CEP290 protein analysis in <i>Cep290</i> mouse models.

<p>Representative CEP290 immunodetection in P150 retinas from the three different mouse models. A CEP290-Flag construct was expressed in HEK293-T cells and used as a positive control. Tubulin was used for normalization.</p

Analysis of the <i>CEP290</i> cryptic exons.

<p>(A) Expected transcripts for each model. (B) Exon Y is expressed in all tissues in either <i>Cep290</i>^{<i>hum/hum</i>} and <i>Cep290</i>^{<i>lca/lca</i>} mice. (C) Only in the <i>Cep290</i>^{<i>lca/lca</i>} model, exon X is expressed, either with exon Y or without, at very low levels. The arrow indicates the band that contains both cryptic exons (exon X and exon Y) in the same transcript. <i>Cep290</i>^lca/lca PCR products were run longer to clearly separate the two different bands. (D) Schematic representation of the different transcripts containing cryptic exons found in the three mouse models. Mouse exons and introns are depicted in black, while human exons and introns are represented in blue. Cryptic exons X and Y are shown in grey and red, respectively. Arrows and letters indicate the position of the oligonucleotides in the cryptic exons. Semi-quantification was performed using ImageJ software [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079369#B21" target="_blank">21</a>]. MQ: H₂O (negative control); F: human LCA fibroblasts; B: Brain; K: Kidney; R: Retina; Lu: Lung; S: Spleen; T: Testis and Li: Liver.</p

Morphological and CEP290 immunolocalization analyses in P150 mouse retinas.

<p>Immunodetection of CEP290 and acetylated tubulin in retinal sections from <i>Cep290</i>^<i>wt/wt</i>, <i>Cep290</i>^{<i>hum/hum</i>} and <i>Cep290</i>^{<i>lca/lca</i>} mice, at P150. Images were taken at 200X (A) and 400x (B) magnifications. CEP290 (green) is located in the connecting cilium (CC) and remains unaltered in the humanized models. Nuclei were marked with DAPI (blue) and CC with acetylated tubulin (red). GCL: ganglion cell layer; IPL: inner plexiform layer; INL: inner nuclear layer; OPL: outer plexiform layer; ONL: outer nuclear layer; PhL: photoreceptor layer; RPE: retinal pigment epithelium; IS: inner segment; CC: connecting cilium; OS: outer segment.</p

Generation of the <i>Cep290</i> humanized mouse models.

<p>(A) Structure of the <i>CEP290</i> gene in human and mouse (drawn to scale). (B) Two mouse models were generated by introducing human exons 26 and 27 and intron 26, either with or without the LCA-causing mutation (depicted with *), to the mouse <i>Cep290</i> gene. Human and mouse loci, targeting vector and recombinant locus are depicted. Arrows and letters indicate the position of the oligonucleotides (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079369#pone.0079369.s001" target="_blank">Table S1</a>). </p

Transcriptional characterisation of <i>Cep290</i> in humanized mouse models.

<p>(A-B) <i>Cep290</i> expression levels in various mouse tissues were assessed by RT-PCR. Regions containing murine exons 10 to 13 (A) and 24 to 27 (B) were analyzed. No differences were observed. (C) Amplification using human primers was also assessed in the three models. Only humanized models showed amplification. (D) Actin was used for normalization and comparison among tissues and models. MQ: H₂O (negative control); B: Brain; K: Kidney; R: Retina; Lu: Lung; S: Spleen; T: Testis and Li: Liver.</p

Analysis of the cryptic exons X and Y in human retina and fibroblast cell lines.

<p>The expression of the <i>CEP290</i> human exons 26 and 27, and the cryptic exons Y and exon X were assessed in tissues derived from both mice and humans. Actin was used for normalization. </p

CC2D2A and NINL co-localize at the ciliary base in hTERT-RPE1 cells and in zebrafish retina.

<p>(<b>a, a’</b> and inset) When expressed alone, eCFP-tagged CC2D2A (green signal) localizes to the ciliary base (basal body, accessory centriole). The cilium is marked by anti-polyglutamylated tubulin (red signal, <b>a’</b> and inset). eCFP-tagged CC2D2A (green signal; <b>b</b>) also (partly) localizes to the ciliary transition zone, which was visualized using anti-RPGRIP1L as a marker (red signal; <b>b</b>). (<b>c, c’</b> and inset) mRFP-tagged NINL isoform B was localized at the ciliary base (cilium in green, <b>c’</b> and inset). (<b>d</b> and inset) mRFP-tagged NINL isoform B (red signal) localizes adjacent to the ciliary transition zone (anti-RPGRIP1L; green signal). (<b>e-e”</b> and inset) Co-expression of mRFP-tagged NINL isoform B (red signal) and eCFP-tagged CC2D2A showed co-localization of both proteins around the ciliary base (yellow signal). (<b>f</b>) In wild-type larval zebrafish retina (4 dpf), Cc2d2a marked by anti-Cc2d2a antibodies (red signal) is localized apically to the photoreceptor basal body (marked by anti-centrin antibodies, green signal). (<b>g</b>) Ninl, stained with anti-Ninl antibodies, (red signal) is localized at the zebrafish photoreceptor ciliary base, partially overlapping with and apical to the green centrin signal. (<b>h</b>) Cc2d2a localization is unaffected by <i>ninl</i> knockdown and (<b>i</b>) Ninl localization is normal in <i>cc2d2a</i>^<b>-/-</b> larvae. (<b>j</b>) Schematic representation of the localization of Ninl and Cc2d2a in zebrafish photoreceptor cells. (<b>f-i</b>) are immunostainings on cryosections from 4 dpf larvae. Nuclei were stained with DAPI (blue signal) in all panels. Scale bars are 10 μm in a-e, and 4 μm in f-i.</p

Ninl is required for correct Rab8A localization.

<p>(<b>a-a’</b>) Expression of a rhodopsin-promoter driven cherry-tagged Rab8a in wild-type photoreceptors is mostly concentrated in one or several puncta (arrows <b>a-a’</b>) whereas it is diffuse in the majority of <i>ninl</i> morphant photoreceptors (<b>b-b’</b>). (<b>c</b>) Proportion of Rab8a-cherry expressing photoreceptors with punctate expression versus diffuse expression (bars represent 95% confidence interval; ** <i>P</i><0.001, <i>Fisher’s</i> exact test). (<b>d-d’</b>) Endogenous Rab8a localization as seen by immunohistochemistry using an anti-Rab8a antibody (green) displays similar puncta (arrowheads) in wild-type photoreceptors, while the number of puncta is decreased in <i>ninl</i> morphant photoreceptors (<b>e-e’</b>). (<b>f</b>) Quantification of the number of Rab8a puncta displayed in the form of a scatter plot indicating that significantly fewer endogenous Rab8 puncta per μm² are present in <i>ninl</i> morphants compared to uninjected controls (*<i>P = 0</i>.<i>01</i>, unpaired Student’s <i>t-</i>test; bars represent standard error of the mean). Scoring was performed blinded as to injection status for (<b>c</b>) and (<b>f</b>). Outer segments are counterstained with bodipy in (<b>d-e</b>). Nuclei are counterstained with DAPI. All images are cryosections of 4 dpf larvae. Scale bars are 4 μm in all panels.</p

NINL and CC2D2A co-localize with MICAL3 and are required for correct MICAL3 localization.

<p>(<b>a</b>) Schematic of a photoreceptor for orientation. (<b>b-d’</b>) Co-localization of endogenous MICAL3 (green signal; <b>b</b>) and polyglutamylated tubulin (red signal; <b>c</b>) in rat retina (P20) by co-immunostaining radial cryo-sections. The yellow signal in the merged image (<b>d’</b>) indicates co-localization at the base of the photoreceptor connecting cilium. (<b>b’-d’</b>) are high magnification images of the boxed areas in (b-d). (<b>e-f”</b>) Centrosomal co-localization of NINL^isoB, CC2D2A and MICAL3 in hTERT-RPE1 cells. mRFP-NINL^isoB (red signal, <b>e</b>) localizes to the basal body of the cilia marked with polyglutamylated tubulin (cyanid signal; <b>e</b>) and overlaps with GFP-tagged MICAL3 (green signal, <b>e’</b>) at the ciliary base when co-expressed (yellow signal, <b>e”</b>). Co-expression of eCFP-CC2D2A (green signal, <b>f</b>) and mRFP-MICAL3 (red signal, f’) resulted in partial overlap at the base of the cilia (yellow signal, <b>f”</b>). (<b>g</b>) Endogenous MICAL3 (green signal) detected by immunostaining clusters at the ciliary base (white arrows; cilium marked with anti-acetylated tubulin in red) of hTERT-RPE1 cells treated with non-targeting siRNA. (<b>h, i</b>) Knockdown of <i>NINL</i> (<b>h</b>) or <i>CC2D2A</i> (<b>i</b>) expression by siRNA results in dispersed distribution of MICAL3 throughout the cell body (brackets) with retention of some MICAL3 puncta at the ciliary base (arrows). qPCR analysis of <i>NINL</i> (<b>j</b>) and <i>CC2D2A</i> (<b>k</b>) siRNA treated hTERT-RPE1 cells. Cells were transfected with 10nM siRNA and all qPCR data were normalized against <i>GUSB</i> levels. Bar and error bars refer to mean and standard deviation, respectively (n = 3, on two biologicial replicates). *: P<0.05; **: P<0.01 versus non targeting siRNA (NT) (student’s <i>t-</i>test). Nuclei are counter stained with DAPI in all panels (blue signal). Scale bars: are 5 μm in d, 1 μm in d’ and 10 μm in e-i.</p

NINL interactome screen identifies MICAL3.

<p>(<b>a</b>) Strep-SILAC and TAP (tandem affinity purification) experiments show that NINL interacts specifically with MICAL3 (Yellow). The solid line between NINL and MICAL3 symbolizes a direct interaction, whereas the dashed lines indicate interactions determined by IP. (<b>b</b>) Co-immunoprecipitation of eGFP-MICAL3 with FLAG-NINL^isoB, but not with FLAG-STRAD. The immunoblot (IB) in the top panel shows that eGFP-tagged MICAL3 co-immunoprecipitated with FLAG -tagged NINL (lane 2), whereas FLAG-tagged STRAD used as a negative control (lane 3) did not. The anti-GFP immunoprecipitates are shown in the middle panel; protein input is shown in the bottom panel. Reciprocal IP experiments using anti-FLAG antibodies confirmed the co-immunoprecipitation of eGFP-tagged MICAL3 with FLAG-tagged NINL^isoB (lane 2) and not with STRAD (lane 3) shown in the top panel. The anti-FLAG immunoprecipitations are shown in the middle panel; protein input is shown in the bottom panel. A co-immunoprecipitation experiment using untagged eGFP as a negative control (right panel) showed that eGFP-tagged MICAL3 immunoprecipitates with FLAG-tagged NINL^isoB but not with untagged eGFP.</p