LCA-associated mutations in lebercilin disrupt the physical connection with IFT.

The effect of LCA mutations on the lebercilin interactome was assessed by quantitative protein complex comparisons of wild-type lebercilin with 2 known human LCA5 mutations (Figure ​(Figure1A).1A). The same setup was used as for the detection of specific protein complex components, with a single modification. Instead of SF-TAP as control, lebercilin-p.P493TfsX1 and lebercilin-p.Q279X were expressed, and their complexes were compared with normal lebercilin in HEK293T cells. The correct production of both mutants was demonstrated by Western blot analysis of HEK293T lysates (Supplemental Figure 1A) as well as by the identification of peptides covering large parts of the theoretical sequence (Supplemental Figure 6, A and B). Both mutations are causative for LCA without obvious phenotypic differences (4). p.P493TfsX1 is located in the last exon of LCA5, and therefore the mRNA should not be subjected to nonsense-mediated mRNA decay (27). This was confirmed by real-time quantitative PCR (qPCR) analysis of EBV-transformed lymphocytes from a LCA patient harboring a homozygous p.P493TfsX1 mutation in LCA5 (family 27240; ref. 4), which showed no significant decrease in LCA5 mRNA expression compared with EBV-transformed lymphocytes of a control subject (Supplemental Figure 1B). Our analyses showed that both mutations resulted in the loss of all identified IFT and IFT-associated proteins from the protein complex (Figure ​(Figure2,2, Supplemental Table 1, and Supplemental Figure 1C). These results were confirmed by Western blot analysis, in which IFT74 and IFT88 could not be detected in SF-TAP eluates of p.P493TfsX1 and p.Q279X, whereas the presence of 14-3-3ε was not altered (Supplemental Figure 1A). Moreover, 6 other proteins previously identified in the lebercilin complex (4) were lost from the complex of both lebercilin mutants: WDR26, KIAA0564, PGAM5, YPEL5, C20orf11, and RANBP9 (Supplemental Table 1). Therefore, these may also be associated to lebercilin function. The more aminoterminally truncated mutant p.Q279X additionally displayed reduced binding to CSNK2A1/2, CSNK2B, DYNLL1, DYNLL2, and USP9X (Figure ​(Figure2B),2B), which suggests that this protein variant has lost additional protein binding domains. This stresses the role of the carboxyterminal part of lebercilin as a crucial and specific interaction domain for IFT proteins.

Open in a separate window

Figure 2

Quantitative protein complex analysis of LCA-causative lebercilin mutations.

Detection of protein complex alterations by comparison of the complexes of SF-TAP tagged lebercilin to either lebercilin-p.P493TfsX1 (A) or lebercilin-p.Q279X (B) in HEK293T cells. Plotted are log₁₀ ratios and log₁₀ intensities as quantified by MaxQuant from 3 independent experiments. For each experiment, label switching was done to exclude label-specific effects. Shown are only proteins quantified and identified in at least 2 of 3 experiments. Highly significant (P < 0.001) altered protein interactions are plotted in red, the majority of them being IFT proteins (Supplemental Table 1). Also shown are representations of the respective protein complexes; all proteins of the IFT protein complex, as well as WDR26, KIAA0564, PGAM5, YPEL5, C20orf11, and RANBP9, were lost for both mutants. CSNK2A1/2, CSNK2B, DYNLL1, DYNLL2, and USP9X only lost association to lebercilin-p.Q279X, not to lebercilin-p.P493TfsX1.

Lebercilin and IFT proteins colocalize in cilia of retinal cells.

To validate the interaction between endogenous lebercilin and IFT proteins in retinal cells, we analyzed the localization of lebercilin and IFT proteins in mouse retina and in ciliated human hTERT-RPE1 cells. Lebercilin localized along the CC of mouse photoreceptors (Figure ​(Figure3A)3A) and in primary cilia of hTERT-RPE1 cells (Figure ​(Figure3C).3C). Colocalization between lebercilin and IFT complex A and B proteins was observed in the CC of mouse photoreceptors (Figure ​(Figure3B)3B) and in primary cilia of hTERT-RPE1 cells (Figure ​(Figure3D). 3D).

Open in a separate window

Figure 3

Endogenous localization of lebercilin and IFTs in mammalian retina and retinal cells.

(A) Immunohistochemistry of mouse BALB/c retina (P20) showed endogenous lebercilin (SN2134) expression in the connecting cilia of mouse photoreceptors, where it colocalized with the ciliary marker polyglutamylated tubulin (GT335). (B) Endogenous lebercilin (SN2134) colocalized with endogenous IFT complex B proteins Traf3ip1, Ift88, and IFT complex A protein Wdr19 in connecting cilia of mouse photoreceptors. (C) Immunocytochemistry of hTERT-RPE1 cells confirmed lebercilin (SN2134) localization in primary cilia (arrow). (D) Colocalization of endogenous lebercilin (SN2134) in the cilium with endogenous TRAF3IP1, IFT88, and WDR19 (arrows). (E) siRNA-mediated knockdown of LCA5 did not affect ciliogenesis compared with nontargeting siRNA–transfected hTERT-RPE1 cells (arrows). GT335 stained the primary cilium. SN2134 antibody was used to stain lebercilin. (F) Knockdown of LCA5 in hTERT-RPE1 cells did not affect ciliary localization of IFT88 (arrows). Scale bars: 5 μm (A–F). Enlarged views are shown in the insets (A and B, ×5; C–F, ×2).

Lebercilin is not a structural component of the IFT machinery.

We performed knockdown of TRAF3IP1, which plays a critical role in cilium assembly in Caenorhabditis elegans (28), to determine whether IFT is involved in the ciliary localization of lebercilin. siRNA-mediated knockdown of TRAF3IP1 in hTERT-RPE1 cells affected ciliogenesis, as well as subsequent ciliary lebercilin localization, compared with nontargeting siRNA–transfected cells (Supplemental Figure 2C). Knockdown of LCA5 in hTERT-RPE1 cells, however, did not affect ciliogenesis or ciliary localization of IFT88 (Figure ​(Figure3,3, E and F), which indicates that lebercilin and lebercilin-associated proteins are likely to be essential for specialized transport by the IFT machinery, rather than a structural component of the IFT core.

Inactivation of Lca5 in mice recapitulates LCA in humans.

To investigate the role of lebercilin in IFT-mediated transport in the retina, we generated mice bearing an inactivating gene trap insertion in intron 3 of Lca5 (Supplemental Figure 3, A and B). Lca5 inactivation in the homozygous Lca5^gt/gt mice was confirmed at the RNA and protein levels (Supplemental Figure 3, C–F). β-Galactosidase staining allowed us to detect Lca5 expression in ciliated tissues of heterozygous Lca5^gt/+ mice (Supplemental Figure 4, A–F). Homozygous F2 Lca5^gt/gt mice were viable up to 1 year of age, the latest time point examined. Mutant males and females both sired offspring successfully. Phenotypically, Lca5^gt/gt mice developed retinal patches of depigmentation and lacked rod and cone ERG responses to light stimuli at a very early stage (Figure ​(Figure4,4, A and B), thereby recapitulating LCA in humans. Despite the broad expression of Lca5 in ciliated tissues (13), the disease phenotype was restricted to the retina; brain histology, hearing assessed by auditory brainstem response (ABR), and olfactory function were similar to those of controls (data not shown). The fundus of Lca5 heterozygous animals was normal at all ages examined (data not shown).

Open in a separate window

Figure 4

Clinical and morphological assessment of Lca5^gt/gt mouse retina.

(A) Fundus photography of Lca5^gt/gt animals and littermate controls. At 2 months of age, depigmented patches were observed. (B) Scotopic and photopic ERG amplitudes of rod (left) and cone (right) waveforms at P25 (n = 3). Data are mean ± SEM. (C) Progressive retinal degeneration demonstrated in Lca5^gt/gt mice. Retinal sections of Lca5^gt/gt mice and wild-type controls, between P12 and 4 months of age, stained with hematoxylin and eosin. At P12, the OS/IS layers were remarkably thinner in Lca5^gt/gt animals, whereas the ONL and INL were comparable to those of controls. At P28, the photoreceptor nuclei in the ONL were significantly reduced compared with controls. By 4 months of age, the photoreceptor layer was completely absent in Lca5^gt/gt mice. (D) Scanning EM of control and Lca5^gt/gt retinas at P10 and P15 from a scleral orientation (left) and fractured view (right). Control OSs at P10 were ovoid and regular in size. Vertically stacked OS discs (double asterisks) were rarely observed. OSs in Lca5^gt/gt retinas were shorter and irregular in size and shape. By P15, OS discs were disorganized and did not reach the length observed in controls. Most OSs were rudimentary or abnormal in shape (asterisk). (E) Transmission EM of Lca5^gt/gt retinas showed that the structure of the CC, including basal body, was preserved at P8. RPE, retinal pigment epithelium; NC, nascent cilia. Scale bars: 50 μm (C); 10 μm (D); 500 nm (E).

Lca5 inactivation results in shorter ISs and OSs.

The retinal phenotype resulting from Lca5 inactivation was studied in greater detail using histology and EM. Retinal sections of Lca5^gt/gt mice at P12 showed shorter ISs and OSs, whereas the outer nuclear layer (ONL) was unaffected. At 4 months of age, the photoreceptor layer was completely absent in these animals (Figure ​(Figure4C).4C). Using transmission EM and scanning EM, we observed that the initiation of OS development in Lca5^gt/gt was delayed and OSs were disorganized (Figure ​(Figure4D);4D); discs in the OS were not oriented properly at P9–P13 (Supplemental Figure 5A). The structure of both the CC and the basal body appeared to be normal at this stage (Figure ​(Figure4,4, D and E, and Supplemental Figure 5A).

Ophthalmic examination and histology.

The methods for fundus photography and ERG have been described previously (12). For histological examination by light microscopy, whole eyes were collected and fixed overnight in methanol and acetic acid (3:1, v/v), washed, and embedded in paraffin. Tissue samples were cut in 4- to 6-μm sections and stained with hematoxylin and eosin. For transmission EM, the cornea and lens were removed, and the posterior retina was fixed overnight in 2.5% glutaraldehyde and 2% PFA. For scanning EM, retinas were dissected and fixed overnight in 3% glutaraldehyde and 1% PFA. Both transmission EM and scanning EM samples were postfixed with osmium tetroxide and stained with uranyl acetate as previously described (12).

Cell culture.

Human B lymphocytes from a LCA patient (family 27240; ref. 4) and an unaffected control were immortalized by transformation with EBV according to established procedures (46) and grown as described previously (35). This study was approved by the Research Ethics Board of McGill University. We obtained blood samples after receiving informed consent from all participating individuals. hTERT-RPE1 cells and HEK293T cells were cultured as described previously (47). For SILAC experiments, HEK293T cells were grown in SILAC DMEM (PAA) supplemented with 3 mM l-glutamine (PAA), 10% dialyzed fetal bovine serum (PAA), 0.55 mM lysine, and 0.4 mM arginine. Light SILAC medium was supplemented with ¹²C₆,¹⁴N₂ lysine and ¹²C₆,¹⁴N₄ arginine. Heavy SILAC medium was supplemented with either ¹³C₆ lysine and ¹³C₆,¹⁵N₄ arginine or ¹³C₆,¹⁵N₂ lysine and ¹³C₆,¹⁵N₄ arginine. 0.5 mM proline was added to all SILAC media to prevent arginine-to-proline conversion (48). All aa were purchased from Silantes. Primary cilium formation in hTERT-RPE1 cells was induced by serum starvation (0.2% serum) for 48 hours. For siRNA experiments, hTERT-RPE1 cells were seeded on glass slides, grown overnight, and then transfected with 3 pooled Silencer Select siRNAs (15 nM final siRNA concentration; Ambion) using Hiperfect (Qiagen) according to the manufacturer’s instructions. Nontargeting siRNA control no. 1 (15 nM final siRNA concentration; Ambion) was included as reference. The targeting sequence for each siRNA is included in Supplemental Table 4. After 72 hours of siRNA transfection, cells were harvested for either knockdown analysis or immunofluorescence. For DNA transfections, HEK293T or hTERT-RPE1 cells were seeded, grown overnight, and then transfected using Effectene (Qiagen) according to the manufacturer’s instructions.

DNA constructs.

We generated IFT cDNA constructs by PCR using IMAGE clones of IFT20 (clone 3609552), IFT27 (clone 3163341), IFT57 (clone 4053749), and IFT88 (clone 4824965; all from Geneservice) cDNA as template. IFT52 was generated via PCR from human marathon-ready testis cDNA (Clontech). Gateway-adapted constructs were created using the Gateway cloning system (Invitrogen) according to the manufacturer’s procedures and were sequence verified. Gateway-adapted full-length human lebercilin, lebercilin-p.P493TfsX1, and lebercilin-p.Q279X have been described previously (4, 49).

AP of protein complexes.

For 1-step Strep purifications, SF-TAP–tagged proteins and associated protein complexes were purified essentially as described previously (4, 14). HEK293T cells transiently expressing the SF-TAP tagged constructs were lysed in lysis buffer containing 0.5% Nonidet-P40, protease inhibitor cocktail (Roche), and phosphatase inhibitor cocktails I and II (Sigma-Aldrich) in TBS (30 mM Tris-HCl, pH 7.4, and 150 mM NaCl) for 20 minutes at 4°C. After sedimentation of nuclei at 10,000 g for 10 minutes, the cleared lysates were transferred to Strep-Tactin-Superflow beads (IBA) and incubated for 1 hour before the resin was washed 3 times with wash buffer (TBS containing 0.1% NP-40 and phosphatase inhibitor cocktails I and II). The protein complexes were eluted by incubation for 10 minutes in Strep-elution buffer (IBA). For quantitative protein complex comparison or detection of specific complex components, protein concentration was determined using a standard Bradford assay. Equal protein amounts were used as input for the experiments to be compared. The protein complex purification was done as described above with 1 modification: the samples were combined before the resin was washed and the protein complexes were eluted. The eluted samples were concentrated using 10-kDa cutoff VivaSpin 500 centrifugal devices (Sartorius Stedim Biotech) and prefractionated using SDS-PAGE and in-gel tryptic cleavage as described elsewhere (47). For SF-TAP analysis, the constructs were expressed and cells were harvested as described above. The cleared supernatant was incubated for 1 hour at 4°C with Strep-Tactin superflow (IBA). Subsequently, the resin was washed 3 times in wash buffer. Protein baits were eluted with Strep-elution buffer. For the second purification step, the eluates were transferred to anti-FlagM2 agarose (Sigma-Aldrich) and incubated for 1 hour at 4°C. The beads were washed 3 times with wash buffer, and proteins were eluted with Flag peptide (200 μg/ml; Sigma-Aldrich) in TBS. After purification, the samples were precipitated with chloroform and methanol and subjected to in-solution tryptic cleavage as described previously (47).

Mass spectrometry and data analysis.

LC-MS/MS analysis was performed on an Ultimate3000 nano HPLC system (Dionex) coupled to a LTQ OrbitrapXL mass spectrometer (Thermo Fisher Scientific) by a nano spray ion source. Tryptic peptide mixtures were automatically injected and loaded at a flow rate of 30 μl/min in 95% buffer C (0.5% trifluoroacetic acid in HPLC-grade water) and 5% buffer B (98% actetonitrile and 0.1% formic acid in HPLC-grade water) onto a nano trap column (100 μm i.d. × 2 cm, packed with Acclaim PepMap100 C18, 5 μm, 100 Å; Dionex). After 5 minutes, peptides were eluted and separated on the analytical column (75 μm i.d. × 15 cm, Acclaim PepMap100 C18, 3 μm, 100 Å; Dionex) by a linear gradient from 5% to 40% of buffer B in buffer A (2% acetonitrile and 0.1% formic acid in HPLC-grade water) at a flow rate of 300 nl/min over 90 minutes. Remaining peptides were eluted by a short gradient from 40% to 100% buffer B in 5 minutes. The eluted peptides were analyzed by the LTQ OrbitrapXL mass spectrometer. From the high-resolution mass spectrometry prescan with a mass range of 300–1,500, the 10 most intense peptide ions were selected for fragment analysis in the linear ion trap if they exceeded an intensity of at least 200 counts and if they were at least doubly charged. The normalized collision energy for collision-induced dissociation was set to a value of 35, and the resulting fragments were detected with normal resolution in the linear ion trap. The lock mass option was activated, the background signal with a mass of 445.12002 was used as lock mass (50). Every ion selected for fragmentation was excluded for 30 seconds by dynamic exclusion. For SILAC experiments, all acquired spectra were processed and analyzed using MaxQuant software (version 1.0.13.13; ref. 16) and the human-specific IPI database version 3.52 ( http://www.maxquant.org/) in combination with Mascot (version 2.2; Matrix Science). Cysteine carbamidomethylation was selected as fixed modification; methionine oxidation and protein acetylation were allowed as variable modifications. The peptide and protein false discovery rates were set to 1%. Contaminants like keratins were removed. Proteins identified and quantified by at least 2 peptides per experiment in at least 2 of 3 independent experiments were considered for further analysis. Each experiment consisted of a forward and a reverse labeling approach to exclude label-specific effects. A P value of 0.001 was selected as threshold for significant enrichment or alteration. Only proteins identified as specific lebercilin complex components by significant enrichment in the comparison of wild-type lebercilin with SF-TAP control were considered for the quantitative protein complex comparisons. As there is little relevant information present in public databases, including STRING, MINT, and IntAct, we annotated and grouped protein interactions manually based on publicly available data and constructed a scale-free protein interaction map representing proteins as nodes and interactions as edges.

For nonquantitative experiments, the raw data were analyzed using Sequest (Thermo Fisher Scientific) or Mascot and Scaffold (Proteome Software) as described previously (47). Proteins were considered to be specific protein complex components if they were not detected in the control and were detected at least twice with 2 or more peptides (peptide probability >80%) in 3 experiments. The protein probability threshold was set to 99%.

Northern blot analysis.

Total retinal RNA was isolated from wild-type, heterozygous, and homozygous Lca5^gt mice at P17 using TRIzol (Invitrogen) reagents, separated in a 1% formamide gel by electrophoresis, and transferred onto a nitrocellulose membrane. The N-terminal probe for Lca5 (860 bp) was generated by RT-PCR amplification using primers E2F1 and E4R1 (see Supplemental Table 5). 18S control probe (Ambion) and Lca5 probe were labeled with ³⁵P using the Redi-prime II DNA labeling kit (Amersham) and incubated with the membrane overnight at 42°C. The resulting images were obtained by autoradiography.

β-Galactosidase assay.

Dissected retina, brain, and testis were incubated in PBS containing 30 mM K₄Fe(CN)₆, 30 mM K₃Fe(CN)₆, 2 mM MgCl₂, 0.01% sodium desoxycholate, and 0.02% NP-40 after 3 hours of fixation with 4% PFA. Additional fixation with fresh fixative overnight was carried out after the blue color developed in the tissue expressing Lca5^gt. The stained tissues were photographed and embedded in paraffin. 6-μm sections were stained with Nuclear Fast Red for nuclear counterstaining.

RT-PCR.

The gene trap insertion site was identified by direct sequencing of the RT-PCR amplicon with primers E3F1 and en2R1 from ES cell cDNA. Retinal cDNA from Lca5^+/+ and Lca5^gt/gt animals was generated using the Retroscript kit (Ambion). Primers used for Lca5 cDNA analysis (E2F1, E4R1, E3F2, and LacZ6R) and input cDNA control (rig/S15; Ambion) are included in Supplemental Table 5.

Quantitative PCR.

Total RNA from ciliated hTERT-RPE1 cells and EBV-transformed cell lines was isolated using TRIzol (Invitrogen) according to the manufacturer’s instructions. Residual traces of genomic DNA were removed via DNase I (Invitrogen) treatment according to the manufacturer’s instructions. 1 μg total RNA was transcribed into cDNA using the iScript cDNA synthesis kit (BioRad Laboratories) according to the manufacturer’s protocol. SYBR Green–based qPCR analysis was performed on a 7500 Fast Real-Time PCR System (Applied Biosystems) with Power SYBR Green PCR Master Mix (Applied Biosystems) according to the manufacturer’s instructions. Primers were developed by the Primer3 program (Supplemental Table 5 and ref. 51). PCR products encompassed at least 1 exon-exon junction. GUSB was used as reference gene. Further analysis was performed as described previously (35).

GST pulldown.

In order to produce GST fusion proteins, BL21-DE3 cells were transformed with either pDEST15-lebercilin or pDEST15 alone. Cells were induced at 30°C for 3 hours with 0.5 mM IPTG (Sigma-Aldrich) and subsequently lysed with STE buffer (10 mM Tris-HCl, pH 8.0; 1 mM EDTA; and 150 mM NaCl) supplemented with 10 mg/ml lysozyme, 0.5% Sarkosyl, 1% Triton X-100, and complete protease inhibitor cocktail (Roche). Lysates were incubated for 2 hours at 4°C with glutathione-sepharose 4B beads (Amersham Biosciences). After incubation, beads were washed with STE and TBSTD (TBS with 1% Triton X-100 and 2 mM DTT). The amount of GST fusion proteins bound to the beads was verified on a NuPAGE Novex 4%–12% Bis-Tris SDS-PAGE gel by staining with SimplyBlue SafeStain (Invitrogen). Beads with bound GST fusion proteins were incubated for 2.5 hours at 4°C with bovine retinal lysates, made by incubating retina in 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 10% glycerol, and 1% Triton-X-100 supplemented with complete protease inhibitors (Roche). Beads with bound protein complexes were washed in lysis buffer, then taken up in 1× NuPage Sample Buffer, heated for 10 minutes at 70°C, precipitated by centrifugation, and subjected to immunoblot analysis.

Immunochemistry.

Mouse retinal sections were obtained either from paraffin-embedded eyes or from fresh-frozen sections, washed in 0.01% Tween-20/PBS for 20 minutes, blocked overnight at 4°C with blocking solution (0.1% ovalbumin and 0.5% fish gelatin in PBS), and incubated for 1 hour with antibodies in the presence of blocking solution. Ciliated hTERT-RPE1 cells were washed briefly in PBS, fixed in 2% PFA/PBS for 10 minutes, treated with 1% Triton-X100/PBS for 5 minutes, and blocked with 2% BSA/PBS for 30 minutes. Mouse retinal sections and fixed hTERT-RPE1 cells were stained using anti-rhodopsin (1:500, Leico Technologies), anti-red/green cone opsin (1:250, Chemicon), anti-transducin (1:200, Santa Cruz Biotechnology Inc.), anti-arrestin (1:1,000, ABR), anti-NPHP4 (1:200, raised against N-terminal recombinant protein), anti-RPGRIP1 (1:1,000, provided by T. Li, National Eye Institute, Bethesda, Maryland, USA), anti-RP1 (1:2,000, gift of E. Pierce, Scheie Eye Institute, Philadelphia, Pennsylvania, USA), anti–γ-tubulin (1:500, Sigma-Aldrich), GT335 (1:1,000, detecting polyglutamylated tubulin, mouse monoclonal; provided by C. Janke, Institut Curie, Orsay, France), anti-acetylated tubulin (1:1,000, Sigma-Aldrich), anti-IFT88 (1:300, Abnova; 1:300, provided by G. Pazour, University of Massachusetts Medical School, Worcester, Massachusetts, USA), anti-TRAF3IP1 (1:300, Abnova), anti-WDR19 (1:300, Abnova), anti-IFT20 and anti-IFT140 (1:300, provided by G. Pazour), anti-RPGRIP1L (1:1,000, SNC040; ref. 52) or anti-lebercilin (1:300, ref. 4; 1:500, SN2134). Rabbit polyclonal antisera to lebercilin (SN2134 and SN2135) were raised against purified C-terminal human lebercilin (aa residues 491–697) by Eurogentec. Following primary antibody incubation, cells and sections were subsequently washed with PBS and incubated for 45 minutes with either Cy3-conjugated (1:250, Jackson Immunoresearch Laboratories) or Alexa Fluor 405, 488, or 568 (1:500, Invitrogen) secondary antibodies and embedded in either Prolong Gold Anti-fade (Invitrogen) for retinal sections or Vectashield with DAPI (Vector Laboratories) for hTERT-RPE1 cells. Cells and retinal sections were analyzed with a Zeiss Axio Imager Z1 fluorescence microscope (Carl Zeiss). Optical sections were generated through structured illumination by inserting an ApoTome slider into the illumination path, followed by processing with AxioVision (Carl Zeiss) and Photoshop CS4 software (Adobe Systems).

Immunoblot analysis.

Dissected retinas were lysed in NP-40 lysis buffer with sodium deoxycholate and EDTA, then supplemented with complete protease inhibitors (Roche). For protein analysis of TAP eluates, cells were lysed in lysis buffer containing 0.5% Nonidet-P40, protease inhibitor cocktail (Roche), and phosphatase inhibitor cocktails I and II (Sigma-Aldrich) in TBS (30 mM Tris-HCl, pH 7.4, and 150 mM NaCl) for 20 minutes at 4°C, and nuclei and cell debris were sedimented by centrifugation at 10,000 g for 10 minutes at 4°C. Equal protein amounts of lysates or eluates were separated by SDS-PAGE and electrophoretically transferred to nitrocellulose membranes. Membranes were blocked in 5% nonfat milk in TBS containing 0.1% Tween20 (Sigma-Aldrich) and incubated with anti-lebercilin (1:500, SN2135 and 1702#2, rabbit polyclonal antibody mix raised using multiple synthetic peptides covering aa 1–20, 493–512, and 667–686 of mouse lebercilin; Peptide Specialty Laboratories GmbH), anti–γ-tubulin (1:2,000, Sigma-Aldrich), anti-IFT74 (1:300, Everest Biotech), anti-IFT88 and anti-IFT57 (1:500, gifts of G. Pazour), anti-TRAF3IP1 (1:500, Abnova), anti–14-3-3ε (1:1,000, Santa Cruz Biotechnology Inc.), anti-GST (1:1,000, Sigma-Aldrich), and anti-FlagM2 (1:1,000, Sigma-Aldrich). Secondary antibodies from Jackson Immunoresearch were used (1:15,000). Protein bands were visualized by ECL plus (GE Healthcare).

This work was supported by the National Genome Research Framework program NGFN-Plus; the Helmholtz Alliance for Mental Health in an Aging Society (HelMA; to M. Ueffing); the European Community’s Seventh Framework Programme FP7/2009 under grant agreement no. 241955; SYSCILIA (to R. Roepman and M. Ueffing); the German Federal Ministry of Education and Research (BMBF, DYNAMO; FKZ, 0315513A; to M. Ueffing); the Kerstan Foundation (to M. Ueffing); grants from the Netherlands Organization for Scientific Research (NWO Vidi-91786396; to R. Roepman); the Foundation for Retinal Research (to A.I. den Hollander, R.K. Koenekoop, J. Bennett, F.P.M. Cremers, P.M. Nishina, and R. Roepman); the Foundation Fighting Blindness (to P.M. Nishina, R.K. Koenekoop, and R. Roepman); and institutional grant CA34196 to The Jackson Laboratory. We thank Chantal Longo-Guess and Kenneth Johnson for ABR tests of hearing function, Moniek Riemersma for help in cloning IFT constructs, Emine Bolat for qPCR assistance, Heleen Arts for help in lebercilin antibody production, and Theo Peters for retinal sectioning. We thank Bernd Wissinger and Nicholas Katsanis for critical reading of the manuscript. We thank Sandra Helm, Silke Becker, and Hakan Sarioglu from the Core Facility Proteomics at Helmholtz-Zentrum München for their excellent technical support.