Protein-protein interactions within the ciliary tip module depend on different functional domains and are disrupted by Joubert syndrome variants

Abstract

Primary cilia are microtubule based organelles found on the surface of many eukaryotic cell types. They are involved in various signalling pathways, for example in hedgehog signalling. Defects to primary cilia underly a subset of disorders, together termed ciliopathies, including the neurodevelopmental disorder Joubert syndrome (1). Ciliary length is directly determined by axonemal microtubule length and is suggested to be crucial for proper cilia functioning, as ciliopathy patient-derived cells typically contain cilia with abnormal lengths (2–4). A recent study characterized how proteins ARMC9, CCDC66, CEP104, mCrescerin1 and CSPP1, the core components of the ciliary tip module, directly affect microtubule dynamics through in vitro reconstitutions (5,6). Unpublished cryo-ET data from our lab shows that these proteins form a single complex at microtubule plus ends. Yet the exact geometry of individual proteins within this complex and how interactions are facilitated remains unclear, especially when proteins are localized on opposite sides of the microtubule wall. In this study, we further map protein-protein interactions within the tip module. By performing multiple co-immunoprecipitations assays, we narrowed down interactions between (I)ARMC9 and mCrescerin1, (II) ARMC9 and CSPP1 and (III) CEP104 and mCrescerin1 to a specific functional domain or protein region and confirmed from literature that interaction of CEP104 with CCDC66 or CSPP1 is dependent on its jelly roll domain. Additionally, we showed that mutations linked to the ciliopathy Joubert syndrome affect ARMC9-mCrescerin1, CEP104-CCDC66 and CEP104-CSPP1 interactions, highlighting the biological relevance of protein-protein interactions within the tip module.