Cilia are microtubule-based cell appendages, serving motility, chemo-/mechano-/photo- sensation, and developmental signaling functions. Cilia are comprised of distinct structural and functional subregions including the basal body, transition zone (TZ) and inversin (Inv) compartments, and defects in this organelle are associated with an expanding spectrum of inherited disorders including Bardet-Biedl syndrome (BBS), Meckel-Gruber Syndrome (MKS), Joubert Syndrome (JS) and Nephronophthisis (NPHP). Despite major advances in understanding ciliary trafficking pathways such as intraflagellar transport (IFT), how proteins are transported to subciliary membranes remains poorly understood. Using Caenorhabditis elegans and mammalian cells, we investigated the transport mechanisms underlying compartmentalization of JS-associated ARL13B/ARL-13, which we previously found is restricted at proximal ciliary membranes. We now show evolutionary conservation of ARL13B/ARL-13 localisation to an Inv-like subciliary membrane compartment, excluding the TZ, in many C. elegans ciliated neurons and in a subset of mammalian ciliary subtypes. Compartmentalisation of C. elegans ARL-13 requires a C-terminal RVVP motif and membrane anchoring to prevent distal cilium and nuclear targeting, respectively. Quantitative imaging in more than 20 mutants revealed differential contributions for IFT and ciliopathy modules in defining the ARL-13 compartment; IFT-A/B, IFT-dynein and BBS genes prevent ARL-13 accumulation at periciliary membranes, whereas MKS/NPHP modules additionally inhibit ARL-13 association with TZ membranes. Furthermore, in vivo FRAP analyses revealed distinct roles for IFT and MKS/NPHP genes in regulating a TZ barrier to ARL-13 diffusion, and intraciliary ARL-13 diffusion. Finally, C. elegans ARL-13 undergoes IFT-like motility and quantitative protein complex analysis of human ARL13B identified functional associations with IFT-B complexes, mapped to IFT46 and IFT74 interactions. Together, these findings reveal distinct requirements for sequence motifs, IFT and ciliopathy modules in defining an ARL-13 subciliary membrane compartment. We conclude that MKS/NPHP modules comprise a TZ barrier to ARL-13 diffusion, whereas IFT genes predominantly facilitate ARL-13 ciliary entry and/or retention via active transport mechanisms.
Protruding from most cells surfaces is a hair-like extension called the primary cilium. This organelle functions as a cellular antenna, receiving physical and chemical signals such as light, odorants, and molecules that coordinate cell growth, differentiation and migration. Underscoring their importance, cilium defects underlie an expanding spectrum of diseases termed ciliopathies, characterised by wide-ranging symptoms such as cystic kidneys, blindness and bone abnormalities. A key question is how ciliary proteins are targeted to and retained within cilia. The best understood system is intraflagellar transport (IFT), thought to ferry proteins between the ciliary base and tip. Also, ciliopathy protein modules organise protein diffusion barriers at the ciliary base transition zone (TZ). Despite major advances, it remains poorly understood how proteins are targeted to cilia, and ciliary membrane subdomains in particular. Here, we investigated how Joubert syndrome-associated ARL13B/ARL-13 is compartmentalized at subciliary membranes. Using C. elegans nematodes and mammalian cell experimental systems, we uncovered differential requirements for sequence motifs, IFT and ciliopathy modules in regulating ARL-13 ciliary restriction, mobility and compartment length. Also, we provide essential insight into how IFT and ciliopathy-associated protein complexes and modules influence ciliary membrane protein transport, diffusion across the TZ, the integrity of the ciliary membrane, and subciliary protein composition.