CCDC66 regulates primary cilium length and signaling via interactions with transition zone and axonemal proteins

Abstract

The primary cilium is a 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 ciliary axoneme 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. Live-cell imaging revealed that CCDC66 compartmentalizes between centrosomes, centriolar satellites, and ciliary axoneme and tip during cilium biogenesis. CCDC66 depletion in human cells causes defects in cilium assembly, length and morphology. Notably, CCDC66 interacts with the ciliopathy-linked MAPs CEP104 and CSPP1 and regulates axonemal length and Hedgehog pathway activation. Moreover, CCDC66 is required for the basal body recruitment of transition zone proteins and IFT-B machinery. Overall, our results establish CCDC66 as a multifaceted regulator of the primary cilium and propose insight into how ciliary MAPs and subcompartments cooperate to ensure assembly of functional cilia.

A network of interacting ciliary tip proteins with opposing activities imparts slow and processive microtubule growth

“…Based on our own and previously published co-immunoprecipitation experiments, the interactions of CCDC66 and CSPP1 with CEP104 depended on its jelly-roll (JR) domain (Fig. S2C) (Frikstad et al, 2019; Odabasi et al, 2023). The deletion of this domain abolished the ability of both proteins to recruit CEP104 to MTs (Fig.…”

“…Since recent studies described functional and biochemical interactions between CEP104, CCDC66, CSPP1 and ARMC9 (Frikstad et al, 2019; Latour et al, 2020; Odabasi et al, 2023), we next tested in vitro the combinations of these proteins. We found that in the presence of 15 nM CCDC66, already 2 nM CEP104 was sufficient to occasionally block MT plus end outgrowth from the seed (Fig.…”

“…Depletion of any of these proteins causes ciliary defects, and mutations in most of them lead to a primary ciliopathy called Joubert syndrome, characterized by brain malformations, breathing problems or more severe developmental disorders (Akizu et al, 2014; Hildebrandt et al, 2011; Latour et al, 2020; Shaheen et al, 2014; Srour et al, 2015; Tuz et al, 2014; Van De Weghe et al, 2017). The proteins comprising the ciliary tip module interact with MTs and with each other, and although they do not form a stoichiometric complex, they are all important for controlling ciliary length and the signaling pathways dependent on primary cilia (Conkar et al, 2017; Das et al, 2015; Frikstad et al, 2019; Jiang et al, 2012; Latour et al, 2020; Odabasi et al, 2023; Patzke et al, 2006; Van De Weghe et al, 2017). In addition to their roles in cilia, some of the tip module proteins also associate with centrioles, centrosomes, centriolar satellites and cytoplasmic MTs, where they participate in cilia-independent processes, such as cell division (Batman et al, 2022; Conkar et al, 2019; Patzke et al, 2006; Ryniawec et al, 2023; Zhu et al, 2015).…”

“…ARMC9 and TOGARAM1 have been localized to motile cilia, where they were found to have opposite functions in regulating MT length (Louka et al, 2018; Perlaza et al, 2022), though mutants of both proteins make mammalian primary cilia shorter (Latour et al, 2020). Finally, CSPP1 and CEP104 interact with CCDC66, and co-depletion of CCDC66 with either CSPP1 or CEP104 results in further reduction of ciliary length compared to the removal of either CSPP1 or CEP104 alone (Odabasi et al, 2023).…”

The miR‐669a‐5p/G3BP/HDAC6/AKAP12 Axis Regulates Primary Cilia Length

“…[ 7 ] Dysregulation of cilium length causes multiple types of organ dysfunction, leading to a class of diseases called ciliopathies. [ 8 ] Therefore, understanding how cilium length is regulated will provide new perspectives for the treatment and prevention of ciliopathies.…”