Dyneins and kinesins are ATPase microtubule motor proteins that promote the rhythmic beating of cilia axonemes. Despite common consensus about the importance of axonemal dynein motor proteins, little is known about how Kinesin motors contribute to cilia motility. Here, we define the function of Kinesin family member 6 (Kif6) using a mutation that lacks a highly conserved C-terminal tail domain (Kif6p.G555fs) and which displays progressive hydrocephalus in mice. An analogous mutation was isolated in a proband displaying macrocephaly, hypotonia, and seizures implicating an evolutionarily conserved function for Kif6 in neurodevelopment. We find that loss of Kif6 function caused decreased ependymal cilia motility and subsequently decreased fluid flow on the surface of brain ventricular walls. Kif6 protein was localized at ependymal cilia and displayed processive motor movement (676 nm/s) on microtubules in vitro. Loss of the Kif6 C-terminal tail domain did not affect the initial ciliogenesis in vivo, but did result in defects in cilia orientation, the formation of robust apical actin networks, and stabilization of basal bodies at the apical surface. This suggests a novel role for the Kif6 motor in maintenance of ciliary homeostasis of ependymal cells. Summary statementWe found that Kif6 is localized to the axonemes of ependymal cells. In vitro analysis shows that Kif6 moves on microtubules and that its loss mice decrease cilia motility and cilia-driven flow, resulting in hydrocephalus.

developmental biology↗