RESUMO
Arl13b belongs to the ADP-ribosylation factor family within the Ras superfamily of regulatory GTPases. Mutations in Arl13b cause Joubert syndrome, which is characterized by congenital cerebellar ataxia, hypotonia, oculomotor apraxia, and mental retardation. Arl13b is highly enriched in cilia and is required for ciliogenesis in multiple organs. Nevertheless, the precise role of Arl13b remains elusive. Here we report that the exocyst subunits Sec8, Exo70, and Sec5 bind preferentially to the GTP-bound form of Arl13b, consistent with the exocyst being an effector of Arl13b. Moreover, we show that Arl13b binds directly to Sec8 and Sec5. In zebrafish, depletion of arl13b or the exocyst subunit sec10 causes phenotypes characteristic of defective cilia, such as curly tail up, edema, and abnormal pronephric kidney development. We explored this further and found a synergistic genetic interaction between arl13b and sec10 morphants in cilia-dependent phenotypes. Through conditional deletion of Arl13b or Sec10 in mice, we found kidney cysts and decreased ciliogenesis in cells surrounding the cysts. Moreover, we observed a decrease in Arl13b expression in the kidneys from Sec10 conditional knockout mice. Taken together, our results indicate that Arl13b and the exocyst function together in the same pathway leading to functional cilia.
Assuntos
Fatores de Ribosilação do ADP/metabolismo , Cílios/metabolismo , Fatores de Ribosilação do ADP/genética , Anormalidades Múltiplas , Animais , Cerebelo/anormalidades , Anormalidades do Olho , Estudos de Associação Genética , Células HeLa , Humanos , Rim/metabolismo , Doenças Renais Císticas , Camundongos , Camundongos Knockout , Microtúbulos/metabolismo , Mutação , Células NIH 3T3 , Retina/anormalidades , Proteínas de Transporte Vesicular/metabolismo , Peixe-Zebra , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
Primary cilia are built and maintained by intraflagellar transport (IFT), whereby the two IFT complexes, IFTA and IFTB, carry cargo via kinesin and dynein motors for anterograde and retrograde transport, respectively. Many signaling pathways, including platelet- derived growth factor (PDGF)-AA/αα, are linked to primary cilia. Active PDGF-AA/αα signaling results in phosphorylation of Akt at two residues: P-Akt(T308) and P-Akt(S473), and previous work showed decreased P-Akt(S473) in response to PDGF-AA upon anterograde transport disruption. In this study, we investigated PDGF-AA/αα signaling via P-Akt(T308) and P-Akt(S473) in distinct ciliary transport mutants. We found increased Akt phosphorylation in the absence of PDGF-AA stimulation, which we show is due to impaired dephosphorylation resulting from diminished PP2A activity toward P-Akt(T308). Anterograde transport mutants display low platelet-derived growth factor receptor (PDGFR)α levels, whereas retrograde mutants exhibit normal PDGFRα levels. Despite this, neither shows an increase in P-Akt(S473) or P-Akt(T308) upon PDGF-AA stimulation. Because mammalian target of rapamycin complex 1 (mTORC1) signaling is increased in ciliary transport mutant cells and mTOR signaling inhibits PDGFRα levels, we demonstrate that inhibition of mTORC1 rescues PDGFRα levels as well as PDGF-AA-dependent phosphorylation of Akt(S473) and Akt(T308) in ciliary transport mutant MEFs. Taken together, our data indicate that the regulation of mTORC1 signaling and PP2A activity by ciliary transport plays key roles in PDGF-AA/αα signaling.
Assuntos
Cílios/metabolismo , Fator de Crescimento Derivado de Plaquetas/farmacologia , Proteína Fosfatase 2/metabolismo , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , Animais , Transporte Biológico/efeitos dos fármacos , Transporte Biológico/genética , Western Blotting , Células Cultivadas , Embrião de Mamíferos/citologia , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos Knockout , Microscopia de Fluorescência , Complexos Multiproteicos/metabolismo , Mutação , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Sirolimo/farmacologiaRESUMO
The construction of cerebral cortex begins with the formation of radial glia. Once formed, polarized radial glial cells divide either symmetrically or asymmetrically to balance appropriate production of progenitor cells and neurons. Following birth, neurons use the processes of radial glia as scaffolding for oriented migration. Radial glia therefore provide an instructive structural matrix to coordinate the generation and placement of distinct groups of cortical neurons in the developing cerebral cortex. We found that Arl13b, a cilia-enriched small GTPase that is mutated in Joubert syndrome, was critical for the initial formation of the polarized radial progenitor scaffold. Using developmental stage-specific deletion of Arl13b in mouse cortical progenitors, we found that early neuroepithelial deletion of ciliary Arl13b led to a reversal of the apical-basal polarity of radial progenitors and aberrant neuronal placement. Arl13b modulated ciliary signaling necessary for radial glial polarity. Our findings indicate that Arl13b signaling in primary cilia is crucial for the initial formation of a polarized radial glial scaffold and suggest that disruption of this process may contribute to aberrant neurodevelopment and brain abnormalities in Joubert syndrome-related ciliopathies.