Endocytosis Genes Facilitate Protein and Membrane Transport in C. elegans Sensory Cilia

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

Active Transport and Diffusion Barriers Restrict Joubert Syndrome-Associated ARl 13B/ARL-13 to an Inv-like Ciliary Membrane Subdomain

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Joubert syndrome Arl13b functions at ciliary membranes and stabilizes protein transport in Caenorhabditis elegans

The small ciliary G protein Arl13b is required for cilium biogenesis and sonic hedgehog signaling and is mutated in patients with Joubert syndrome (JS). In this study, using Caenorhabditis elegans and mammalian cell culture systems, we investigated the poorly understood ciliary and molecular basis of Arl13b function. First, we show that Arl13b/ARL-13 localization is frequently restricted to a proximal ciliary compartment, where it associates with ciliary membranes via palmitoylation modification motifs. Next, we find that loss-of-function C. elegans arl-13 mutants possess defects in cilium morphology and ultrastructure, as well as defects in ciliary protein localization and transport; ciliary transmembrane proteins abnormally accumulate, PKD-2 ciliary abundance is elevated, and anterograde intraflagellar transport (IFT) is destabilized. Finally, we show that arl-13 interacts genetically with other ciliogenic and ciliary transport–associated genes in maintaining cilium structure/morphology and anterograde IFT stability. Together, these data implicate a role for JS-associated Arl13b at ciliary membranes, where it regulates ciliary transmembrane protein localizations and anterograde IFT assembly stability

RVVP and palmitoylation modification motifs prevent targeting of ARL-13 to ciliary distal segments and the nucleus.

<p>(<b>A–E</b>) Shown are worms expressing a GFP-tagged ARL-13 sequence variant alone (left-hand images) or together with a CHE-13/IFT57::mCherry transgene (right-hand cilium images). Note that CHE-13 ciliary levels are highly reduced in Δ285–370 and ΔRVVP variants. rPal; replacement of N-terminal palmitoylation modification motif cysteines with Ser-Ala <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003977#pgen.1003977-Cevik1" target="_blank">[35]</a>. prs; proline-rich sequence. DS; distal segment. MS; middle segment. TZ; transition zone. BB; basal body. N; nucleus. Bars; 1 µm. (<b>F</b>) Plots of ARL-13 compartment length in phasmid cilia, at all larval stages, for worms expressing the indicated GFP-tagged ARL-13 variant or wild-type (WT) protein.</p

Differential requirements of ciliary transport and ciliopathy modules for ARL-13 ciliary compartmentalisation.

<p>(<b>A</b>) Phasmid cilia of worms expressing ARL-13::GFP in the indicated genotype. Arrowheads; transition zone (TZ). MS; middle segment. BB; basal body. PCM; periciliary membrane (bracket). Bars; 1 µm. (<b>B</b>) Box and whisker plot distribution of ARL-13 signal ratio in the cilium versus total (cilium+PCM). Measurements represent absolute signal intensities (arbitrary units) within both compartments, adjusted for background. (<b>C</b>) Box and whisker (min to max) distribution plots of ARL-13::GFP ciliary compartment length in phasmid neurons of the indicated mutant genotype. *p<0.001 (vs WT).</p

ARL-13/ARL13b localisation and mobility within an Inversin-like ciliary compartment.

<p>(<b>A</b>) Staining of mouse oviduct and tracheal tissue for endogenous Arl13b and acetylated tubulin shows proximal ciliary enrichment of Arl13b. Graph; line intensity profiles of Arl13b and acetylated tubulin (AcTub) signals from cilia denoted by white arrows. Bars; 5 µm (<b>B</b>) Co-expression of ARL-13::GFP with CHE-13/IFT57::mCherry, or ARL-13::tdTomato with either OSM-6::GFP or MKSR-1/B9D1::GFP, show that <i>C. elegans</i> ARL-13 is excluded from the transition zone (TZ). DS; distal segment. MS; middle segment. BB; basal body. PCMC; periciliary membrane compartment. Bars; 1 µm. (<b>C</b>) Staining of human hTERT-RPE1 cells shows that endogenous ARL13B does not colocalise with endogenous RPGRIP1L at the TZ. Bar; 10 µm (<b>D</b>) Phasmid cilia from L1 worms co-expressing ARL-13::GFP with CHE-13/IFT57::mCherry show that the ARL-13 compartment extends to the ciliary tips in young larva. Graph shows ARL-13::GFP, KAP-1::GFP (kinesin-II subunit) and OSM-6/IFT52::GFP ciliary compartment lengths in larval and adult stages of transgenic worms. Bar; 1 µm. (<b>E</b>) Fluorescence recovery after photobleaching (FRAP) curves after quenching 100%, or proximal-most 40%, of ARL-13::GFP ciliary signals in wild-type phasmid neurons. Signal ratio (au; arbitrary units) calculated from the average intensity of ARL-13 signal in the photobleached region compared to the non-photobleached region. All measurements are background subtracted and normalised to a pre-bleach ratio of 1.0. Each data point reports mean ± SEM. (<b>F</b>) Time-lapse images taken from a recording of an amphid channel cilium from worms expressing ARL-13::GFP show processive retrograde movement of an ARL-13::GFP-associated particle. Kymograph and associated schematic derived from one such recording show multiple moving anterograde and retrograde particles. Bar; 1 µm.</p

Identification of human ARL13B complex proteins.

<p>Shown are average tandem affinity purification (TAP) peptide counts (4 independent experiments; details in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003977#pgen.1003977.s021" target="_blank">Table S2</a>) and SILAC enrichment factors (4 independent experiments; details in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003977#pgen.1003977.s022" target="_blank">Table S3</a>) of proteins co-immunoprecipitating with SF-tagged ARL13B(WT) or ARL13B(T35N). This list contains all 47 proteins uncovered by the TAP experiments, with an average peptide count >1.5. Note only 6 of these proteins were not detected (nd) using the more sensitive SILAC approach.</p