RESUMEN
Axon regeneration in response to injury has been documented in many animals over several hundred years. In contrast, how neurons respond to dendrite injury has been examined only in the last decade. So far, dendrite regeneration after injury has been documented in invertebrate model systems, but has not been assayed in a vertebrate. In this study, we use zebrafish motor neurons to track neurons after dendrite injury. We address two major gaps in our knowledge of dendrite regeneration: 1) whether post-synaptic dendrites can regenerate and 2) whether vertebrate dendrites can regenerate. We find that motor neurons survive laser microsurgery to remove one or all dendrites. Outgrowth of new dendrites typically initiated one to three days after injury, and a new, stable dendrite arbor was in place by five days after injury. We conclude that zebrafish motor neurons have the capacity to regenerate a new dendrite arbor.
Asunto(s)
Dendritas , Regeneración de la Medula Espinal , Animales , Axones , Dendritas/fisiología , Neuronas Motoras , Regeneración Nerviosa/fisiología , Médula Espinal , Pez CebraRESUMEN
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
Microtubule minus ends are thought to be stable in cells. Surprisingly, in Drosophila and zebrafish neurons, we observed persistent minus end growth, with runs lasting over 10 min. In Drosophila, extended minus end growth depended on Patronin, and Patronin reduction disrupted dendritic minus-end-out polarity. In fly dendrites, microtubule nucleation sites localize at dendrite branch points. Therefore, we hypothesized minus end growth might be particularly important beyond branch points. Distal dendrites have mixed polarity, and reduction of Patronin lowered the number of minus-end-out microtubules. More strikingly, extra Patronin made terminal dendrites almost completely minus-end-out, indicating low Patronin normally limits minus-end-out microtubules. To determine whether minus end growth populated new dendrites with microtubules, we analyzed dendrite development and regeneration. Minus ends extended into growing dendrites in the presence of Patronin. In sum, our data suggest that Patronin facilitates sustained microtubule minus end growth, which is critical for populating dendrites with minus-end-out microtubules.
Asunto(s)
Dendritas/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/crecimiento & desarrollo , Proteínas Asociadas a Microtúbulos/genética , Neuronas/metabolismo , Animales , Polaridad Celular/genética , Drosophila melanogaster/genética , Embrión no Mamífero , Desarrollo Embrionario/genética , Cinesinas/genética , Microtúbulos/genéticaRESUMEN
In Drosophila neurons, uniform minus-end-out polarity in dendrites is maintained in part by kinesin-2-mediated steering of growing microtubules at branch points. Apc links the kinesin motor to growing microtubule plus ends and Apc2 recruits Apc to branch points where it functions. Because Apc2 acts to concentrate other steering proteins to branch points, we wished to understand how Apc2 is targeted. From an initial broad candidate RNAi screen, we found Miro (a mitochondrial transport protein), Ank2, Axin, spastin and Rac1 were required to position Apc2-GFP at dendrite branch points. YFP-Ank2-L8, Axin-GFP and mitochondria also localized to branch points suggesting the screen identified relevant proteins. By performing secondary screens, we found that energy production by mitochondria was key for Apc2-GFP positioning and spastin acted upstream of mitochondria. Ank2 seems to act independently from other players, except its membrane partner, Neuroglian (Nrg). Rac1 likely acts through Arp2/3 to generate branched actin to help recruit Apc2-GFP. Axin can function in a variety of wnt signaling pathways, one of which includes heterotrimeric G proteins and Frizzleds. Knockdown of Gαs, Gαo, Fz and Fz2, reduced targeting of Apc2 and Axin to branch points. Overall our data suggest that mitochondrial energy production, Nrg/Ank2, branched actin generated by Arp2/3 and Fz/G proteins/Axin function as four modules that control localization of the microtubule regulator Apc2 to its site of action in dendrite branch points.