Abstract
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.
Highlights
Primary cilia are organized into specific subcompartments, defined by distinct ultrastructure, protein and lipid compositions, and include the basal body (BB), the adjacent transition zone (TZ), and axonemal regions consisting of doublet and singlet microtubules [1]
We found that the C. elegans ARL-13 proximal ciliary domain in amphid and phasmid channel cilia corresponds to the middle segment (MS) and does not include the transition zone (TZ); ARL-13 is juxtaposed to MKSR-1/B9D1 at the TZ, and OSM-6/IFT52 basal body signals are separated from ARL-13 signals by an,1 mm ‘gap’, which corresponds to the TZ length (Figure 1B)
We show that ARL13B/ARL-13 is compartmentalised within an evolutionarily conserved Inversin-like ciliary membrane subdomain and requires palymitoylation modification and RVVP motifs to prevent inappropriate targeting of C. elegans ARL-13 to the nucleus and distal ciliary regions
Summary
Primary cilia are organized into specific subcompartments, defined by distinct ultrastructure, protein and lipid compositions, and include the basal body (BB), the adjacent transition zone (TZ), and axonemal regions consisting of doublet and singlet microtubules [1]. Multiple proteins linked to ciliopathies such as Meckel-Gruber syndrome (MKS), nephronophthisis (NPHP), oral-facial digital syndrome (OFD) and Joubert syndrome (JS) are sequestered within specific ciliary subdomains These include at least twenty MKS/NPHP/JSassociated proteins concentrated at the TZ, multiple ciliopathy proteins targeted at the BB such as OFD1, and proteins such as NPHP2/INVS confined to a proximal ciliary subdomain called the Inversin compartment [3,4,5]. Many of these proteins regulate cilium-based signaling (e.g., via Sonic hedgehog and Wnt) that probably occurs at specific subciliary domains
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