CCDC66 regulates primary cilium length and signaling competence via multi-site interactions with transition zone and axonemal proteins

The primary cilium is a conserved microtubule-based organelle that serves as a hub for many signaling pathways. It functions as part of the centrosome/cilium complex, which also contains the basal body and the centriolar satellites. Little is known about the mechanisms by which the microtubule-based axoneme of the cilium is assembled with proper length and structure, particularly in terms of the activity of microtubule-associated proteins (MAPs) and the crosstalk between the different compartments of the centrosome/cilium complex. Here, we analyzed CCDC66, a MAP implicated in cilium biogenesis and ciliopathies affecting eye and brain. Live-cell imaging revealed that CCDC66 compartmentalizes between centrosomes, centriolar satellites, and the ciliary axoneme and tip during cilium assembly and disassembly. CCDC66 loss-of-function in human cells causes defects in cilium assembly, length and morphology. Notably, CCDC66 interacts with the MAPs and ciliopathy proteins CEP104 and CSPP1 and cooperates with them during axonemal length regulation. Moreover, CCDC66 interacts with the transition zone protein CEP290 selectively at the centriolar satellites. Its loss disrupts basal body recruitment of transition zone proteins and IFT-B machinery and causes defective Hedgehog signaling. Overall, our results establish CCDC66 as a multifaceted regulator of the primary cilium, and propose a mechanistic insight into how the cooperation of ciliary MAPs as well as subcompartments ensures assembly of a functional cilium.