Ccdc66 regulates primary cilium stability, disassembly and signaling important for epithelial organization

The primary cilium is a conserved, microtubule-based organelle that transduces signaling pathways essential for development and homeostasis. It is a dynamic structure that assembles and disassembles in response to intrinsic and extrinsic stimuli while maintaining remarkable stability and tightly controlled length. Although cilium assembly is well-understood, less is known about the molecular players and pathways governing their stability, length and disassembly. Here, we elucidated the function of Ccdc66, a microtubule-associated protein linked to ciliopathies, in cilium maintenance and disassembly in mouse epithelial cells. We found that Ccdc66 depletion disrupts cilium disassembly, length and stability, but does not affect assembly in these cells. Live imaging of these processes revealed that cilia in Ccdc66-depleted cells frequently fluctuate in length and exhibit increased ectocytosis from the cilium tip. Phenotypic rescue experiments and in vitro microtubule stabilization assays showed that Ccdc66 mediates these functions via regulating the stability of microtubules. Temporal proximity mapping of CCDC66 identified potential new regulators and molecular pathways involved in cilium disassembly. Additionally, depletion of CCDC66 compromised Hedgehog and Wnt pathway activation and disrupted epithelial cell organization and polarity in two-dimensional and three-dimensional cultures. Collectively, our results define Ccdc66 as a new microtubule-stabilizing factor that regulates cilium stability and disassembly, providing insights into the mechanisms of cilium homeostasis and the pathologies associated with Ccdc66.