RESUMEN
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.
Asunto(s)
Cilios/genética , Distrofias de Conos y Bastones/genética , Proteínas del Ojo/genética , Segmento Externo de la Célula en Bastón/metabolismo , Familia de Proteínas del Síndrome de Wiskott-Aldrich/genética , Complejo 2-3 Proteico Relacionado con la Actina/genética , Actinas/genética , Animales , Cilios/patología , Distrofias de Conos y Bastones/patología , Modelos Animales de Enfermedad , Regulación de la Expresión Génica/genética , Humanos , Ratones , Ratones Noqueados , ARN Interferente Pequeño/genética , Células Fotorreceptoras Retinianas Conos/metabolismo , Células Fotorreceptoras Retinianas Conos/patología , Segmento Externo de la Célula en Bastón/patologíaRESUMEN
The Rab GTPase family comprises â¼70 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.
Asunto(s)
Epilepsia/genética , Discapacidad Intelectual/genética , Hipotonía Muscular/genética , Mutación , Enfermedades del Nervio Óptico/congénito , Proteínas de Unión al GTP rab/genética , Adolescente , Secuencia de Aminoácidos , Sitios de Unión , Vermis Cerebeloso/diagnóstico por imagen , Vermis Cerebeloso/metabolismo , Vermis Cerebeloso/patología , Niño , Preescolar , Cuerpo Calloso/diagnóstico por imagen , Cuerpo Calloso/metabolismo , Cuerpo Calloso/patología , Epilepsia/diagnóstico por imagen , Epilepsia/patología , Femenino , Expresión Génica , Guanosina Difosfato/química , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/química , Guanosina Trifosfato/metabolismo , Humanos , Discapacidad Intelectual/diagnóstico por imagen , Discapacidad Intelectual/patología , Imagen por Resonancia Magnética , Masculino , Modelos Moleculares , Hipotonía Muscular/diagnóstico por imagen , Hipotonía Muscular/patología , Enfermedades del Nervio Óptico/diagnóstico por imagen , Enfermedades del Nervio Óptico/genética , Enfermedades del Nervio Óptico/patología , Fenotipo , Unión Proteica , Sustancia Blanca/diagnóstico por imagen , Sustancia Blanca/metabolismo , Sustancia Blanca/patología , Proteínas de Unión al GTP rab/química , Proteínas de Unión al GTP rab/deficienciaRESUMEN
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.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Mutación Missense , Retinitis Pigmentosa/genética , Anomalías Múltiples/genética , Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras del Transporte Vesicular , Adulto , Cerebelo/anomalías , Anomalías del Ojo/genética , Femenino , Humanos , Enfermedades Renales Quísticas/genética , Masculino , Persona de Mediana Edad , Linaje , Dominios Proteicos/genética , Retina/anomalíasRESUMEN
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.
Asunto(s)
Proteínas Portadoras/genética , Dineínas/genética , Larva/genética , Proteínas Asociadas a Microtúbulos/genética , Proteínas Nucleares/genética , Células Fotorreceptoras de Vertebrados , Retina/crecimiento & desarrollo , Proteínas de Pez Cebra/genética , Animales , Transporte Biológico/genética , Cilios/genética , Células HEK293 , Humanos , Larva/crecimiento & desarrollo , Neurogénesis/genética , Proteómica , Transducción de Señal , Pez Cebra/genética , Pez Cebra/crecimiento & desarrolloRESUMEN
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.
Asunto(s)
Cerebelo/anomalías , Trastornos de la Motilidad Ciliar/genética , Encefalocele/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Oxigenasas de Función Mixta/genética , Proteínas Nucleares/metabolismo , Enfermedades Renales Poliquísticas/genética , Proteínas/genética , Retina/anomalías , Proteínas de Unión al GTP rab/genética , Anomalías Múltiples/genética , Anomalías Múltiples/metabolismo , Anomalías Múltiples/patología , Animales , Cerebelo/metabolismo , Cerebelo/patología , Cilios/genética , Cilios/metabolismo , Cilios/patología , Trastornos de la Motilidad Ciliar/metabolismo , Trastornos de la Motilidad Ciliar/patología , Proteínas del Citoesqueleto , Encefalocele/metabolismo , Encefalocele/patología , Anomalías del Ojo/genética , Anomalías del Ojo/metabolismo , Anomalías del Ojo/patología , Técnicas de Silenciamiento del Gen , Humanos , Enfermedades Renales Quísticas/genética , Enfermedades Renales Quísticas/metabolismo , Enfermedades Renales Quísticas/patología , Proteínas Asociadas a Microtúbulos/genética , Oxigenasas de Función Mixta/metabolismo , Mutación , Proteínas Nucleares/genética , Enfermedades Renales Poliquísticas/metabolismo , Enfermedades Renales Poliquísticas/patología , Transporte de Proteínas/genética , Proteínas/metabolismo , Retina/metabolismo , Retina/patología , Retinitis Pigmentosa , Transducción de Señal , Pez Cebra , Proteínas de Unión al GTP rab/metabolismoRESUMEN
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.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Cilio Conector de los Fotorreceptores/patología , Proteínas/metabolismo , Células Fotorreceptoras Retinianas Bastones/patología , Animales , Apoptosis , Bovinos , Proteínas del Citoesqueleto , Proteínas de Unión al ADN/genética , Eliminación de Gen , Humanos , Ratones , Ratones Mutantes , Cilio Conector de los Fotorreceptores/metabolismo , Transporte de Proteínas , Células Fotorreceptoras Retinianas Conos/patología , Células Fotorreceptoras Retinianas Bastones/metabolismo , Rodopsina/metabolismoRESUMEN
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.
Asunto(s)
Factores de Ribosilacion-ADP/genética , Caenorhabditis elegans/genética , Enfermedades Cerebelosas/genética , Cilios/genética , Anomalías del Ojo/genética , Enfermedades Renales Quísticas/genética , Retina/anomalías , Factores de Ribosilacion-ADP/metabolismo , Anomalías Múltiples , Animales , Síndrome de Bardet-Biedl/genética , Síndrome de Bardet-Biedl/patología , Transporte Biológico Activo/genética , Caenorhabditis elegans/metabolismo , Enfermedades Cerebelosas/patología , Cerebelo/anomalías , Cilios/metabolismo , Trastornos de la Motilidad Ciliar/genética , Trastornos de la Motilidad Ciliar/patología , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/metabolismo , Encefalocele/genética , Encefalocele/patología , Anomalías del Ojo/patología , Humanos , Enfermedades Renales Quísticas/patología , Membranas/metabolismo , Enfermedades Renales Poliquísticas/genética , Enfermedades Renales Poliquísticas/patología , Retina/patología , Retinitis Pigmentosa , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Protein-protein interaction analyses have uncovered a ciliary and basal body protein network that, when disrupted, can result in nephronophthisis (NPHP), Leber congenital amaurosis, Senior-Løken syndrome (SLSN) or Joubert syndrome (JBTS). However, details of the molecular mechanisms underlying these disorders remain poorly understood. RPGRIP1-like protein (RPGRIP1L) is a homolog of RPGRIP1 (RPGR-interacting protein 1), a ciliary protein defective in Leber congenital amaurosis. We show that RPGRIP1L interacts with nephrocystin-4 and that mutations in the gene encoding nephrocystin-4 (NPHP4) that are known to cause SLSN disrupt this interaction. RPGRIP1L is ubiquitously expressed, and its protein product localizes to basal bodies. Therefore, we analyzed RPGRIP1L as a candidate gene for JBTS and identified loss-of-function mutations in three families with typical JBTS, including the characteristic mid-hindbrain malformation. This work identifies RPGRIP1L as a gene responsible for JBTS and establishes a central role for cilia and basal bodies in the pathophysiology of this disorder.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Enfermedades Cerebelosas/genética , Cilios/genética , Trastornos de la Motilidad Ciliar/genética , Oftalmopatías/genética , Enfermedades Renales/genética , Proteínas/genética , Proteínas/metabolismo , Adulto , Animales , Línea Celular , Proteínas del Citoesqueleto , Femenino , Humanos , Masculino , Datos de Secuencia Molecular , Linaje , Ratas , SíndromeRESUMEN
Leber congenital amaurosis (LCA) causes blindness or severe visual impairment at or within a few months of birth. Here we show, using homozygosity mapping, that the LCA5 gene on chromosome 6q14, which encodes the previously unknown ciliary protein lebercilin, is associated with this disease. We detected homozygous nonsense and frameshift mutations in LCA5 in five families affected with LCA. In a sixth family, the LCA5 transcript was completely absent. LCA5 is expressed widely throughout development, although the phenotype in affected individuals is limited to the eye. Lebercilin localizes to the connecting cilia of photoreceptors and to the microtubules, centrioles and primary cilia of cultured mammalian cells. Using tandem affinity purification, we identified 24 proteins that link lebercilin to centrosomal and ciliary functions. Members of this interactome represent candidate genes for LCA and other ciliopathies. Our findings emphasize the emerging role of disrupted ciliary processes in the molecular pathogenesis of LCA.
Asunto(s)
Proteínas del Ojo/genética , Proteínas Asociadas a Microtúbulos/genética , Atrofia Óptica Hereditaria de Leber/genética , Animales , Células COS , Línea Celular , Chlorocebus aethiops , Cilios/genética , Codón sin Sentido , Proteínas del Ojo/metabolismo , Femenino , Mutación del Sistema de Lectura , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Proteínas Asociadas a Microtúbulos/metabolismo , Datos de Secuencia Molecular , Linaje , Ratas , Ratas WistarRESUMEN
Mutations in PDE6D impair the function of its cognate protein, phosphodiesterase 6D (PDE6D), in prenylated protein trafficking towards the ciliary membrane, causing the human ciliopathy Joubert Syndrome (JBTS22) and retinal degeneration in mice. In this study, we purified the prenylated cargo of PDE6D by affinity proteomics to gain insight into PDE6D-associated disease mechanisms. By this approach, we have identified a specific set of PDE6D-interacting proteins that are involved in photoreceptor integrity, GTPase activity, nuclear import, or ubiquitination. Among these interacting proteins, we identified novel ciliary cargo proteins of PDE6D, including FAM219A, serine/threonine-protein kinase NIM1 (NIM1K), and ubiquitin-like protein 3 (UBL3). We show that NIM1K and UBL3 localize inside the cilium in a prenylation-dependent manner. Furthermore, UBL3 also localizes in vesicle-like structures around the base of the cilium. Through affinity proteomics of UBL3, we confirmed its strong interaction with PDE6D and its association with proteins that regulate small extracellular vesicles (sEVs) and ciliogenesis. Moreover, we show that UBL3 localizes in specific photoreceptor cilium compartments in a prenylation-dependent manner. Therefore, we propose that UBL3 may play a role in the sorting of proteins towards the photoreceptor outer segment, further explaining the development of PDE6D-associated retinal degeneration.
Asunto(s)
Cilios , Degeneración Retiniana , Humanos , Animales , Ratones , Cilios/metabolismo , Degeneración Retiniana/metabolismo , Proteínas/metabolismo , Retina/metabolismo , Transporte de Proteínas , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 6/metabolismoRESUMEN
Establishment and maintenance of the primary cilium as a signaling-competent organelle requires a high degree of fine tuning, which is at least in part achieved by a variety of post-translational modifications. One such modification is ubiquitination. The small and highly conserved ubiquitin protein possesses a unique versatility in regulating protein function via its ability to build mono and polyubiquitin chains onto target proteins. We aimed to take an unbiased approach to generate a comprehensive blueprint of the ciliary ubiquitinome by deploying a multi-proteomics approach using both ciliary-targeted ubiquitin affinity proteomics, as well as ubiquitin-binding domain-based proximity labelling in two different mammalian cell lines. This resulted in the identification of several key proteins involved in signaling, cytoskeletal remodeling and membrane and protein trafficking. Interestingly, using two different approaches in IMCD3 and RPE1 cells, respectively, we uncovered several novel mechanisms that regulate cilia function. In our IMCD3 proximity labeling cell line model, we found a highly enriched group of ESCRT-dependent clathrin-mediated endocytosis-related proteins, suggesting an important and novel role for this pathway in the regulation of ciliary homeostasis and function. In contrast, in RPE1 cells we found that several structural components of caveolae (CAV1, CAVIN1, and EHD2) were highly enriched in our cilia affinity proteomics screen. Consistently, the presence of caveolae at the ciliary pocket and ubiquitination of CAV1 specifically, were found likely to play a role in the regulation of ciliary length in these cells. Cilia length measurements demonstrated increased ciliary length in RPE1 cells stably expressing a ubiquitination impaired CAV1 mutant protein. Furthermore, live cell imaging in the same cells revealed decreased CAV1 protein turnover at the cilium as the possible cause for this phenotype. In conclusion, we have generated a comprehensive list of cilia-specific proteins that are subject to regulation via ubiquitination which can serve to further our understanding of cilia biology in health and disease.
RESUMEN
BACKGROUND: Sensenbrenner syndrome is a heterogeneous ciliopathy that is characterised by skeletal and ectodermal anomalies, accompanied by chronic renal failure, heart defects, liver fibrosis and other features. OBJECTIVE: To identify an additional causative gene in Sensenbrenner syndrome. METHODS: Single nucleotide polymorphism array analysis and standard sequencing techniques were applied to identify the causative gene. The effect of the identified mutation on protein translation was determined by western blot analysis. Antibodies against intraflagellar transport (IFT) proteins were used in ciliated fibroblast cell lines to investigate the molecular consequences of the mutation on ciliary transport. RESULTS: Homozygosity mapping and positional candidate gene sequence analysis were performed in two siblings with Sensenbrenner syndrome of a consanguineous Moroccan family. In both siblings, a homozygous mutation in the initiation codon of C14ORF179 was identified. C14ORF179 encodes IFT43, a subunit of the IFT complex A (IFT-A) machinery of primary cilia. Western blots showed that the mutation disturbs translation of IFT43, inducing the initiation of translation of a shorter protein product from a downstream ATG. The IFT-A protein complex is implicated in retrograde ciliary transport along axonemal microtubules. It was shown that in fibroblasts of one of the siblings affected by Sensenbrenner syndrome, disruption of IFT43 disturbs this transport from the ciliary tip to its base. As anterograde transport in the opposite direction apparently remains functional, the IFT complex B proteins accumulate in the ciliary tip. Interestingly, similar results were obtained using fibroblasts from a patient with Sensenbrenner syndrome with mutations in WDR35/IFT121, encoding another IFT-A subunit. CONCLUSIONS: The results indicate that Sensenbrenner syndrome is caused by disrupted IFT-A-mediated retrograde ciliary transport.
Asunto(s)
Proteínas Portadoras/genética , Cilios/metabolismo , Anomalías Craneofaciales/genética , Displasia Ectodérmica/genética , Flagelos/metabolismo , Transporte de Proteínas/genética , Proteínas Recombinantes/genética , Animales , Secuencia de Bases , Proteínas Portadoras/metabolismo , Niño , Cilios/genética , Anomalías Craneofaciales/etnología , Displasia Ectodérmica/etnología , Fibroblastos/fisiología , Flagelos/genética , Células HEK293 , Humanos , Masculino , Datos de Secuencia Molecular , Marruecos/etnología , Mutación , Países Bajos/epidemiología , Análisis de Secuencia por Matrices de Oligonucleótidos , Polimorfismo de Nucleótido Simple , Proteínas Recombinantes/metabolismo , Hermanos , Síndrome , TransfecciónRESUMEN
Joubert syndrome and related disorders (JSRD) are primarily autosomal-recessive conditions characterized by hypotonia, ataxia, abnormal eye movements, and intellectual disability with a distinctive mid-hindbrain malformation. Variable features include retinal dystrophy, cystic kidney disease, and liver fibrosis. JSRD are included in the rapidly expanding group of disorders called ciliopathies, because all six gene products implicated in JSRD (NPHP1, AHI1, CEP290, RPGRIP1L, TMEM67, and ARL13B) function in the primary cilium/basal body organelle. By using homozygosity mapping in consanguineous families, we identify loss-of-function mutations in CC2D2A in JSRD patients with and without retinal, kidney, and liver disease. CC2D2A is expressed in all fetal and adult tissues tested. In ciliated cells, we observe localization of recombinant CC2D2A at the basal body and colocalization with CEP290, whose cognate gene is mutated in multiple hereditary ciliopathies. In addition, the proteins can physically interact in vitro, as shown by yeast two-hybrid and GST pull-down experiments. A nonsense mutation in the zebrafish CC2D2A ortholog (sentinel) results in pronephric cysts, a hallmark of ciliary dysfunction analogous to human cystic kidney disease. Knockdown of cep290 function in sentinel fish results in a synergistic pronephric cyst phenotype, revealing a genetic interaction between CC2D2A and CEP290 and implicating CC2D2A in cilium/basal body function. These observations extend the genetic spectrum of JSRD and provide a model system for studying extragenic modifiers in JSRD and other ciliopathies.
Asunto(s)
Anomalías Múltiples/genética , Antígenos de Neoplasias/metabolismo , Mutación , Proteínas de Neoplasias/metabolismo , Proteínas/genética , Proteínas/metabolismo , Antígenos de Neoplasias/genética , Ataxia/genética , Proteínas de Ciclo Celular , Cerebelo/anomalías , Cerebelo/diagnóstico por imagen , Mapeo Cromosómico , Cromosomas Humanos Par 4 , Cilios/genética , Estudios de Cohortes , Consanguinidad , Proteínas del Citoesqueleto , Exones , Marcadores Genéticos , Haplotipos , Homocigoto , Humanos , Inmunohistoquímica , Enfermedades Renales Quísticas/genética , Masculino , Repeticiones de Microsatélite , Hipotonía Muscular/genética , Proteínas de Neoplasias/genética , Trastornos de la Motilidad Ocular/genética , Linaje , Polimorfismo de Nucleótido Simple , Radiografía , Proteínas Recombinantes/metabolismo , Análisis de Secuencia de ADN , Síndrome , Técnicas del Sistema de Dos HíbridosRESUMEN
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.
Asunto(s)
Anomalías Múltiples , Proteínas del Dominio Armadillo , Cerebelo/anomalías , Cilios , Anomalías del Ojo , Enfermedades Renales Quísticas , Retina/anomalías , Proteínas de Pez Cebra , Pez Cebra , Anomalías Múltiples/genética , Anomalías Múltiples/metabolismo , Acetilación , Animales , Proteínas del Dominio Armadillo/genética , Proteínas del Dominio Armadillo/metabolismo , Sistemas CRISPR-Cas , Cerebelo/metabolismo , Cilios/genética , Cilios/metabolismo , Modelos Animales de Enfermedad , Anomalías del Ojo/genética , Anomalías del Ojo/metabolismo , Humanos , Enfermedades Renales Quísticas/genética , Enfermedades Renales Quísticas/metabolismo , Péptidos/genética , Péptidos/metabolismo , Retina/metabolismo , Pez Cebra/genética , Pez Cebra/metabolismo , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
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/.
Asunto(s)
Cilios/genética , Genómica , Animales , Teorema de Bayes , Caenorhabditis elegans/citología , Caenorhabditis elegans/genética , Anotación de Secuencia Molecular , Fenotipo , Reproducibilidad de los Resultados , Células Receptoras Sensoriales/metabolismo , Pez Cebra/genéticaRESUMEN
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.
Asunto(s)
Cilios/metabolismo , Ciliopatías/genética , Enanismo/genética , Hipotonía Muscular/genética , Mapas de Interacción de Proteínas , Proteínas/metabolismo , Columna Vertebral/anomalías , Transporte Biológico/fisiología , Cromatografía de Afinidad/métodos , Ciliopatías/patología , Ciliopatías/terapia , Análisis Mutacional de ADN , Conjuntos de Datos como Asunto , Enanismo/patología , Enanismo/terapia , Fibroblastos , Células HEK293 , Humanos , Espectrometría de Masas , Terapia Molecular Dirigida/métodos , Hipotonía Muscular/patología , Hipotonía Muscular/terapia , Mapeo de Interacción de Proteínas/métodos , Proteínas/genética , Proteínas/aislamiento & purificación , Proteómica/métodos , Columna Vertebral/patología , Análisis de SistemasRESUMEN
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.
Asunto(s)
Factores de Ribosilacion-ADP/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Sitios de Unión , Línea Celular , Cilios/metabolismo , Ratones , Datos de Secuencia Molecular , Unión Proteica , Transporte de Proteínas , ProteínasRESUMEN
The analysis of individuals with ciliary chondrodysplasias can shed light on sensitive mechanisms controlling ciliogenesis and cell signalling that are essential to embryonic development and survival. Here we identify TCTEX1D2 mutations causing Jeune asphyxiating thoracic dystrophy with partially penetrant inheritance. Loss of TCTEX1D2 impairs retrograde intraflagellar transport (IFT) in humans and the protist Chlamydomonas, accompanied by destabilization of the retrograde IFT dynein motor. We thus define TCTEX1D2 as an integral component of the evolutionarily conserved retrograde IFT machinery. In complex with several IFT dynein light chains, it is required for correct vertebrate skeletal formation but may be functionally redundant under certain conditions.
Asunto(s)
Dineínas/genética , Síndrome de Ellis-Van Creveld/genética , Flagelos/fisiología , Animales , Chlamydomonas reinhardtii , Proteínas del Citoesqueleto , Técnicas de Silenciamiento del Gen , Células HEK293 , Humanos , Ratones , Mutación , Penetrancia , Pez CebraRESUMEN
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.
Asunto(s)
Cuerpos Basales/metabolismo , Cerebelo/anomalías , Proteínas Asociadas a Microtúbulos/genética , Mutación , Retina/anomalías , Factores de Ribosilacion-ADP/metabolismo , Anomalías Múltiples/genética , Anomalías Múltiples/patología , Adenosina Trifosfatasas/metabolismo , Adulto , Animales , Cuerpos Basales/patología , Encéfalo/metabolismo , Encéfalo/patología , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Células Cultivadas , Cerebelo/patología , Niño , Preescolar , Cilios/genética , Cilios/patología , Exoma , Anomalías del Ojo/genética , Anomalías del Ojo/patología , Femenino , Humanos , Katanina , Enfermedades Renales Quísticas/genética , Enfermedades Renales Quísticas/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Linaje , Unión Proteica , Retina/patologíaRESUMEN
Leber congenital amaurosis (LCA) is the most severe form of retinal dystrophy with an onset in the first year of life. The most frequent genetic cause of LCA, accounting for up to 15% of all LCA cases in Europe and North-America, is a mutation (c.2991+1655AG) in intron 26 of CEP290. This mutation generates a cryptic splice donor site resulting in the insertion of an aberrant exon (exon X) containing a premature stop codon to CEP290 mRNA. In order to study the pathophysiology of the intronic CEP290 mutation, we generated two humanized knock-in mouse models each carrying ~6.3 kb of the human CEP290 gene, either with or without the intronic mutation. Transcriptional characterization of these mouse models revealed an unexpected splice pattern of CEP290 mRNA, especially in the retina. In both models, a new cryptic exon (coined exon Y) was identified in ~5 to 12% of all Cep290 transcripts. This exon Y was expressed in all murine tissues analyzed but not detected in human retina or fibroblasts of LCA patients. In addition, exon x that is characteristic of LCA in humans, was expressed at only very low levels in the retina of the LCA mouse model. Western blot and immunohistochemical analyses did not reveal any differences between the two transgenic models and wild-type mice. Together, our results show clear differences in the recognition of splice sites between mice and humans, and emphasize that care is warranted when generating animal models for human genetic diseases caused by splice mutations.