Mutations in WHRN lead to Usher syndrome type 2d or to non-syndromic hearing impairment. The WHRN-encoded gene product whirlin directly interacts with the intracellular regions of the other two Usher syndrome type 2-associated proteins, usherin and ADGRV1. In photoreceptor cells, this protein complex constitutes fibrous links between the periciliary membrane and the connecting cilium. However, the molecular mechanism(s) of retinal degeneration due to compromised formation and function of the USH2-associated protein complex remains elusive. To unravel this pathogenic mechanism, we isolated and characterized whirlin-associated protein complexes from zebrafish photoreceptor cells. We generated transgenic zebrafish that express Strep/FLAG-tagged Whrna, a zebrafish ortholog of human whirlin, under the control of a photoreceptor-specific promoter. Affinity purification of Strep/FLAG-tagged Whrna and associated proteins from adult transgenic zebrafish retinas followed by mass spectrometry identified 19 novel candidate associated proteins. Pull down experiments and dedicated yeast two-hybrid assays confirmed the association of Whrna with 7 of the co-purified proteins. Several of the co-purified proteins are part of the synaptic proteome, which indicates a role for whirlin in the photoreceptor synapse. Future studies will elucidate which of the newly identified protein-protein interactions contribute to the development of the retinal phenotype observed in USH2d patients. SIGNIFICANCE: Since protein-protein interactions identified using targeted in vitro studies do not always recapitulate interactions that are functionally relevant in vivo, we established a transgenic zebrafish line that stably expresses a Strep/FLAG-tagged ortholog of human whirlin (SF-Whrna) in photoreceptor cells. Affinity purification of in vivo-assembled SF-Whrna-associated protein complexes from retinal lysates followed by mass spectrometry, identified 19 novel candidate interaction partners, many of which are enriched in the synaptic proteome. Two human orthologs of the identified candidate interaction partners, FRMPD4 and Kir2.3, were validated as direct interaction partners of human whirlin using a yeast two-hybrid assay. The strong connection of whirlin with postsynaptic density proteins was not identified in previous in vitro protein-protein interaction assays, presumably due to the absence of a biologically relevant context. Isolation and identification of in vivo-assembled whirlin-associated protein complexes from the tissue of interest is therefore a powerful methodology to obtain novel insight into tissue specific protein-protein interactions and has the potential to improve significantly our understanding of the function of whirlin and the molecular pathogenesis underlying Usher syndrome type 2.

Mutations in USH2A are the most frequent cause of Usher syndrome and autosomal recessive nonsyndromic retinitis pigmentosa. To unravel the pathogenic mechanisms underlying USH2A-associated retinal degeneration and to evaluate future therapeutic strategies that could potentially halt the progression of this devastating disorder, an animal model is needed. The available Ush2a knock-out mouse model does not mimic the human phenotype, because it presents with only a mild and late-onset retinal degeneration. Using CRISPR/Cas9-technology, we introduced protein-truncating germline lesions into the zebrafish ush2a gene (ush2armc1: c.2337_2342delinsAC; p.Cys780GlnfsTer32 and ush2ab1245: c.15520_15523delinsTG; p.Ala5174fsTer). Homozygous mutants were viable and displayed no obvious morphological or developmental defects. Immunohistochemical analyses with antibodies recognizing the N- or C-terminal region of the ush2a-encoded protein, usherin, demonstrated complete absence of usherin in photoreceptors of ush2armc1, but presence of the ectodomain of usherin at the periciliary membrane of ush2ab1245-derived photoreceptors. Furthermore, defects of usherin led to a reduction in localization of USH2 complex members, whirlin and Adgrv1, at the photoreceptor periciliary membrane of both mutants. Significantly elevated levels of apoptotic photoreceptors could be observed in both mutants when kept under constant bright illumination for three days. Electroretinogram (ERG) recordings revealed a significant and similar decrease in both a- and b-wave amplitudes in ush2armc1 as well as ush2ab1245 larvae as compared to strain- and age-matched wild-type larvae. In conclusion, this study shows that mutant ush2a zebrafish models present with early-onset retinal dysfunction that is exacerbated by light exposure. These models provide a better understanding of the pathophysiology underlying USH2A-associated RP and a unique opportunity to evaluate future therapeutic strategies.

The frequent deep-intronic c.7595-2144A>G mutation in intron 40 of USH2A generates a high-quality splice donor site, resulting in the incorporation of a pseudoexon (PE40) into the mature transcript that is predicted to prematurely terminate usherin translation. Aberrant USH2A pre-mRNA splicing could be corrected in patient-derived fibroblasts using antisense oligonucleotides. With the aim to study the effect of the c.7595-2144A>G mutation and USH2A splice redirection on retinal function, a humanized zebrafish knockin model was generated, in which 670 basepairs of ush2a intron 40 were exchanged for 557 basepairs of the corresponding human sequence using an optimized CRISPR/Cas9-based protocol. However, in the retina of adult homozygous humanized zebrafish, only 7.4% +/- 3.9% of ush2a transcripts contained the human PE40 sequence and immunohistochemical analyses revealed no differences in the usherin expression and localization between the retina of humanized and wild-type zebrafish larvae. Nevertheless, we were able to partially correct aberrant ush2a splicing using a PE40-targeting antisense morpholino. Our results indicate a clear difference in splice-site recognition by the human and zebrafish splicing machinery. Therefore, we propose a protocol in which the effect of human splice-modulating mutations is studied in a zebrafish-specific cell-based splice assay before the generation of a humanized zebrafish knockin model.

Program Number: 2485 Presentation Time: 8:30 AM–8:45 AM Photoreceptor-specific transition zone (PSTZ), a novel sub-region of the connecting cilium (CC), is maintained by retinal ciliopathy protein SPATA7 Rachayata Dharmat1, 2, Aiden Eblimit2, Yumei Li2, Michael Robichaux3, Zhixian Zhang3, Feng He3, Antrix Jain3, Graeme Mardon1, Sung Yun Jung3, Theodore G. Wensel3, Rui Chen1, 2. 1Molecular and Human Genetics, Baylor college of medicine, Houston, TX; 2HGSC, Baylor College of Medicine, Houston, TX; 3Dept of Biochemistry, Baylor College of Medicine, Houston, TX. Purpose: A hallmark of the photoreceptor sensory cilium is the presence of a specialized structural homolog of transition zone called the connecting cilium (CC). Interestingly, certain transition zone genes, such as SPATA7, specifically impairs the function of the CC without affecting the transition zone of primary cilia when mutated. To determine how the CC is functionally distinct from the transition zone, we investigated the impact of the loss of a photoreceptorspecific ciliary protein to probe the differences between the CC and the transition zone using Spata7 KO mice as the model. Methods: To understand the function of SPATA7 at the CC, we performed IP-MS based proteomic profiling to identify SPATA7-interacting proteins. We next assessed the localization of interacting partners in the absence of Spata7 and Sdccag8 using immunohistofluorescence and confirmed it using super-resolution STORM microscopy on the photoreceptors of P15 Spata7 KO mice. Since we observed morphological defects, we further assessed structural alterations of the microtubules using cryo-tomography. Results: SPATA7 interacts with the RPGR and NPHP complex and localizes throughout the length of the CC. In the absence of SPATA7, its interacting proteins are specifically absent (or excluded) from the distal CC which we named the photoreceptor-specific transition zone (“PSTZ”). However, the localization of CC proteins in the proximal CC (pCC) remains unaffected. In contrast, this peculiar phenotype is not observed in the absence of a pCC protein, SDCCAG8. Functionally, the PSTZ complex is important for stabilization of the CC structure as its absence leads to destabilization of ciliary microtubules specifically in the distal CC. This destabilization is caused due to the absence of CEP290, from the PSTZ region, a component of Y-links that are essential for the integrity of microtubules. Conclusions: Our data displays a novel photoreceptor-specific sub-region in the distal CC, termed the PSTZ, which plays a critical role in the functioning of the CC. Cilia-related proteins at the PSTZ are essential for maintaining the integrity of the microtubule core thereby stabilizing the CC. Hence we propose that this unique PSTZ region makes the CC functionally and structurally distinct from the transition zone found in other primary cilia. Commercial Relationships: Rachayata Dharmat, None; Aiden Eblimit, None; Yumei Li, None; Michael Robichaux, None; Zhixian Zhang, None; Feng He, None; Antrix Jain, None; Graeme Mardon, None; Sung Yun Jung, None; Theodore G. Wensel, None; Rui Chen, None Support: Grants from National Eye Institute (R01EY022356) and Retinal Research Foundation to R.C Program Number: 2486 Presentation Time: 8:45 AM–9:00 AM Contribution of autophagy to Usher syndrome pathogenesis Erik de Vrieze1, Ralph Slijkerman1, Margo Dona1, Sanne Broekman1, 2, Lisette Hetterschijt1, Theo Peters1, Hannie Kremer1, 2, Erwin van Wijk1. 1Otorhinolaryngology, Radboudumc, Nijmegen, Netherlands; 2Human Genetics, Radboudumc, Nijmegen, New Caledonia. Purpose: Usher syndrome (USH) is the most common cause of hereditary deaf-blindness. USH patients are born with congenital hearing impairment, and suffer from progressive vision loss (retinitis pigmentosa, RP), a combination that puts them at risk for social isolation and loss of independence. Mutations in the USH2A gene are the most frequent cause of USH, explaining up to 50% of all cases. Mutations in USH2A can also result in non-syndromic RP. Currently, virtually nothing is known about the pathogenesis of USH2A-associated RP. Methods: We used complementary proteomics techniques to identify novel interaction partners of USH2A. Using CRISPR/Cas9-mediated genome editing, we generated an ush2a zebrafish knockout model, carrying a protein-truncating mutation in exon 13. Guided by our proteomic studies, we deep-phenotyped our ush2a-/zebrafish. Results: We identified and validated interactions between multiple subunits of the Cop9 signalosome (CSN) and members of the USH protein complex. CSN subunit 8 (COPS8) is a direct interactor of USH2A. CSN/COPS8 is well documented to play a role in two proteostatic pathways: ubiquitin proteasome system (UPS) and autophagy. Immunohistochemistry showed that Cops8 localization in the photoreceptor overlaps with Ush2a, allowing interactions in vivo. Phenotypic analyses of the ush2a-/retina showed increased levels of photoreceptor apoptosis, as well as an increase in autophagosomes. However, we did not find evidence of increased activity of the UPS system. We also identified mislocalization of rhodopsin-containing transport vesicles. Surprisingly, ER stress (protein-accumulation in the endoplasmic reticulum) appears diminished. Conclusions: Using zebrafish as a model organism, we have identified that elevated levels of autophagy might be the pathogenic mechanism underlying USH2A-associated retinal degeneration. Whether this is a direct consequence of misregulated autophagy (through CSN), or is a response to the mislocalized transport vesicles, remains to be established. Both prolonged elevation of autophagy and activation of mislocalized photopigments can lead to the observed apoptosis of photoreceptors. The pathways that we have identified explain the slow progressive nature of the retinal degeneration in USH2A patients. This brings us closer to understanding the pathogenesis of USH2A-associated RP, which is also important for the development of future therapies. Commercial Relationships: Erik de Vrieze; Ralph Slijkerman, None; Margo Dona, None; Sanne Broekman, None; Lisette Hetterschijt, None; Theo Peters, None; Hannie Kremer, None; Erwin van Wijk, None

BACKGROUND:Recent findings suggesting that Abelson helper integration site 1 (AHI1) is involved in non-syndromic retinal disease have been debated, as the functional significance of identified missense variants was uncertain. We assessed whether AHI1 variants cause non-syndromic retinitis pigmentosa (RP). METHODS:Exome sequencing was performed in three probands with RP. The effects of the identified missense variants in AHI1 were predicted by three-dimensional structure homology modelling. Ciliary parameters were evaluated in patient's fibroblasts, and recombinant mutant proteins were expressed in ciliated retinal pigmented epithelium cells. RESULTS:In the three patients with RP, three sets of compound heterozygous variants were detected in AHI1 (c.2174G>A; p.Trp725* and c.2258A>T; p.Asp753Val, c.660delC; p.Ser221Glnfs*10 and c.2090C>T; p.Pro697Leu, c.2087A>G; p.His696Arg and c.2429C>T; p.Pro810Leu). All four missense variants were present in the conserved WD40 domain of Jouberin, the ciliary protein encoded by AHI1, with variable predicted implications for the domain structure. No significant changes in the percentage of ciliated cells, nor in cilium length or intraflagellar transport were detected. However, expression of mutant recombinant Jouberin in ciliated cells showed a significantly decreased enrichment at the ciliary base. CONCLUSIONS:This report confirms that mutations in AHI1 can underlie autosomal recessive RP. Moreover, it structurally and functionally validates the effect of the RP-associated AHI1 variants on protein function, thus proposing a new genotype-phenotype correlation for AHI1 mutation associated retinal ciliopathies.

Usher syndrome (USH) is the most common cause of combined deaf-blindness in man. The hearing loss can be partly compensated by providing patients with hearing aids or cochlear implants, but the loss of vision is currently untreatable. In general, mutations in the USH2A gene are the most frequent cause of USH explaining up to 50% of all patients worldwide. The first deep-intronic mutation in the USH2A gene (c.7595-2144A>G) was reported in 2012, leading to the insertion of a pseudoexon (PE40) into the mature USH2A transcript. When translated, this PE40-containing transcript is predicted to result in a truncated non-functional USH2A protein. In this study, we explored the potential of antisense oligonucleotides (AONs) to prevent aberrant splicing of USH2A pre-mRNA as a consequence of the c.7595-2144A>G mutation. Engineered 2'-O-methylphosphorothioate AONs targeting the PE40 splice acceptor site and/or exonic splice enhancer regions displayed significant splice correction potential in both patient derived fibroblasts and a minigene splice assay for USH2A c.7595-2144A>G, whereas a non-binding sense oligonucleotide had no effect on splicing. Altogether, AON-based splice correction could be a promising approach for the development of a future treatment for USH2A-associated retinitis pigmentosa caused by the deep-intronic c.7595-2144A>G mutation.

Cellular organelles provide opportunities to relate biological mechanisms to disease. Here we use affinity proteomics, genetics and cell biology to interrogate cilia: poorly understood organelles, where defects cause genetic diseases. Two hundred and seventeen tagged human ciliary proteins create a final landscape of 1,319 proteins, 4,905 interactions and 52 complexes. Reverse tagging, repetition of purifications and statistical analyses, produce a high-resolution network that reveals organelle-specific interactions and complexes not apparent in larger studies, and links vesicle transport, the cytoskeleton, signalling and ubiquitination to ciliary signalling and proteostasis. We observe sub-complexes in exocyst and intraflagellar transport complexes, which we validate biochemically, and by probing structurally predicted, disruptive, genetic variants from ciliary disease patients. The landscape suggests other genetic diseases could be ciliary including 3M syndrome. We show that 3M genes are involved in ciliogenesis, and that patient fibroblasts lack cilia. Overall, this organelle-specific targeting strategy shows considerable promise for Systems Medicine.

Results of the SF-TAP analysis with over-expressed N-terminally SF-TAP-tagged KIAA0556 in HEK293T cells. Shown is the number of unique identified peptides as well as the sequence coverage for each protein detected by mass spectrometry. Proteins identified in >1 out 17 SF-TAP control experiments (empty vector) were removed. (XLSX 28 kb)

Background Joubert syndrome (JBTS) and related disorders are defined by cerebellar malformation (molar tooth sign), together with neurological symptoms of variable expressivity. The ciliary basis of Joubert syndrome related disorders frequently extends the phenotype to tissues such as the eye, kidney, skeleton and craniofacial structures. Results Using autozygome and exome analyses, we identified a null mutation in KIAA0556 in a multiplex consanguineous family with hallmark features of mild Joubert syndrome. Patient-derived fibroblasts displayed reduced ciliogenesis potential and abnormally elongated cilia. Investigation of disease pathophysiology revealed that Kiaa0556 -/- null mice possess a Joubert syndrome-associated brain-restricted phenotype. Functional studies in Caenorhabditis elegans nematodes and cultured human cells support a conserved ciliary role for KIAA0556 linked to microtubule regulation. First, nematode KIAA0556 is expressed almost exclusively in ciliated cells, and the worm and human KIAA0556 proteins are enriched at the ciliary base. Second, C. elegans KIAA0056 regulates ciliary A-tubule number and genetically interacts with an ARL13B ( JBTS8 ) orthologue to control cilium integrity. Third, human KIAA0556 binds to microtubules in vitro and appears to stabilise microtubule networks when overexpressed. Finally, human KIAA0556 biochemically interacts with ciliary proteins and p60/p80 katanins. The latter form a microtubule-severing enzyme complex that regulates microtubule dynamics as well as ciliary functions. Conclusions We have identified KIAA0556 as a novel microtubule-associated ciliary base protein mutated in Joubert syndrome. Consistent with the mild patient phenotype, our nematode, mice and human cell data support the notion that KIAA0556 has a relatively subtle and variable cilia-related function, which we propose is related to microtubule regulation.

Ciliopathies are a group of human disorders caused by dysfunction of primary cilia, ubiquitous microtubule-based organelles involved in transduction of extra-cellular signals to the cell. This function requires the concentration of receptors and channels in the ciliary membrane, which is achieved by complex trafficking mechanisms, in part controlled by the small GTPase RAB8, and by sorting at the transition zone located at the entrance of the ciliary compartment. Mutations in the transition zone gene CC2D2A cause the related Joubert and Meckel syndromes, two typical ciliopathies characterized by central nervous system malformations, and result in loss of ciliary localization of multiple proteins in various models. The precise mechanisms by which CC2D2A and other transition zone proteins control protein entrance into the cilium and how they are linked to vesicular trafficking of incoming cargo remain largely unknown. In this work, we identify the centrosomal protein NINL as a physical interaction partner of CC2D2A. NINL partially co-localizes with CC2D2A at the base of cilia and ninl knockdown in zebrafish leads to photoreceptor outer segment loss, mislocalization of opsins and vesicle accumulation, similar to cc2d2a-/- phenotypes. Moreover, partial ninl knockdown in cc2d2a-/- embryos enhances the retinal phenotype of the mutants, indicating a genetic interaction in vivo, for which an illustration is found in patients from a Joubert Syndrome cohort. Similar to zebrafish cc2d2a mutants, ninl morphants display altered Rab8a localization. Further exploration of the NINL-associated interactome identifies MICAL3, a protein known to interact with Rab8 and to play an important role in vesicle docking and fusion. Together, these data support a model where CC2D2A associates with NINL to provide a docking point for cilia-directed cargo vesicles, suggesting a mechanism by which transition zone proteins can control the protein content of the ciliary compartment.

Ciliopathies are Mendelian disorders caused by dysfunction of cilia, ubiquitous organelles involved in fluid propulsion (motile cilia) or signal transduction (primary cilia). Retinal dystrophy is a common phenotypic characteristic of ciliopathies since photoreceptor outer segments are specialized primary cilia. These ciliary structures heavily rely on intracellular minus-end directed transport of cargo, mediated at least in part by the cytoplasmic dynein 1 motor complex, for their formation, maintenance and function. Ninein-like protein (NINL) is known to associate with this motor complex and is an important interaction partner of the ciliopathy-associated proteins lebercilin, USH2A and CC2D2A. Here, we scrutinize the function of NINL with combined proteomic and zebrafish in vivo approaches. We identify Double Zinc Ribbon and Ankyrin Repeat domains 1 (DZANK1) as a novel interaction partner of NINL and show that loss of Ninl, Dzank1 or both synergistically leads to dysmorphic photoreceptor outer segments, accumulation of trans-Golgi-derived vesicles and mislocalization of Rhodopsin and Ush2a in zebrafish. In addition, retrograde melanosome transport is severely impaired in zebrafish lacking Ninl or Dzank1. We further demonstrate that NINL and DZANK1 are essential for intracellular dynein-based transport by associating with complementary subunits of the cytoplasmic dynein 1 motor complex, thus shedding light on the structure and stoichiometry of this important motor complex. Altogether, our results support a model in which the NINL-DZANK1 protein module is involved in the proper assembly and folding of the cytoplasmic dynein 1 motor complex in photoreceptor cells, a process essential for outer segment formation and function.

Exome sequencing revealed a homozygous missense mutation (c.317C>G [p.Arg106Pro]) in POC1B, encoding POC1 centriolar protein B, in three siblings with autosomal-recessive cone dystrophy or cone-rod dystrophy and compound-heterozygous POC1B mutations (c.199_201del [p.G1n67del] and c.810+1G>T) in an unrelated person with cone-rod dystrophy. Upon overexpression of POC1B in human TERT-immortalized retinal pigment epithelium 1 cells, the encoded wild-type protein localized to the basal body of the primary cilium, whereas this localization was lost for p.Arg106Pro and p.G1n67del variant forms of POC1B. Morpholino-oligonucleotide-induced knockdown of poc1b translation in zebrafish resulted in a dose-dependent small-eye phenotype, impaired optokinetic responses, and decreased length of photoreceptor outer segments. These ocular phenotypes could partially be rescued by wild-type human POC1B mRNA, but not by c.199_201del and c.317C>G mutant human POC1B mRNAs. Yeast two-hybrid screening of a human retinal cDNA library revealed FAM161A as a binary interaction partner of POC1B. This was confirmed in coimmunoprecipitation and colocalization assays, which both showed loss of FAM161A interaction with p.Arg106Pro and p.G1n67del variant forms of POC1B. FAM161A was previously implicated in autosomal-recessive retinitis pigmentosa and shown to be located at the base of the photoreceptor connecting cilium, where it interacts with several other ciliopathy-associated proteins. Altogether, this study demonstrates that POC1B mutations result in a defect of the photoreceptor sensory cilium and thus affect cone and rod photoreceptors.

Familial exudative vitreoretinopathy (FEVR) is a genetically heterogeneous disorder characterized by abnormal vascularization of the peripheral retina, which can result in retinal detachment and severe visual impairment. In a large Dutch FEVR family, we performed linkage analysis, exome sequencing, and segregation analysis of DNA variants. We identified putative disease-causing DNA variants in proline-alanine-rich ste20-related kinase (c.791dup; p.Ser265ValfsX64) and zinc finger protein 408 (ZNF408) (c.1363C>T; p.His455Tyr), the latter of which was also present in an additional Dutch FEVR family that subsequently appeared to share a common ancestor with the original family. Sequence analysis of ZNF408 in 132 additional individuals with FEVR revealed another potentially pathogenic missense variant, p.Ser126Asn, in a Japanese family. Immunolocalization studies in COS-1 cells transfected with constructs encoding the WT and mutant ZNF408 proteins, revealed that the WT and the p.Ser126Asn mutant protein show complete nuclear localization, whereas the p.His455Tyr mutant protein was localized almost exclusively in the cytoplasm. Moreover, in a cotransfection assay, the p.His455Tyr mutant protein retains the WT ZNF408 protein in the cytoplasm, suggesting that this mutation acts in a dominant-negative fashion. Finally, morpholino-induced knockdown of znf408 in zebrafish revealed defects in developing retinal and trunk vasculature, that could be rescued by coinjection of RNA encoding human WT ZNF408 but not p.His455Tyr mutant ZNF408. Together, our data strongly suggest that mutant ZNF408 results in abnormal retinal vasculogenesis in humans and is associated with FEVR.

Cilia are microtubule-based cell appendages, serving motility, chemo-/mechano-/photo- sensation, and developmental signaling functions. Cilia are comprised of distinct structural and functional subregions including the basal body, transition zone (TZ) and inversin (Inv) compartments, and defects in this organelle are associated with an expanding spectrum of inherited disorders including Bardet-Biedl syndrome (BBS), Meckel-Gruber Syndrome (MKS), Joubert Syndrome (JS) and Nephronophthisis (NPHP). Despite major advances in understanding ciliary trafficking pathways such as intraflagellar transport (IFT), how proteins are transported to subciliary membranes remains poorly understood. Using Caenorhabditis elegans and mammalian cells, we investigated the transport mechanisms underlying compartmentalization of JS-associated ARL13B/ARL-13, which we previously found is restricted at proximal ciliary membranes. We now show evolutionary conservation of ARL13B/ARL-13 localisation to an Inv-like subciliary membrane compartment, excluding the TZ, in many C. elegans ciliated neurons and in a subset of mammalian ciliary subtypes. Compartmentalisation of C. elegans ARL-13 requires a C-terminal RVVP motif and membrane anchoring to prevent distal cilium and nuclear targeting, respectively. Quantitative imaging in more than 20 mutants revealed differential contributions for IFT and ciliopathy modules in defining the ARL-13 compartment; IFT-A/B, IFT-dynein and BBS genes prevent ARL-13 accumulation at periciliary membranes, whereas MKS/NPHP modules additionally inhibit ARL-13 association with TZ membranes. Furthermore, in vivo FRAP analyses revealed distinct roles for IFT and MKS/NPHP genes in regulating a TZ barrier to ARL-13 diffusion, and intraciliary ARL-13 diffusion. Finally, C. elegans ARL-13 undergoes IFT-like motility and quantitative protein complex analysis of human ARL13B identified functional associations with IFT-B complexes, mapped to IFT46 and IFT74 interactions. Together, these findings reveal distinct requirements for sequence motifs, IFT and ciliopathy modules in defining an ARL-13 subciliary membrane compartment. We conclude that MKS/NPHP modules comprise a TZ barrier to ARL-13 diffusion, whereas IFT genes predominantly facilitate ARL-13 ciliary entry and/or retention via active transport mechanisms.

Mutations in the gene for Usher syndrome 2A (USH2A) are causative for non-syndromic retinitis pigmentosa and Usher syndrome, a condition that is the most common cause of combined deaf-blindness. To gain insight into the molecular pathology underlying USH2A-associated retinal degeneration, we aimed to identify interacting proteins of USH2A isoform B (USH2AisoB) in the retina.

Retinitis pigmentosa (RP) is a retinal degenerative disease characterized by the progressive loss of photoreceptors. We have previously demonstrated that RP can be caused by recessive mutations in the human FAM161A gene, encoding a protein with unknown function that contains a conserved region shared only with a distant paralog, FAM161B. In this study, we show that FAM161A localizes at the base of the photoreceptor connecting cilium in human, mouse and rat. Furthermore, it is also present at the ciliary basal body in ciliated mammalian cells, both in native conditions and upon the expression of recombinant tagged proteins. Yeast two-hybrid analysis of binary interactions between FAM161A and an array of ciliary and ciliopathy-associated proteins reveals direct interaction with lebercilin, CEP290, OFD1 and SDCCAG8, all involved in hereditary retinal degeneration. These interactions are mediated by the C-terminal moiety of FAM161A, as demonstrated by pull-down experiments in cultured cell lines and in bovine retinal extracts. As other ciliary proteins, FAM161A can also interact with the microtubules and organize itself into microtubule-dependent intracellular networks. Moreover, small interfering RNA-mediated depletion of FAM161A transcripts in cultured cells causes the reduction in assembled primary cilia. Taken together, these data indicate that FAM161A-associated RP can be considered as a novel retinal ciliopathy and that its molecular pathogenesis may be related to other ciliopathies.

Mutations in LCA5 are causative for Leber congenital amaurosis, a severe hereditary retinal dystrophy in humans. Lebercilin, encoded by LCA5, localizes to connecting cilia of photoreceptor cells in the retina and specifically interacts with the intraflagellar transport (IFT) machinery. Bioinformatic analysis has identified lebercilin-like protein, previously known as C21orf13, as a lebercilin homolog in humans. In this study, we have characterized the molecular properties of lebercilin-like protein by defining the lebercilin-like interactome and assessing its (sub)cellular localization in ciliated cells. We show that lebercilin-like protein is embedded in a ciliary protein network and specifically localizes at the basal body and ciliary axoneme of ciliated cells, like lebercilin. mRNA expression studies indicate that lebercilin-like protein is preferentially expressed in tissues featuring motile cilia and/or flagella. Based on these data and bioinformatic co-expression profiling, we suggest that LCA5L is a likely candidate gene for motile ciliopathies such as Primary Ciliary Dyskinesia (PCD).