RESUMEN
While many regulators of axon regeneration have been identified, very little is known about mechanisms that allow dendrites to regenerate after injury. Using a Drosophila model of dendrite regeneration, we performed a candidate screen of receptor tyrosine kinases (RTKs) and found a requirement for RTK-like orphan receptor (Ror). We confirmed that Ror was required for regeneration in two different neuron types using RNA interference (RNAi) and mutants. Ror was not required for axon regeneration or normal dendrite development, suggesting a specific role in dendrite regeneration. Ror can act as a Wnt coreceptor with frizzleds (fzs) in other contexts, so we tested the involvement of Wnt signaling proteins in dendrite regeneration. We found that knockdown of fz, dishevelled (dsh), Axin, and gilgamesh (gish) also reduced dendrite regeneration. Moreover, Ror was required to position dsh and Axin in dendrites. We recently found that Wnt signaling proteins, including dsh and Axin, localize microtubule nucleation machinery in dendrites. We therefore hypothesized that Ror may act by regulating microtubule nucleation at baseline and during dendrite regeneration. Consistent with this hypothesis, localization of the core nucleation protein γTubulin was reduced in Ror RNAi neurons, and this effect was strongest during dendrite regeneration. In addition, dendrite regeneration was sensitive to partial reduction of γTubulin. We conclude that Ror promotes dendrite regeneration as part of a Wnt signaling pathway that regulates dendritic microtubule nucleation.
Asunto(s)
Dendritas/fisiología , Proteínas de Drosophila/metabolismo , Regeneración Nerviosa/fisiología , Receptores Huérfanos Similares al Receptor Tirosina Quinasa/metabolismo , Animales , Drosophila , Proteínas de Drosophila/genética , Microtúbulos/genética , Microtúbulos/metabolismo , Mutación , Neuronas/fisiología , Interferencia de ARN , Receptores Huérfanos Similares al Receptor Tirosina Quinasa/genética , Receptores Wnt/genética , Receptores Wnt/metabolismo , Vía de Señalización WntRESUMEN
Dendrite microtubules are polarized with minus-end-out orientation in Drosophila neurons. Nucleation sites concentrate at dendrite branch points, but how they localize is not known. Using Drosophila, we found that canonical Wnt signaling proteins regulate localization of the core nucleation protein γTubulin (γTub). Reduction of frizzleds (fz), arrow (low-density lipoprotein receptor-related protein [LRP] 5/6), dishevelled (dsh), casein kinase Iγ, G proteins, and Axin reduced γTub-green fluorescent protein (GFP) at branch points, and two functional readouts of dendritic nucleation confirmed a role for Wnt signaling proteins. Both dsh and Axin localized to branch points, with dsh upstream of Axin. Moreover, tethering Axin to mitochondria was sufficient to recruit ectopic γTub-GFP and increase microtubule dynamics in dendrites. At dendrite branch points, Axin and dsh colocalized with early endosomal marker Rab5, and new microtubule growth initiated at puncta marked with fz, dsh, Axin, and Rab5. We propose that in dendrites, canonical Wnt signaling proteins are housed on early endosomes and recruit nucleation sites to branch points.
Asunto(s)
Dendritas/metabolismo , Proteínas de Drosophila/metabolismo , Endosomas/metabolismo , Microtúbulos/metabolismo , Proteínas Wnt/metabolismo , Animales , Complejo de Señalización de la Axina/genética , Complejo de Señalización de la Axina/metabolismo , Axones/metabolismo , Polaridad Celular , Dendritas/genética , Drosophila , Proteínas de Drosophila/genética , Endosomas/genética , Microtúbulos/genética , Mutación , Receptores Wnt/genética , Receptores Wnt/metabolismo , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo , Proteínas Wnt/genética , Vía de Señalización Wnt/genética , Proteínas de Unión al GTP rab5/genética , Proteínas de Unión al GTP rab5/metabolismoRESUMEN
Kinetochores connect centromeric chromatin to spindle microtubules during mitosis. Neurons are postmitotic, so it was surprising to identify transcripts of structural kinetochore (KT) proteins and regulatory chromosome passenger complex (CPC) and spindle assembly checkpoint (SAC) proteins in Drosophila neurons after dendrite injury. To test whether these proteins function during dendrite regeneration, postmitotic RNA interference (RNAi) was performed and dendrites or axons were removed using laser microsurgery. Reduction of KT, CPC, and SAC proteins decreased dendrite regeneration without affecting axon regeneration. To understand whether neuronal functions of these proteins rely on microtubules, we analyzed microtubule behavior in uninjured neurons. The number of growing plus, but not minus, ends increased in dendrites with reduced KT, CPC, and SAC proteins, while axonal microtubules were unaffected. Increased dendritic microtubule dynamics was independent of dual leucine zipper kinase (DLK)-mediated stress but was rescued by concurrent reduction of γ-tubulin, the core microtubule nucleation protein. Reduction of γ-tubulin also rescued dendrite regeneration in backgrounds containing kinetochore RNAi transgenes. We conclude that kinetochore proteins function postmitotically in neurons to suppress dendritic microtubule dynamics by inhibiting nucleation.