RESUMEN
Axons elongate and perform steering reactions with their growth cones constantly undergoing local collapse and stabilization. Our previous studies have shown that a type-1 phosphorylated form of microtubule-associated protein 1B, recognized by monoclonal antibody 1E11 (mab1E11), is present in stable regions and absent from unstable regions of turning growth cones of retinal ganglion cells. In contrast, the total population of microtubule-associated protein 1B is present in the entire growth cone. Here we demonstrate that inhibition of cyclin-dependent kinase 5 (Cdk5) results in loss of mab1E11 binding whereas inhibition of glycogen synthase kinase 3 has no such effect, revealing that mab1E11 recognizes a Cdk5 phosphorylation site on type-1 phosphorylated form of microtubule-associated protein 1B. We moreover show that kinase Cdk5 as well as its activator P35 is present in retinal ganglion cells in the early developing chick embryo retina and enriched in their extending axons. Cdk5 and P35 are concentrated in the youngest, distal axon region and the growth cone as also seen for Cdk5-phosphorylated type-1 phosphorylated form of microtubule-associated protein 1B. Inhibition of Cdk5 by antibodies or inhibitor Roscovitine results in growth cone collapse and axon retraction and prevents substantial axon outgrowth. In contrast, glycogen synthase kinase 3 inhibition causes only a transient axon retraction which is soon recovered and allows for axon formation. In growth cones induced to turn at substrate borders, where stable and instable parts of the growth cone are clearly defined, Cdk5 is present in the entire growth cone. P35, in contrast, is restricted to the stable parts of the growth cone, which do not collapse but instead transform into new distal axon. The local presence of Cdk5-phosphorylated type-1 phosphorylated form of microtubule-associated protein 1B in stabilized growth cone areas can be therefore attributed to the local activation of Cdk5 by P35 in these regions. Together our data demonstrate a crucial role of Cdk5 and its activator P35 in elongation and maintenance of axons as well as for stability and steering of their growth cones.
Asunto(s)
Axones/enzimología , Quinasas Ciclina-Dependientes/metabolismo , Células Ganglionares de la Retina/enzimología , Animales , Anticuerpos Monoclonales , Axones/ultraestructura , Embrión de Pollo , Quinasa 5 Dependiente de la Ciclina , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Conos de Crecimiento/enzimología , Conos de Crecimiento/metabolismo , Conos de Crecimiento/ultraestructura , Inmunohistoquímica , Proteínas Asociadas a Microtúbulos/metabolismo , Purinas/farmacología , Retina/embriología , Roscovitina , Especificidad por SustratoRESUMEN
For the development of the nervous system it is crucial that growth cones detect environmental information and react by altering their growth direction. The latter process is thought to depend on local stabilization of growth cone microtubules. We have obtained evidence of a role for the microtubule-associated protein MAP1B, in particular a mode 1 phosphoisoform of the molecule, P1-MAP1B, in this process. P1-MAP1B is tightly associated with the cytoskeleton and is present at highest concentrations in the distal axon and the growth cone of chick retinal ganglion cells. In growth cones turning at nonpermissive substrate borders, P1-MAP1B is restricted to regions which are stabilized. Unilateral neutralization of P1-MAP1B in one-half the growth cone by microscale chromophore-assisted laser inactivation changes growth cone motility, morphology, and growth direction. The results indicate a functional role for P1-MAP1B in local growth cone stabilization and thus growth cone steering.