RESUMO
Nephronophthisis (NPHP), Joubert (JBTS), and Meckel-Gruber (MKS) syndromes are autosomal-recessive ciliopathies presenting with cystic kidneys, retinal degeneration, and cerebellar/neural tube malformation. Whether defects in kidney, retinal, or neural disease primarily involve ciliary, Hedgehog, or cell polarity pathways remains unclear. Using high-confidence proteomics, we identified 850 interactors copurifying with nine NPHP/JBTS/MKS proteins and discovered three connected modules: "NPHP1-4-8" functioning at the apical surface, "NPHP5-6" at centrosomes, and "MKS" linked to Hedgehog signaling. Assays for ciliogenesis and epithelial morphogenesis in 3D renal cultures link renal cystic disease to apical organization defects, whereas ciliary and Hedgehog pathway defects lead to retinal or neural deficits. Using 38 interactors as candidates, linkage and sequencing analysis of 250 patients identified ATXN10 and TCTN2 as new NPHP-JBTS genes, and our Tctn2 mouse knockout shows neural tube and Hedgehog signaling defects. Our study further illustrates the power of linking proteomic networks and human genetics to uncover critical disease pathways.
Assuntos
Doenças Renais Císticas/genética , Proteínas de Membrana/genética , Transdução de Sinais , Animais , Ataxina-10 , Centrossomo/metabolismo , Cílios/metabolismo , Transtornos da Motilidade Ciliar/genética , Encefalocele/genética , Proteínas Hedgehog/metabolismo , Humanos , Doenças Renais Císticas/metabolismo , Camundongos , Células NIH 3T3 , Proteínas do Tecido Nervoso/genética , Doenças Renais Policísticas/genética , Retinose Pigmentar , Peixe-ZebraRESUMO
We describe a method for the highly efficient and precise targeted modification of gene trap loci in mouse embryonic stem cells (ESCs). Through the Floxin method, gene trap mutations were reverted and new DNA sequences inserted using Cre recombinase and a shuttle vector, pFloxin. Floxin technology is applicable to the existing collection of 24,149 compatible gene trap cell lines, which should enable high-throughput modification of many genes in mouse ESCs.
Assuntos
Células-Tronco Embrionárias/metabolismo , Engenharia Genética/métodos , Alelos , Animais , Sítios de Ligação Microbiológicos/genética , Sequência de Bases , Linhagem Celular , Células-Tronco Embrionárias/citologia , Elementos Facilitadores Genéticos , Etiquetas de Sequências Expressas , Marcadores Genéticos , Vetores Genéticos , Integrases/genética , Integrases/metabolismo , Camundongos , Camundongos Transgênicos , MutaçãoRESUMO
Mutations affecting ciliary components cause ciliopathies. As described here, we investigated Tectonic1 (Tctn1), a regulator of mouse Hedgehog signaling, and found that it is essential for ciliogenesis in some, but not all, tissues. Cell types that do not require Tctn1 for ciliogenesis require it to localize select membrane-associated proteins to the cilium, including Arl13b, AC3, Smoothened and Pkd2. Tctn1 forms a complex with multiple ciliopathy proteins associated with Meckel and Joubert syndromes, including Mks1, Tmem216, Tmem67, Cep290, B9d1, Tctn2 and Cc2d2a. Components of this complex co-localize at the transition zone, a region between the basal body and ciliary axoneme. Like Tctn1, loss of Tctn2, Tmem67 or Cc2d2a causes tissue-specific defects in ciliogenesis and ciliary membrane composition. Consistent with a shared function for complex components, we identified a mutation in TCTN1 that causes Joubert syndrome. Thus, a transition zone complex of Meckel and Joubert syndrome proteins regulates ciliary assembly and trafficking, suggesting that transition zone dysfunction is the cause of these ciliopathies.
Assuntos
Membrana Celular/fisiologia , Cílios/metabolismo , Cílios/patologia , Proteínas de Membrana/fisiologia , Mutação/genética , Anormalidades Múltiplas , Animais , Doenças Cerebelares/genética , Cerebelo/anormalidades , Galinhas , Transtornos da Motilidade Ciliar/genética , Encefalocele/genética , Anormalidades do Olho/genética , Humanos , Doenças Renais Císticas/genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Morfogênese , Especificidade de Órgãos , Fragmentos de Peptídeos/imunologia , Doenças Renais Policísticas/genética , Coelhos , Retina/anormalidades , Retinose Pigmentar , Transdução de Sinais , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Primary cilia are present on most mammalian cells and are implicated in transducing Hedgehog (Hh) signals during development; however, the prevalence of cilia on human tumors remains unclear, and the role of cilia in cancer has not been examined. Here we show that human basal cell carcinomas (BCCs) are frequently ciliated, and we test the role of cilia in BCC by conditionally deleting Kif3a (encoding kinesin family member 3A) or Ift88 (encoding intraflagellar transport protein 88), genes required for ciliogenesis, in two Hh pathway-dependent mouse tumor models. Ciliary ablation strongly inhibited BCC-like tumors induced by an activated form of Smoothened. In contrast, removal of cilia accelerated tumors induced by activated Gli2, a transcriptional effector of Hh signaling. These seemingly paradoxical effects are consistent with a dual role for cilia in mediating both the activation and the repression of the Hh signaling pathway. Our findings demonstrate that cilia function as unique signaling organelles that can either mediate or suppress tumorigenesis depending on the nature of the oncogenic initiating event.