Azi1 localises to centriolar satellites and the transition zone, and traffics along microtubules.

<p>(A–F) hTERT-RPE1 cells, (A) stained with anti-polyglutamylated tubulin (PGT), marking the cilium and basal body, but absent from the transition zone and anti-AZI1 (Abcam; ab110018). AZI1 localises to the transition zone (TZ). (B) Average intensity profiling confirms localisation of AZI1 at the TZ (n = 30). (C) AZI1 localisation at the TZ was further confirmed by co-staining with anti-NPHP1, which marks the TZ and anti-acetylated α-Tubulin (AcTub) marking the axoneme. (D) Average intensity profiles show AZI1 colocalises with NPHP1 (n = 10). (E) Centriolar satellite marker PCM1 also localises to the TZ, identified by the absence of anti-PGT staining between the axoneme and the basal body. (F) Average intensity profiling shows PCM1 localisation to the TZ (n = 12). (A, C, E) Enlargements highlight the cilium and show separate channels. Arrowheads highlight the TZ. Below is an intensity graph, highlighting the enrichment of AZI1 (A, C) or PCM1 (E) at the transition zone. (B, D, F) Plotted is mean +/− SEM. BB: basal body, Ax: axoneme, AU: arbitrary units. (G) Azi1-GFP (green) traffics along microtubules marked by Map4-RFP (grayscale) in NIH-3T3 cells. Images were taken at 600 ms intervals (t: time, s: seconds). (H) Anti-acetylated α-Tubulin marks the ciliary axoneme, basal bodies and the centrosomes of unciliated cells. Anti-Azi1 staining is more intense at the basal body in ciliated cells (arrowheads) than the centrosome of unciliated cells (arrows). Mean intensity of pericentrosomal Azi1 staining is quantified in (I). * <i>P</i><0.05, Student's t-test, n = 139 cells, two independent experiments. (J) AZI1 protein levels (anti-Azi1 SF91) in unsynchronised (us) or serum starved (ss) hTERT-RPE cells. Levels of Azi1 (arrow) remain unchanged when cells are serum starved to induce ciliogenesis. A presumed non-specific band at 150 kDa also detected by anti-Azi1 SF91, is more prominent in human cells. Scale bars represent 10 µm (A, C, E and H) or 1 µm (G).</p

Mutant spermatids show defective manchette structure, and abnormal head morphologies, suggesting defects in IMT.

<p>(A and B) TEM of elongating spermatids, highlighting the manchette with brackets. The manchette forms in <i>Azi1^Gt/Gt</i> spermatids, although it often appears kinked (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003928#pgen.1003928.s007" target="_blank">Figure S7A</a>), or is misnucleated away from the spermatid head (B, black arrows, also see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003928#pgen.1003928.s007" target="_blank">Figure S7B</a>). (C–F) Centrioles and the head tail coupling apparatus (HTCA) in early (C and D) or later stage (E and F) elongating spermatids from control (<i>Azi1^+/+</i> or <i>Azi1^Gt/+</i>) (C and E) or <i>Azi1^Gt/Gt</i> testes (D and F). The centrioles implant normally at the early stages of <i>Azi1^Gt/Gt</i> spermatid elongation, and some normal looking later stage HTCA can be found (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003928#pgen.1003928.s008" target="_blank">Fig. S8D</a>), although in many cases late stage HTCA are misaligned, with the implantation fossa or centrioles off-centre (F, brackets and arrow). (G–J) Elongating spermatids prepared by testes squashes from <i>Azi1^+/+</i> or <i>Azi1^Gt/Gt</i> animals were stained with anti-Azi1. Azi1 localises to the acrosome in Step 10–12 spermatids (G), then to punctae concentrated around centrosomes within the HTCA in control Step 13–14 spermatids (I). Importantly no Azi1 was detected in <i>Azi1^Gt/Gt</i> spermatids, demonstrating the staining is specific. (K–P) Elongating spermatids in wax sections stained with anti-acetylated α-Tubulin and anti-Hook1. The manchette is not labelled by anti-acetylated α-Tubulin in wild type spermatids (K–M). In contrast, mutant manchettes display high levels of acetylated α-Tubulin (N and O), indicating altered microtubule dynamics. Hook1 is absent from late stage mutant spermatids, suggesting defects in intramanchette transport (IMT) (P). CA: centriolar adjunct, PC: proximal centriole, DC: Distal centriole, BP: basal plate, CB: centriolar body, IF: implantation fossa, A: annulus. Scale bars represent 2 µm (A and B), 1 µm (C–F), 10 µm (G–J) and 5 µm (K–P).</p

Model of acute versus chronic loss of mammalian <i>Azi1</i> results in distinct ciliary phenotypes.

<p>(A) In mammalian somatic cells, Azi1/Cep131 is a highly conserved and ubiquitously expressed coiled-coil protein found associated with centriolar satellites. During ciliogenesis it becomes enriched during basal body maturation and is found at the transition zone, or ciliary gate, suggesting it plays a role in membrane docking, axoneme extension and/or cargo delivery to nascent cilia. It is one component of a complex and dynamic network required to build and maintain cilia. (B) Transient Azi1/Cep131 knock-down (<b>acute depletion</b>) abruptly reduces levels of this protein and destabilises the entire ciliogenic network, resulting in lack of cilia in somatic cells. (C) Knock-out of Azi1/Cep131 (<b>chronic depletion</b>) in somatic cells allows the system to re-equilibrate the ciliogenic network through compensation and continue to build functional cilia. (D) Knock-out of Azi1/Cep131 (<b>chronic depletion</b>) in developing sperm cannot be compensated for and in its absence, the network never assembles properly. This results in defects in basal body maturation affecting axoneme extension and IFT-dependent cargo delivery necessary to build a functional flagellum. This <i>Azi1/Cep131</i> null sperm phenotype is similar to <i>Azi1</i> mutations in other model organisms.</p

<i>Azi1^Gt/Gt</i> MEF have normal cilia and centriole numbers.

<p><i>Azi1^Gt/Gt</i> MEFs have normal numbers of cilia, centrosomes and centrioles. <i>Azi1^Gt/Gt</i> MEFs have normal cilia compartmentalisation as shown by localisation of ciliary membrane protein Arl13b (A and F), IFT-B protein Ift88 (B and G), transition zone markers Nphp1 and Mks1 (C, D, H and I) as well as normal localisation of small GTPase Rab8, involved in trafficking to the cilium (E and J) (GT: anti-γ-Tubulin, AT: anti-acetylated α-Tubulin, PGT: anti-polyglutamylated Tubulin). (K) Quantification of percentage of cells with a cilium, stained with anti-Arl13b (A and F), showing no difference between <i>Azi1^+/+</i> and <i>Azi1^Gt/Gt</i> MEFs. (L) Quantification of the number of cells with >2 centrosomes stained by anti-γ-Tubulin (A and F) again showing no difference between <i>Azi1^+/+</i> and <i>Azi1^Gt/Gt</i> MEFs. (M–R) <i>Azi1^Gt/Gt</i> MEFs have normal numbers of centrioles, marked with either Centrin2-GFP (M and O) or anti-Centrin 3 (N and P), quantified in (Q) and (R), respectively. (K, L, Q and R) Shown is the mean +/−standard deviation, n = 3 cell lines, passage number <7. Scale bars represent 1 µm (A–J) or 10 µm (M–P).</p

<i>Azi1</i> knock-down leads to reduced ciliogenesis.

<p>Mouse ShhLIGHT-II fibroblast cells were transfected with siRNA (a non-targeting control siRNA (Ctrl), two positive control siRNAs targeting <i>Ift88</i> (Ift88 #3 and #4) or a pool of four siRNAs targeting the 3′ UTR of <i>Azi1</i> (<i>Azi1</i> 3′UTR)), along with plasmids encoding either <i>GFP</i> or <i>Azi1-GFP</i> (which lacks the 3′UTR of <i>Azi1</i>). (A) Western blot probed with anti-Azi1 antibody (SF91) shows endogenous Azi1 levels are reduced upon <i>Azi1</i> 3′UTR siRNA addition (lower arrow). Tagged Azi1-GFP protein is also detected (upper arrow). The blot was reprobed with anti-α Tubulin as a loading control. Below is quantification of total Azi1 levels (endogenous plus overexpressed), relative to α-Tubulin. (B–E) Cells were stained with anti-Arl13b to mark the cilia and GFP Booster (Chromotek) to enhance the GFP signal. Transfected cells are highlighted with arrowheads; closed arrowheads highlight cells with cilia, open arrowheads highlight cells without cilia. (F) Addition of <i>Azi1</i> 3′UTR siRNA significantly reduced the percentage of cells with cilia, and this reduction is rescued to wild type levels by co-transfection with <i>Azi1-GFP</i>. Shown is the mean +/− SEM of two technical and two biological duplicates (*** <i>P</i><0.001, chi-squared test). Scale bars represent 10 µm.</p

<i>Azi1^Gt</i> is a null allele of <i>Azi1</i>.

<p>(A) Schematic of the <i>Azi1</i> gene trap inserted in intron 2 of <i>Azi1</i>. Exons are shown as boxes with translated transcript shaded. Arrows indicate primers used to characterise mRNA expression in <i>Azi1^Gt/Gt</i> mice. SA: Splice acceptor, T2A: self-cleaving peptide, pA: polyA. (B) <i>Azi1</i> mRNA expression in kidneys (K) and ovaries (O) from <i>Azi1^+/+</i> and <i>Azi1^Gt/Gt</i> mice was examined. No expression was detected across the gene trap insertion site in <i>Azi1^Gt/Gt</i> mice. (C) The lack of wild type transcription across the gene trap insertion site was confirmed by qPCR on testes cDNA. (D) LacZ staining of E11.5 <i>Azi1^Gt/+</i> embryos demonstrates the gene trap-βGeo cassette is expressed. <i>Azi1</i> expression is ubiquitous during development with higher expression in the limbs, somite derivatives, eyes and brain. (E) No full length or truncated Azi1 protein expression was detected in <i>Azi1^Gt/Gt</i> mutant testes. A strong band was detected in wild type and at lower levels in <i>Azi1^+/Gt</i> testes extract using an antibody raised to the C-terminal of Azi1 (SF91) (See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003928#pgen.1003928.s003" target="_blank">Figure S3A</a>). Densitometry was used to give Azi1 levels relative to α-Tubulin, normalised to 1 in wild type (lower of panel D). <i>Azi1^Gt/Gt</i> mice are null for <i>Azi1</i>. (F–H) Endogenous Azi1 is detected around centrosomes of wild type MEFs (F) and multiciliated airway epithelial cells (G and H). No Azi1 expression was detected by immunofluorescent staining of MEFs or nasal brush biopsies from <i>Azi1^Gt/Gt</i> mice, further confirming these mice are null for Azi1. Bottom panel shows the secondary only control. Inset in F highlights the centrosome. Arrowheads in G and H highlight the apical surface where γ-Tubulin positive centrosomes dock, and where Azi1 is localised in wild type cells. Scale bars represent 10 µm (F–H).</p

Remaining <i>Azi1^Gt/Gt</i> sperm are immotile, with shortened flagella.

<p>(A and B) Sperm dissected from the cauda epididymis of <i>Azi1^+/+</i> and <i>Azi1^Gt/Gt</i> mice. <i>Azi1^+/+</i> sperm was diluted 1∶5 in 1% methyl cellulose to slow movement to aid imaging. A projection of time points from high-speed video imaging shows the movement of <i>Azi1^+/+</i> sperm was easily detectable (A). (B) No movement was detected in <i>Azi1^Gt/Gt</i> sperm in methyl cellulose (not shown), so it was imaged undiluted without methyl cellulose. (B) A projection of time points shows the lack of movement of <i>Azi1^Gt/Gt</i> sperm. Insets highlight rare sperm with intact flagella. (C and D) TEM of <i>Azi1^+/+</i> and <i>Azi1^Gt/Gt</i> tubules, showing an open lumen filled with sperm flagella in an <i>Azi1^+/+</i> tubule (C), whereas <i>Azi1^Gt/Gt</i> tubule lumens were filled with cellular debris and sperm flagella were difficult to find (D). (E and F) TEM of sperm flagella. (F) In <i>Azi1^Gt/Gt</i> flagella that remain, the central axonemal structure appears generally normal, with the correct 9 + 2 microtubule structure in most remaining flagella. However evidence of abnormal trafficking was observed such as bulging membranes, ectopic outer dense fibres, or occasionally extra microtubules (arrow). MTD: microtubule doublet, ODF: Outer Dense Fibre (G–H). Sperm isolated from <i>Azi1^+/+</i> and <i>Azi1^Gt/Gt</i> testes was fixed and stained with anti-acetylated α-Tubulin and anti-Ift88. The number of <i>Azi1^Gt/Gt</i> sperm was greatly reduced compared to <i>Azi1^+/+</i>. All remaining <i>Azi1^Gt/Gt</i> sperm had shortened and often kinked flagella, with increased anti-acetylated α-Tubulin in all flagella and abnormal distribution of anti-Ift88 staining in a subset of mutant flagella (H and I), suggestive of defects in IFT. (I) Quantification of flagella length from sperm isolated from the testes, the caput or the cauda epididymus, measured using both anti-acetylated α-Tubulin (anti-Acet α-tub) staining and transmitted light. Scale bars represent 10 µm (C, D, G and H) or 100 nm (E and F).</p

<i>CG31320</i> is required for mechanosensory structure and function in Ch neurons.

<p>(A–C) Control and (D–F) Sensory-neural specific <i>CG31320</i> RNAi knock-down (<i>UAS-Dcr2; scaGal4/UAS-CG31320 RNAi</i>). (A,D) <i>In-situ</i> hybridisation confirms that substantial loss of <i>CG31320</i> mRNA was achieved by the knock-down. (Scale bar: 50 µm). (B,E) Immunofluorescence of larval Ch neurons using the pan-neuronal marker (anti-HRP: green, neuronal glycoproteins marking luminal bands at level of basal body and close to ciliary dilation) and the ion channel NompC/TRPN1, (anti-NompC; magenta; marks the distal non-motile cilium tip) shows that loss of <i>CG31320</i> results in no gross cilia dysmorphology or loss of compartmentalization of ciliated Ch structures. (Scale bar:10 µm). (C,F) TEM of Ch cilia cross-sections from adult antennae (Johnston's organ), showing nine axonemal microtubule doublets shown schematically in (G). (C) The electron-dense structures corresponding to inner (red arrowhead) and outer (blue arrowhead) axonemal dynein arms are clearly seen in wild-type. (F) These are not observed in <i>CG31320</i> knock-down cilia. (Scale bar: 100 nm). (G) Schematic illustration of Drosophila Ch neurons showing the localisation of markers in the cilia and the presence of dynein arms in the proximal motile zone. (H) Ch neuronal function is measured by the negative geotaxis climbing assay for adult flies. The height climbed by control (n = 43) versus <i>CG31320</i> RNAi flies (n = 64) reveals the latter to be uncoordinated. (Mann-Whitney U test: <i>P</i>≤0.0001). (I) <i>CG31320</i> RNAi knock-down larvae do not respond in an auditory assay. Retraction score is the number of larvae (in a sample of 5) exhibiting head shortening during a 0.5 second time window. Shown is the mean retraction score for several tests (control: n = 15; RNAi: n = 16); error bars are standard error of the mean. Statistical analysis was performed using the Friedman test followed by Dunn's multiple comparison post-hoc test. (** represents <i>P</i>≤0.01; **** represents <i>P</i>≤0.0001).</p

Cytoplasmic HEATR2 is expressed during early ciliogenesis.

<p>(A–C) Immunostaining of control human nasal brush epithelia reveals endogenous HEATR2 (red: Novus (A), Proteintech (C)) is highly enriched in the cytoplasm of developing MMC when components of outer dynein arms (B: DNAH5, red; C: DNAI2: green) as well as inner dynein arms (A–C: DNALI1, purple) are predominantly cytoplasmic. Arrowheads highlight fully mature MMCs where these components are exclusively axonemal and with relatively lower levels of HEATR2. Arrows highlight immature MMCs for comparison. Nuclei are stained with DAPI (blue). (Scale bar: A–C, 10 µm) (D) Double immunofluorescence of 22C10 (magenta: Futsch, cytoplasmic/membrane marker, but not cilium, of all sensory neurons) and <i>CG31320::mVenus</i> (green) indicates there is cytoplasmic but no ciliary localization of CG31320 in stage 16 Ch neurons (Ci: cilia, marked with square bracket). As this construct uses the upstream regulatory region of <i>CG31320</i> containing the X and Fox motifs to drive reporter expression, it further supports regulation occurs via these sites. (See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004577#pgen.1004577.s007" target="_blank">Figure S7</a>).</p

<i>CG31320</i>/<i>HEATR2</i> orthologues share conserved upstream regulatory FOX motifs and X-boxes of a master cilia motility transcriptional programme.

<p>(A) Using the human upstream epigenetic markings and conservation to mouse and rat to define conserved predicted regulatory elements, we focused analysis on the 500 bp upstream of the <i>HEATR2</i> ATG and syntenic regions in other species to identify X-box sequences, along with the nearest conserved FOX motifs. These sequences are coloured where they conform to recognized core consensus sequences for generic FOX proteins (RYMAAYA <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004577#pgen.1004577-Kaufmann1" target="_blank">[71]</a>) and RFX (RYYRYYN_(1–3)RRNRAC <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004577#pgen.1004577-Laurenon1" target="_blank">[42]</a>). Nucleotides are shown in grey if they vary from the consensus. Note for the second identified X-box site the 3′ site is extremely well-matched whilst the 5′ half-site is often more degenerate <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004577#pgen.1004577-ElZein1" target="_blank">[37]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004577#pgen.1004577-Newton1" target="_blank">[58]</a>. The distance from the Fox motif and X-box to the transcription start site is indicated, or else the distance to the ATG is indicated if a sizeable 5′UTR is present (<i>i.e. D. melanogaster, C. lupus</i>). An expanded table of the analysis is provided in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004577#pgen.1004577.s010" target="_blank">Table S2</a>. (B) ChIP-Seq data reveals a single, specific RFX3 peak 200 bp upstream from the transcriptional start site in OF1 mouse primary differentiated ependymal cell culture. Insert illustrates the two X-boxes bioinformatically predicted within the peak sequence. (C) Directed ChIP-qPCR data validates RFX3 occupancy is enriched at <i>Heatr2</i> promoter in OF1 cells, normalized to known target gene <i>Dyn2li1</i> and relative to a control sequence, downstream region in the <i>Dync2li1</i> gene. (D) <i>Heatr2</i> expression is ≈55% reduced in <i>Rfx3^−/−</i> ependymal cells similar to reductions in expression observed for two known direct Rfx3 targets, <i>Dync2li1</i> and <i>Bbs5</i>. qPCR data represent the average of three different assays performed in triplicate ± SEM. All data are considered significant using Student's t-test. (<i>Heatr2 P</i> = 0.003652632; <i>Dync2li P</i> = 0.013123897; <i>Bbs5 P</i> = 0.022511438).</p