GEF activity of RINL for Rab5 subfamily proteins.

<p>(A–D) HEK293T cells expressing myc-Rab5b (A), Rab21 (B), Rab22 (C), or Rab31 (D) and FLAG-mock, RINL, RIN3, or Rabex-5 were metabolically radiolabeled with ³²P_i for 4 hours. Myc-Rab5 subfamily proteins were immunoprecipitated with an anti-myc monoclonal antibody, and nucleotides associating with each Rab protein were separated by thin-layer chromatography. The radioactivity of GTP and GDP was quantified, and the percentages (%) of each GTP-bound Rab are shown. Total lysates (bottom) and immunoprecipitated samples (middle) from the radiolabeled cells were separated by SDS-PAGE and immunoblotted with anti-FLAG and anti-myc antibodies, respectively. *p<0.05 vs. mock-transfected cells. (E) Myc-Rab3a, 7a, or 11a was co-transfected with FLAG-mock or RINL into HEK293T cells. The percentages of each GTP-bound Rab member in the metabolically radiolabeled cells are shown as described in (A). (F) Myc-Rab5b was co-transfected with wild type (WT), or the DP_AA or YT_AA mutant of FLAG-RINL into HEK293T cells. The percentages of GTP-Rab5b in the metabolically radiolabeled cells are shown as described in (A). Total lysates (bottom) and immunoprecipitated samples (middle) from the radiolabeled cells were separated by SDS-PAGE and immunoblotted with anti-FLAG and anti-myc antibodies, respectively. All data were obtained from more than three independent experiments and are shown as the mean ± S.E. (error bars). **p<0.01 vs. mock-transfected cells.</p

Identification of odin/Anks1a as an interacting molecule with RINL.

<p>(A) HeLa cell lysates were immunoprecipitated with normal rat IgG or anti-odin antibody, followed by immunoblotting with antibodies as indicated. (B) FLAG-RIN family or FLAG-mock were transfected into HEK293T cells. Cells lysates were immunoprecipitated with anti-FLAG antibody, followed by immunoblotting with antibodies as indicated. (C) FLAG-RINL and the indicated deletion mutants of myc-odin were transiently transfected into HEK293T cells. Cells lysates were immunoprecipitated with anti-myc antibody, followed by immunoblotting with antibodies as shown. (D) The indicated deletion mutants of FLAG-RINL were transiently transfected into HEK293T cells. Cells lysates were immunoprecipitated with anti-FLAG antibody, followed by immunoblotting with antibodies as indicated. (E) Myc-odin and V5-RINL were co-transfected with FLAG-tagged constitutively active (CA, lanes 2 and 4) or mock (lanes 1 and 3) into HEK293T cells. Cell lysates were immunoprecipitated with anti-myc antibody, followed by immunoblotting with antibodies as indicated. Aliquots of total lysates were also immunoblotted with antibodies as indicated.</p

RINL forms a ternary complex with odin and EphA8, and RINL affects the degradation of the EphA8 receptor.

<p>(A) HEK293T cells were co-transfected with EphA8-FLAG, HaloTag-odin, and myc-RINL (+) or mock (−) plasmids as indicated, and cell lysates were immunoprecipitated with anti-myc antibody. Immunoprecipitated fractions and total lysates were immunoblotted with antibodies as indicated. (B and C) HeLa cells were transfected with EphA8-FLAG and myc-RINL or mock plasmids, and total lysates were immunoblotted with antibodies as indicated. ΔSH2; SH2 domain-deleted mutant. The data obtained from three independent experiments are shown (C) as the mean ± S.E. (error bars). *, p<0.05 vs. mock-transfected cells. N.S., not significant. (D and E) HEK293T cells were transfected with EphA8-FLAG and myc-RINL or mock plasmids, and total lysates were immunoblotted with antibodies as indicated. WT; wild type. The data obtained from three independent experiments are shown (E) as the mean ± S.E. (error bars). *, p<0.05 vs. mock-transfected cells. (F and G) HeLa cells were transfected with 30 pmol scrambled negative control (NC) or RINL-specific siRNA. 24 hours after the transfection, these cells were transfected with EphA8-FLAG and siRNA-resistant FLAG-RINL, and incubated for 48 hours. Total proteins from the cell lysates were subjected to SDS-PAGE and immunoblotted (IB) with antibodies as indicated. The data obtained from three independent experiments are shown (G) as the mean ± S.E. (error bars). **, p<0.01 vs. NC-transfected cells. *, p<0.05 vs. siRNA-transfected cells with FLAG-mock plasmid transfection. (H and I) HeLa cells were transfected with EphA8-FLAG and FLAG-RINL (+, lanes 2–5) or mock plasmids (−, lane 1), and total lysates were immunoblotted with antibodies as indicated. These cells were non-treated (NT, lanes 1 and 2), or treated with MG132 (20 µM, lane 3), leupeptin (100 µg/ml, lane 4), or bafilomycin (200 nM, lane 5) for 3 hours. Total lysates were immunoblotted with antibodies as indicated. The data obtained from three independent experiments are shown (I) as the mean ± S.E. (error bars). **, p<0.01 vs. mock-transfected cells. *, p<0.05 vs. non-treatment cells transfected with RINL.</p

Structure of RINL.

<p>(A) Diagram of the structural features of RIN family members. The lower numbers represent the amino acid residues. (B) FLAG-RIN1, RIN2, RIN3, and RINL were transiently co-transfected with myc-amphiphysin II (amph II) into HEK293T cells. Cells lysates were immunoprecipitated with anti-FLAG antibody, followed by immunoblotting with anti-myc and anti-FLAG antibodies. Total lysates were immunoblotted with anti-myc antibody. (C) Cell lysates from HEK293T cells were applied to a Superdex 200 Prep Grade gel filtration column. The elution position was compared with those of the globular size markers (upper panel). The fractions (0.5 ml) eluted from the column and total lysate (tot.) were analyzed by SDS-PAGE, and proteins were immunoblotted with anti-RINL antibody.</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

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

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