CCDC66 regulation of cytoskeleton and cilia stability is important for signaling and epithelial organization.

The primary cilium is a conserved, microtubule-based organelle that transduces signaling pathways essential for development and homeostasis. It dynamically assembles and disassembles in response to intrinsic and extrinsic stimuli while maintaining remarkable structural stability and tightly regulated length. The mechanisms underlying this stability and length control are not well understood. Here, we characterized CCDC66, a microtubule-associated protein linked to ciliopathies, as an important regulator of cilium maintenance and disassembly in mouse epithelial cells. Live imaging revealed that cilia in CCDC66-depleted cells frequently fluctuate in length and exhibit increased cilium disassembly and ectocytosis. Phenotypic rescue experiments and in vitro assays showed that microtubule stabilization activity of CCDC66 is required for these functions. Temporal proximity mapping identified potential new regulators and molecular pathways involved in cilium disassembly. Further characterization revealed actin cytoskeleton and vesicular trafficking as additional mechanisms by which CCDC66 may mediate its ciliary functions. Finally, depletion of CCDC66 compromised Hedgehog and Wnt pathway activation and disrupted epithelial cell organization and polarity in two- and three-dimensional cultures. Collectively, we showed that CCDC66 regulates both ciliary and non-ciliary processes through diverse mechanisms involving microtubules, actin, and vesicular trafficking, providing insights into the pathologies associated with CCDC66.