Identification of Proteins Required for Precise Positioning of Apc2 in Dendrites

Abstract

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.

Endosomal Wnt signaling proteins control microtubule nucleation in dendrites

“…Consistent with a role in localizing γTub, a functional tagged Axin transgene [53]) concentrates at dendrite branch points (Fig 2A and [27]). We previously showed that fz, fz2, and Gαo are necessary for Axin-GFP branch point localization [27]. After finding that other Wnt signaling proteins act upstream of γTub localization, we wished to confirm that they also play a role in Axin targeting.…”

“…Cells also expressed either UAS-Rtnl2 RNAi (control 1) (VDRC 33320) (left) or UAS-Axin RNAi (VDRC 7748) (right). (C) A summary of data in earlier figures as well as some previous data [27] is shown. (D) Quantification of EB1-GFP comet direction in the main trunk of the ddaE dendrite in animals expressing hairpin RNAi's.…”

“…Within Drosophila dendrites, γTub is concentrated at branch points [26]. In a previous study, we identified proteins that localize a different microtubule regulator, adenomatous polyposis coli (Apc) 2, to branch points [27]. We reasoned that some or all of this machinery might be used to position γTub to the same region.…”

“…The branch point localization of γTub-GFP is reminiscent of another microtubule regulator, Apc2 [31]. We previously identified proteins required for Apc2 localization to branch points [27] and hypothesized that some of these may also target γTub-GFP. Reduction of mitochondria and actin regulators as well as Wnt signaling proteins disrupted Apc2-GFP localization [27].…”

“…We previously identified proteins required for Apc2 localization to branch points [27] and hypothesized that some of these may also target γTub-GFP. Reduction of mitochondria and actin regulators as well as Wnt signaling proteins disrupted Apc2-GFP localization [27]. RNAi lines that targeted proteins in the Wnt signaling group but not the other groups reduced γTub-GFP at dendrite branch points (Figs 1A-1C and S1B-S1E).…”