Fluorescence Lifetime Imaging.

<p>(<b>A</b>) Simple kinetic scheme for decay of excited FRET probes that are either capable (F_Y^*) or not capable of FRET (F_N^*). The molecules can relax by fluorescence (<i>k_f</i>) or by FRET (<i>k_et</i>). (<b>B</b>) Various proportions of mTFP1 and mTFP1-Venus were mixed in solution and their fluorescence lifetimes analyzed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0027823#s2" target="_blank">Methods</a>. The concentration of mTFP1 is denoted as [T] and mTFP1-Venus as [T-V]. We verified that adding corresponding concentrations of pure mVenus to decreasing amounts mTFP1 did not change the fluorescence lifetime of mTFP1, showing that this lifetime did not depend on mTFP1 concentration and that mTFP1 did not undergo significant intermolecular FRET given the concentrations used (0.1 mg/mL). Error bars denote 95% confidence intervals.</p

Ratiometric FRET.

<p>(<b>A</b>) Simulations for how the ratio R depends on the FRET efficiency <i>φ_F</i> and the relative overlap of the donor fluorescence into the acceptor channel (<i>f_AD</i>/<i>f_DD</i>). The simulations were performed in MATLAB and are based on Eq. 11. Pseudo-color is indicative of z-axis (R) value. (<b>B</b>) Observed ratiometric FRET (y-axis) from various mixtures of GDP and GTP bound mTFP1-RaichuRhoA-mVenus. The proportion of GTP bound molecules is plotted on the x-axis. Data are representative of three independent experiments, and the observed ratio is based on the standard FRET channel (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0027823#s2" target="_blank">Methods</a>). Surface plots were not created to make the linear dependence clearer. (<b>C</b>) Observed ratiometric FRET from various mixtures of mTFP1 ([T]) and mTFP1-mVenus ([T-V]). The overlap index is a measure of how much mTFP1 fluorescence appears in the FRET channel (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0027823#s2" target="_blank">Methods</a>), and is proportional to the quantity <i>f_AD/f_DD</i> plotted in Panel A. Data are representative of three independent experiments. Pseudo-color is indicative of z-axis (R) value. (<b>D</b>) Observed ratiometric FRET, based on <i>R_alt</i>, from various mixtures of mTFP1 ([T]), mTFP1-mVenus ([T-V]), and mVenus. Error bars denote standard error from 3 independent experiments.</p

Kinetic Scheme for FRET Probe Activation.

<p>A forward enzyme F catalyzes the conversion of the FRET probe into an active state, and a reverse enzyme R catalyzes the conversion of the probe into an inactive state.</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

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

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