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

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

<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

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

<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

Precocious capacitation and increased spontaneous acrosome reaction in sperm from <i>DefbΔ9/DefbΔ9</i> mice.

<p>Figure 4A: FITC-conjugated Pisum sativum (PSA) lectin labelling of acrosome reacted sperm. Percentage of spontaneous AR in wild-type (+/+) and <i>DefbΔ9/DefbΔ9</i> (−/−) sperm determined by PSA-FITC lectin labelling before (T0) and after 90 minutes incubation (T90) in complete capacitation medium (mean ±SD; n = 4). *, p<0.05. Figure 4B: Zonadhesin antibody binding and quantification. Images show representative ZAN antibody binding to a sperm from <i>DefbΔ9</i> (−/−) male after capacitation, visualised with a goat anti-rabbit IgG conjugated to Alexa Fluor 594 around the sperm head. Left panel (bright field) and right panel (fluorescent image). Lower panel shows percentages of ZAN exposure evaluated on live spermatozoa from +/+ and −/− before (T0) and after (T90) incubation in capacitation medium. Figure 4C: Acrosome integrity following Coomassie blue G250 staining of fixed sperm. Right panel shows representative image of sperm that is acrosome reacted (arrow) lacking staining and other non-AR sperm with intense staining. Left panel graph shows average percentage of acrosome reacted sperm from three independent experiments where over 150 sperm were counted per sample before and after incubation in complete capacitation medium (mean ± SD; n = 3). p<0.03. Figure 4D: Percentage of capacitation evaluated by the ability of sperm to undergo the AR. AR induced by 10 µM calcium ionophore A23187 in spermatozoa from mutant (−/−) and wild-type (+/+) animals and level of PSA-FITC used to determine AR directly after the treatment (T0) and 90 minutes after (T90) (mean ±SD; n = 4). *, p<0.01. Figure 4E: Sperm-egg binding assay. Light microscopy images show cumulus-free eggs from superovulated CD1 females with sperm from wild-type animals (+/+) (upper left panel) and no sperm from <i>DefbΔ9−/−</i> males bound to the eggs (upper right panel). Sperm were also incubated with 2-cell embryos as a control for non-specific binding (left egg in upper left panel). A range of 47–86 eggs per genotype were used for each set of experiments (n = 3). Original magnification ×20. Graph shows comparison of the average number of sperm from wild-type (+/+) and <i>DefbΔ9</i> (−/−) males bound eggs following 45 minute incubation (mean ± SD; n = 3). **, p<0.001.</p

Ultrastructure of spermatozoa from cauda, caput and testis from wild-type littermates (+/+) and <i>DefbΔ9/DefbΔ9</i> (−/−) male mice reveals a defect in microtubule structure.

<p>Figure 5A: Transmission Electron Microscopy (TEM) of sperm from cauda, caput and testis from <i>DefbΔ9/DefbΔ9</i> (−/−) and wild-type littermates (+/+) mice. Top and middle horizontal panels show overviews of cross section of sperm from cauda of +/+ and −/− mice respectively at various levels of the tail (a). Higher magnification of cross section of principal piece (b–d) and mid-piece (e–f) of sperm tails. Upper panel (f) shows normal axoneme (9+2 microtubules, MT), mitochondrial sheath (M), outer dense fibres (ODF) in wild-type mouse sperm. Middle panel (f) shows clear disruption and disintegration of the MT (arrowhead) in sperm from <i>DefbΔ9</i> (−/−). Middle panel (b) shows example of additional microtubules (*) other than the classical 9+2 arrangement. Bottom panel shows TEM of caput epididymal (a, b) and testis (c, d) sperm from wild type (+/+) and <i>DefbΔ9</i>(−/−) mice. No obvious microtubule disruptions were observed in sperm within the caput or testis of the mutant mice. Bars: 500 nm and 100 nm as labelled (a–f). Figure 5B: Fluorescent intensity of total intracellular calcium of wild-type (+/+) littermates and <i>DefbΔ9/DefbΔ9</i> (−/−) sperm using Fluo3 AM ester assay. The calcium levels were measured using Fluo-3 AM ester calcium fluorescent indicator at 5 µM concentration incubated with spermatozoa at 20 million/ml for 30 mins at 37°C, samples were washed and loaded onto 96-well plate in duplicates at 100 µl/well, and the plates were read by BMG Labtech FluoSTAR Omega fluorescent reader (mean ± SD; n = 3).*, p<0.002. Values given are after subtracting the background levels of the DMSO controls.</p

<i>DefbΔ9/DefbΔ9</i> male mice have more fragile sperm with reduced motility.

<p>Figure 3A: Cauda epididymal sperm from <i>DefbΔ9/DefbΔ9</i> (−/−) show an increased number of detached heads compared to wild-types (+/+). Photomicrographs represented are in phase-contrast microscopy at original magnification of ×40. Fragility of the mutant vs wild-type derived sperm isolated from cauda was determined by dropping the suspensions onto a glass slide and analysing the number of detached heads (arrowheads). A total of 200 sperm were analysed for each slide, which represented one animal. An average of 3 pairs was analysed (mean ± SD; n = 3). p = 0.048. Figure 3B: Spermatozoa from <i>DefbΔ9/DefbΔ9</i> mice have reduced motility. Percentage of total (left) and progressive (right) spermatozoa motility in <i>DefbΔ9</i> +/+ and <i>DefbΔ9</i> −/− mice using CASA before (time 0 mins, T0) and after (time 90 mins, T90) sperm capacitation (mean ±SD; n = 4). *, p<0.001.</p