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

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

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

ARL13B associates with IFT-B complex via IFT46 and IFT74 interactions.

<p>(<b>A</b>) Number of proteins found to associate with SF (Strep-Flag)-tagged human ARL13B using TAP (tandem affinity chromatography) and SILAC (stable isotope labelling affinity chromatography) approaches in HEK293 cells. (<b>B</b>) ARL13B complexes possess IFT-B proteins. White number; average peptide count from 4 independent tandem-affinity purification experiments (details in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003977#pgen-1003977-t001" target="_blank">Table 1</a>). Blue number; average enrichment scores from 4 independent experiments employing a single purification/SILAC quantitative approach. TTC26 also known as DYF-13 in <i>C. elegans</i>, and TTC30A also known as IFT70 in <i>Chlamydomonas</i>. (<b>C</b>) Western blot from HEK293T cells transfected with Arl13b-Flag vector or Flag vector ‘mock’ control (Flag) showing that immunoprecipitated Flag-tagged ARL13B associates with IFT-B proteins (IFT88, IFT52) but not Inversin (INVS). (<b>D</b>) Dedicated yeast-two hybrid one-on-one analysis reveals direct interactions between human ARL13B and both IFT46 and IFT74. WL; minimal media lacking Trp and Leu. WLHA; minimal media lacking Trp, Leu, His and Ade.</p