The small GTPase Arl13b regulates ciliary transmembrane protein localizations and anterograde IFT assembly stability.

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.

Tubulin polyglutamylation is a reversible post-translational modification, serving important roles in microtubule (MT)-related processes. Polyglutamylases of the tubulin tyrosine ligaselike (TTLL) family add glutamate moieties to specific tubulin glutamate residues, whereas as yet unknown deglutamylases shorten polyglutamate chains. First we investigated regulatory machinery of tubulin glutamylation in MT-based sensory cilia of the roundworm Caenorhabditis elegans. We found that ciliary MTs were polyglutamylated by a process requiring ttll-4. Conversely, loss of ccpp-6 gene function, which encodes one of two cytosolic carboxypeptidases (CCPs), resulted in elevated levels of ciliary MT polyglutamylation. Consistent with a deglutamylase function for ccpp-6, overexpression of this gene in ciliated cells decreased polyglutamylation signals. Similarly, we confirmed that overexpression of murine CCP5, one of two sequence orthologs of nematode ccpp-6, caused a dramatic loss of MT polyglutamylation in cultured mammalian cells. Finally, using an in vitro assay for tubulin glutamylation, we found that recombinantly expressed Myc-tagged CCP5 exhibited deglutamylase biochemical activities. Together, these data from two evolutionarily divergent systems identify C. elegans CCPP-6 and its mammalian ortholog CCP5 as a tubulin deglutamylase. The microtubule (MT)2 cytoskeleton plays critical roles in multiple cellular processes such as chromosome segregation, intracellular transport, cell morphogenesis, and polarity. Tubulin, which is the major protein subunit of MT fibers, exhibits polymorphic protein variation and undergoes a variety of unique post-translational modifications at its C-terminal tail, such as detyrosination/tyrosination (1, 2), polyglycylation (3), and polyglutamylation (4). These modifications regulate tubulin and MT function. Polyglutamylation enzymes add multiple glutamate moieties to specific glutamate residues within substrate proteins. Physiological roles of tubulin polyglutamylation for MT-related processes were reported recently (5, 6) and reviewed by Ikegami and Setou (7).Polyglutamylase enzymes have recently been identified as belonging to the tubulin tyrosine ligase-like (TTLL) protein family (8 -10) (supplemental Fig. 1). In contrast, the enzyme(s) underlying deglutamylation have not been discovered yet, although such proteins are suggested to be present in mouse brain neurons and in cultured cells (11,12).In this study, we employed the genetically tractable nematode, Caenorhabditis elegans, to identify genes that regulate tubulin polyglutamylation/deglutamylation. Specifically, we investigated tubulin post-translational modification in C. elegans sensory cilia, which are MT-based organelles that extend from the distal dendrite tips of 60 sensory neurons found in sensory organs named amphid (head) and phasmid (tail) sensilla (see Fig. 1A). In this system, we identified a cytosolic carboxypeptidase (ccpp) gene, ccpp-6, as a candidate tubulin deglutamylase gene, whose functional properties are opposite ...

SUMMARY Background Multiple intracellular transport pathways drive the formation, maintenance and function of cilia, a compartmentalised organelle associated with motility, chemo-/mechano-/photo-sensation, and developmental signaling. These pathways include cilium-based intraflagellar transport (IFT) and poorly understood membrane trafficking events. Defects in ciliary transport contribute to the aetiology of human ciliary disease such as Bardet-Biedl syndrome (BBS). In this study, we employ the genetically tractable nematode Caenorhabditis elegans to investigate if endocytosis genes function in cilium formation and/or the transport of ciliary membrane or ciliary proteins. Results Here we show that localisation of the clathrin light chain, AP-2 clathrin adaptor, dynamin and RAB-5 endocytic proteins overlaps with a morphologically discrete periciliary membrane compartment associated with sensory cilia. In addition, ciliary transmembrane proteins such as G protein-coupled receptors concentrate at periciliary membranes. Disruption of endocytic gene function causes expansion of ciliary and/or periciliary membranes as well as defects in the ciliary targeting and/or transport dynamics of ciliary transmembrane and IFT proteins. Finally, genetic analyses reveal that the ciliary membrane expansions in dynamin and AP-2 mutants require bbs-8 and rab-8 function, and that sensory signaling and endocytic genes may function in a common pathway to regulate ciliary membrane volume. Conclusions These data implicate C. elegans endocytosis proteins localized at the ciliary base in regulating ciliary and periciliary membrane volume, and suggest that membrane retrieval from these compartments is counter-balanced by BBS-8 and RAB-8-mediated membrane delivery.