RESUMEN
Humans with mutations in either DCX or LIS1 display nearly identical neuronal migration defects, known as lissencephaly. To define subcellular mechanisms, we have combined in vitro neuronal migration assays with retroviral transduction. Overexpression of wild-type Dcx or Lis1, but not patient-related mutant versions, increased migration rates. Dcx overexpression rescued the migration defect in Lis1+/- neurons. Lis1 localized predominantly to the centrosome, and after disruption of microtubules, redistributed to the perinuclear region. Dcx outlined microtubules extending from the perinuclear "cage" to the centrosome. Lis1+/- neurons displayed increased and more variable separation between the nucleus and the preceding centrosome during migration. Dynein inhibition resulted in similar defects in both nucleus-centrosome (N-C) coupling and neuronal migration. These N-C coupling defects were rescued by Dcx overexpression, and Dcx was found to complex with dynein. These data indicate Lis1 and Dcx function with dynein to mediate N-C coupling during migration, and suggest defects in this coupling may contribute to migration defects in lissencephaly.
Asunto(s)
Núcleo Celular/metabolismo , Centrosoma/metabolismo , Dineínas/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Neuronas/metabolismo , Neuropéptidos/metabolismo , 1-Alquil-2-acetilglicerofosfocolina Esterasa , Animales , Encéfalo/anomalías , Encéfalo/citología , Encéfalo/metabolismo , Diferenciación Celular/genética , Movimiento Celular/genética , Núcleo Celular/ultraestructura , Células Cultivadas , Centrosoma/ultraestructura , Proteínas de Dominio Doblecortina , Proteína Doblecortina , Dineínas/genética , Regulación del Desarrollo de la Expresión Génica/genética , Sustancias Macromoleculares , Ratones , Proteínas Asociadas a Microtúbulos/genética , Microtúbulos/genética , Microtúbulos/metabolismo , Microtúbulos/ultraestructura , Mutación/genética , Malformaciones del Sistema Nervioso/genética , Malformaciones del Sistema Nervioso/metabolismo , Malformaciones del Sistema Nervioso/fisiopatología , Neuronas/citología , Neuropéptidos/genética , Regulación hacia Arriba/genéticaRESUMEN
Mutations in the doublecortin (DCX) gene in human or targeted disruption of the cdk5 gene in mouse lead to similar cortical lamination defects in the developing brain. Here we show that Dcx is phosphorylated by Cdk5. Dcx phosphorylation is developmentally regulated and corresponds to the timing of expression of p35, the major activating subunit for Cdk5. Mass spectrometry and Western blot analysis indicate phosphorylation at Dcx residue Ser297. Phosphorylation of Dcx lowers its affinity to microtubules in vitro, reduces its effect on polymerization, and displaces it from microtubules in cultured neurons. Mutation of Ser297 blocks the effect of Dcx on migration in a fashion similar to pharmacological inhibition of Cdk5 activity. These results suggest that Dcx phosphorylation by Cdk5 regulates its actions on migration through an effect on microtubules.
Asunto(s)
Quinasas Ciclina-Dependientes/fisiología , Proteínas Asociadas a Microtúbulos/fisiología , Neuronas/fisiología , Neuropéptidos/fisiología , Serina/fisiología , Animales , Western Blotting , Movimiento Celular/fisiología , Corteza Cerebral/citología , Corteza Cerebral/metabolismo , Quinasa 5 Dependiente de la Ciclina , Quinasas Ciclina-Dependientes/metabolismo , Proteínas de Dominio Doblecortina , Proteína Doblecortina , Humanos , Procesamiento de Imagen Asistido por Computador , Inmunoprecipitación , Hibridación in Situ , Ratones , Ratones Noqueados , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Mutagénesis Sitio-Dirigida , Proteínas del Tejido Nervioso/fisiología , Sistema Nervioso/crecimiento & desarrollo , Neuropéptidos/metabolismo , Fenotipo , Fosforilación , Purinas/farmacología , Retroviridae/genética , Roscovitina , Serina/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , TransfecciónRESUMEN
The axonal shafts of neurons contain bundled microtubules, whereas extending growth cones contain unbundled microtubule filaments, suggesting that localized activation of microtubule-associated proteins (MAP) at the transition zone may bundle these filaments during axonal growth. Dephosphorylation is thought to lead to MAP activation, but specific molecular pathways have remained elusive. We find that Spinophilin, a Protein-phosphatase 1 (PP1) targeting protein, is responsible for the dephosphorylation of the MAP Doublecortin (Dcx) Ser 297 selectively at the "wrist" of growing axons, leading to activation. Loss of activity at the "wrist" is evident as an impaired microtubule cytoskeleton along the shaft. These findings suggest that spatially restricted adaptor-specific MAP reactivation through dephosphorylation is important in organization of the neuronal cytoskeleton.