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Significance The photoreceptor outer segments are primary cilia, modified for phototransduction by incorporation of stacked opsin-loaded membrane disks that are continuously regenerated. This process is disrupted in several types of inherited retinal dystrophy, but the driving force remained unclear. We show that C2orf71/PCARE (photoreceptor cilium actin regulator), associated with inherited retinal dystrophy subtype RP54, efficiently recruits the Arp2/3 complex activator WASF3 to the cilium. This activates an actin dynamics-driven expansion of the ciliary tip, resembling membrane evagination in lamellipodia formation. Colocalization of this actin dynamics module to the base of the outer segments, and absence thereof in Pcare−/− mice, suggests PCARE-regulated actin dynamics as a critical process in outer segment disk formation., The outer segments (OS) of rod and cone photoreceptor cells are specialized sensory cilia that contain hundreds of opsin-loaded stacked membrane disks that enable phototransduction. The biogenesis of these disks is initiated at the OS base, but the driving force has been debated. Here, we studied the function of the protein encoded by the photoreceptor-specific gene C2orf71, which is mutated in inherited retinal dystrophy (RP54). We demonstrate that C2orf71/PCARE (photoreceptor cilium actin regulator) can interact with the Arp2/3 complex activator WASF3, and efficiently recruits it to the primary cilium. Ectopic coexpression of PCARE and WASF3 in ciliated cells results in the remarkable expansion of the ciliary tip. This process was disrupted by small interfering RNA (siRNA)-based down-regulation of an actin regulator, by pharmacological inhibition of actin polymerization, and by the expression of PCARE harboring a retinal dystrophy-associated missense mutation. Using human retinal organoids and mouse retina, we observed that a similar actin dynamics-driven process is operational at the base of the photoreceptor OS where the PCARE module and actin colocalize, but which is abrogated in Pcare−/− mice. The observation that several proteins involved in retinal ciliopathies are translocated to these expansions renders it a potential common denominator in the pathomechanisms of these hereditary disorders. Together, our work suggests that PCARE is an actin-associated protein that interacts with WASF3 to regulate the actin-driven expansion of the ciliary membrane at the initiation of new outer segment disk formation.

Contains fulltext : 225431.pdf (Publisher’s version ) (Closed access) Joubert syndrome (JBTS) is a recessive neurodevelopmental ciliopathy characterized by a pathognomonic hindbrain malformation. All known JBTS genes encode proteins involved in the structure or function of primary cilia, ubiquitous antenna-like organelles essential for cellular signal transduction. Here, we used the recently identified JBTS-associated protein armadillo repeat motif-containing 9 (ARMC9) in tandem-affinity purification and yeast 2-hybrid screens to identify a ciliary module whose dysfunction underlies JBTS. In addition to the known JBTS-associated proteins CEP104 and CSPP1, we identified coiled-coil domain containing 66 (CCDC66) and TOG array regulator of axonemal microtubules 1 (TOGARAM1) as ARMC9 interaction partners. We found that TOGARAM1 variants cause JBTS and disrupt TOGARAM1 interaction with ARMC9. Using a combination of protein interaction analyses, characterization of patient-derived fibroblasts, and analysis of CRISPR/Cas9-engineered zebrafish and hTERT-RPE1 cells, we demonstrated that dysfunction of ARMC9 or TOGARAM1 resulted in short cilia with decreased axonemal acetylation and polyglutamylation, but relatively intact transition zone function. Aberrant serum-induced ciliary resorption and cold-induced depolymerization in ARMC9 and TOGARAM1 patient cell lines suggest a role for this new JBTS-associated protein module in ciliary stability.

Joubert syndrome (JBTS) is a recessive neurodevelopmental ciliopathy, characterized by a pathognomonic hindbrain malformation. All known JBTS-genes encode proteins involved in the structure or function of primary cilia, ubiquitous antenna-like organelles essential for cellular signal transduction. Here, we use the recently identified JBTS-associated protein ARMC9 in tandem-affinity purification and yeast two-hybrid screens to identify a novel ciliary module composed of ARMC9-TOGARAM1-CCDC66-CEP104- CSPP1. TOGARAM1-variants cause JBTS and disrupt its interaction with ARMC9. Using a combination of protein interaction analyses and characterization of patient-derived fibroblasts, CRISPR/Cas9-engineered zebrafish and hTERT-RPE1 cells, we demonstrate that dysfunction of ARMC9 or TOGARAM1 results in short cilia with decreased axonemal acetylation and glutamylation, but relatively intact transition zone function. Aberrant serum-induced ciliary resorption and cold-induced depolymerization in both ARMC9 and TOGARAM1 patient cells lines suggest a role for this new JBTS-associated protein complex in ciliary stability.

The cilium is an essential organelle at the surface of mammalian cells whose dysfunction causes a wide range of genetic diseases collectively called ciliopathies. The current rate at which new ciliopathy genes are identified suggests that many ciliary components remain undiscovered. We generated and rigorously analyzed genomic, proteomic, transcriptomic and evolutionary data and systematically integrated these using Bayesian statistics into a predictive score for ciliary function. This resulted in 285 candidate ciliary genes. We generated independent experimental evidence of ciliary associations for 24 out of 36 analyzed candidate proteins using multiple cell and animal model systems (mouse, zebrafish and nematode) and techniques. For example, we show that OSCP1, which has previously been implicated in two distinct non-ciliary processes, causes ciliogenic and ciliopathy-associated tissue phenotypes when depleted in zebrafish. The candidate list forms the basis of CiliaCarta, a comprehensive ciliary compendium covering 956 genes. The resource can be used to objectively prioritize candidate genes in whole exome or genome sequencing of ciliopathy patients and can be accessed at http://bioinformatics.bio.uu.nl/john/syscilia/ciliacarta/. This work was supported by the European Community’s Seventh Framework Programme [241955, 278568 to MU and KB, 602273 to RS]; the Virgo consortium, funded by the Dutch government [FES0908 to TvD, RvdL and MAH]; the Netherlands Genomics Initiative [050-060-452 to TvD, RvdL and MAH]; the Canadian Institutes of Health Research [MOP-142243, MOP-82870 and PJT-156042 to MRL]; Michael Smith Foundation for Health Research to MRL and VLJ; Kidney Research Scientist Core Education and National Training fellowship to VLJ; The Foundation Fighting Blindness [PPA-0717-0719-RAD to UW, RR, and MU]; the Dutch Kidney Foundation “KOUNCIL” consortium [CP11.18 to RHG, PLB and RR]; The Deutsche Forschungsgemeinschaft [Excellence grant CellNetworks to RBR and QL, CRC1140 “Kidney Disease – From Genes to Mechanisms” to MS, collaborative research center grant SFB-1411 KIDGEM to MS]; Metakids Foundation to RS; the National Institute for Health Research to PLB and VH-H. PLB is an NIHR Senior Investigator; Radboudumc Hypatia Tenure Track Fellowship, Radboud Universiteit excellence fellowship, ERC starting grant TREATCilia, grant agreement no. 716344 to MS; and the Netherlands Organization for Scientific Research [NWO Vici-865.12.005 to RR].

Contains fulltext : 182652.pdf (Publisher’s version ) (Open Access) The Rab GTPase family comprises approximately 70 GTP-binding proteins, functioning in vesicle formation, transport and fusion. They are activated by a conformational change induced by GTP-binding, allowing interactions with downstream effectors. Here, we report five individuals with two recurrent de novo missense mutations in RAB11B; c.64G>A; p.Val22Met in three individuals and c.202G>A; p.Ala68Thr in two individuals. An overlapping neurodevelopmental phenotype, including severe intellectual disability with absent speech, epilepsy, and hypotonia was observed in all affected individuals. Additionally, visual problems, musculoskeletal abnormalities, and microcephaly were present in the majority of cases. Re-evaluation of brain MRI images of four individuals showed a shared distinct brain phenotype, consisting of abnormal white matter (severely decreased volume and abnormal signal), thin corpus callosum, cerebellar vermis hypoplasia, optic nerve hypoplasia and mild ventriculomegaly. To compare the effects of both variants with known inactive GDP- and active GTP-bound RAB11B mutants, we modeled the variants on the three-dimensional protein structure and performed subcellular localization studies. We predicted that both variants alter the GTP/GDP binding pocket and show that they both have localization patterns similar to inactive RAB11B. Evaluation of their influence on the affinity of RAB11B to a series of binary interactors, both effectors and guanine nucleotide exchange factors (GEFs), showed induction of RAB11B binding to the GEF SH3BP5, again similar to inactive RAB11B. In conclusion, we report two recurrent dominant mutations in RAB11B leading to a neurodevelopmental syndrome, likely caused by altered GDP/GTP binding that inactivate the protein and induce GEF binding and protein mislocalization.

Contains fulltext : 177221.pdf (Publisher’s version ) (Open Access) 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.

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.

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., Author Summary The cytoplasmic dynein 1 motor complex is known to be essential for photoreceptor outer segment formation and function. NINL, an important interaction partner of three ciliopathy-associated proteins (lebercilin, USH2A and CC2D2A), was previously shown to associate with this motor complex. In this work, we scrutinize the role of NINL using a combination of affinity proteomics and zebrafish studies, in order to gain insight into the pathogenic mechanisms underlying these three associated hereditary disorders. We identify DZANK1 as an important interaction partner of NINL and show that loss of Ninl, Dzank1, or a combination of both synergistically results in impaired transport of trans Golgi-derived vesicles and, as a consequence, defective photoreceptor outer segment formation. Using affinity proteomics, we demonstrate that NINL and DZANK1 associate with complementary subunits of the cytoplasmic dynein 1 complex. Our results support a model in which the NINL-DZANK1 protein module is essential for the proper assembly and folding of the cytoplasmic dynein 1 motor complex, shedding light on the structure and stoichiometry of this important motor complex.

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., Author Summary Ciliopathies are a group of disorders caused by dysfunction of primary cilia, ubiquitous organelles involved in signal transduction. Mutations in CC2D2A cause two ciliopathies, Joubert and Meckel syndromes, and result in loss of ciliary protein localization. The mechanism by which CC2D2A, located at the ciliary transition zone, controls ciliary protein composition and its link to vesicular trafficking of incoming cargo remain largely unknown. Here, we identify a series of physical interactions linking CC2D2A to vesicular trafficking controlled by the small GTPase RAB8, suggesting a new model, whereby CC2D2A provides a specific docking point for ciliary-bound vesicles at the entrance to the ciliary compartment. We first identify NINL as a physical and genetic interaction partner of CC2D2A, show that both proteins co-localize at the entrance to the cilium and demonstrate that absence of Ninl or Cc2d2a result in similar retinal phenotypes in zebrafish, including mislocalization of Rab8. We further identify MICAL3, a protein known to bind RAB8, as another NINL interaction partner, thus linking CC2D2A to RAB8A-controlled trafficking. Finally, we describe an individual with Joubert syndrome, in whom combined CC2D2A and NINL mutations result in an enhanced phenotype, illustrating the impact of the detected interaction on the disease.

Summary Cilia are small antenna-like cellular protrusions critical for many developmental signaling pathways. The ciliary protein Arl3 has been shown to act as a specific release factor for myristoylated and farnesylated ciliary cargo molecules by binding to the effectors Unc119 and PDE6δ. Here we describe a newly identified Arl3 binding partner, CCDC104/CFAP36. Biochemical and structural analyses reveal that the protein contains a BART-like domain and is called BARTL1. It recognizes an LLxILxxL motif at the N-terminal amphipathic helix of Arl3, which is crucial for the interaction with the BART-like domain but also for the ciliary localization of Arl3 itself. These results seem to suggest a ciliary role of BARTL1, and possibly link it to the Arl3 transport network. We thus speculate on a regulatory mechanism whereby BARTL1 aids the presentation of active Arl3 to its GTPase-activating protein RP2 or hinders Arl3 membrane binding in the area of the transition zone., Graphical Abstract, Highlights • Identification of a BART-like protein, CCDC104/BARTL1, as Arl3 binding partner • BARTL1 is a ciliary protein with transition zone localization • Complex structure shows BARTL1 binding to conserved LLxILxxL N-term motif of Arl3 • N-terminal amphipathic helix of Arl3 is crucial for its ciliary localization, Lokaj et al. biochemically and structurally characterize BARTL1 as an Arl3 effector and speculate on its function in cilia.

Item does not contain fulltext Leber congenital amaurosis (LCA) and juvenile retinitis pigmentosa (RP) are severe hereditary diseases that causes visual impairment in infants and children. SPATA7 has recently been identified as the LCA3 and juvenile RP gene in humans, whose function in the retina remains elusive. Here, we show that SPATA7 localizes at the primary cilium of cells and at the connecting cilium (CC) of photoreceptor cells, indicating that SPATA7 is a ciliary protein. In addition, SPATA7 directly interacts with the retinitis pigmentosa GTPase regulator interacting protein 1 (RPGRIP1), a key connecting cilium protein that has also been linked to LCA. In the retina of Spata7 null mutant mice, a substantial reduction of RPGRIP1 levels at the CC of photoreceptor cells is observed, suggesting that SPATA7 is required for the stable assembly and localization of the ciliary RPGRIP1 protein complex. Furthermore, our results pinpoint a role of this complex in protein trafficking across the CC to the outer segments, as we identified that rhodopsin accumulates in the inner segments and around the nucleus of photoreceptors. This accumulation then likely triggers the apoptosis of rod photoreceptors that was observed. Loss of Spata7 function in mice indeed results in a juvenile RP-like phenotype, characterized by progressive degeneration of photoreceptor cells and a strongly decreased light response. Together, these results indicate that SPATA7 functions as a key member of a retinal ciliopathy-associated protein complex, and that apoptosis of rod photoreceptor cells triggered by protein mislocalization is likely the mechanism of disease progression in LCA3/ juvenile RP patients.