Kinesin‐2 and Apc function at dendrite branch points to resolve microtubule collisions

Abstract

In Drosophila neurons, kinesin-2, EB1 and Apc are required to maintain minus-end-out dendrite microtubule polarity, and we previously proposed they steer microtubules at branch points. Motor-mediated steering of microtubule plus ends could be accomplished in two ways: 1) by linking a growing microtubule tip to the side of an adjacent microtubule as it navigates the branch point (bundling), or 2) by directing a growing microtubule after a collision with a stable microtubule (collision resolution). Using live imaging to distinguish between these two mechanisms, we found that reduction of kinesin-2 did not alter the number of microtubules that grew along the edge of the branch points where stable microtubules are found. However, reduction of kinesin-2 or Apc did affect the number of microtubules that slowed down or depolymerized as they encountered the side of the branch opposite to the entry point. These results are consistent with kinesin-2 functioning with Apc to resolve collisions. However, they do not pinpoint stable microtubules as the collision partner as stable microtubules are typically very close to the membrane. To determine whether growing microtubules were steered along stable ones after a collision, we analyzed the behavior of growing microtubules at dendrite crossroads where stable microtubules run through the middle of the branch point. In control neurons, microtubules turned in the middle of the crossroads. However, when kinesin-2 was reduced some microtubules grew straight through the branch point and failed to turn. We propose that kinesin-2 functions to steer growing microtubules along stable ones following collisions.

Microtubules originate asymmetrically at the somatic Golgi and are guided via Kinesin2 to maintain polarity within neurons

“…Our data therefore suggests that Kinesin-2 guides growing plus ends within the soma, similar to how it guides microtubule turning events within dendrites 47,49 , to help somatic microtubules grow into the axon and to help maintain minus-end-out microtubule polarity within dendrites. Our data also helps to resolve the current controversy regarding whether γ-TuRCs function at Golgi outposts or at dendritic branch points in a Golgi independent manner -we find that both situations exist, but are rarer than previously thought and occur in a neuronal-class-specific and spatially-restricted manner.…”

“…7A -see for example time 04:00; Movie S4), suggesting that growing microtubules might be directed along pre-existing microtubules towards the axon. Turning events similar to this have been observed within dendrites, where it has been proposed that the plus-end-directed motor Kinesin-2 associates via its tail with the plus end of a growing microtubule and that Kinesin-2's motor domains simultaneously engage with the shaft of an adjacent microtubule, therefore guiding the plus end of the growing microtubule towards the plus end of the adjacent microtubule 47,49 . Indeed, most turning events within the soma no longer occurred when we knocked down the regulatory subunit of Kinesin-2, Kap3 (Movie S5); comets grew in straight lines unless they collided with the nuclear envelope or cell cortex (although they could still traverse the edge of the cell) ( Fig.…”

“…The plus-enddirected motor Kinesin-2 is thought to associate via its tail region with growing plus ends due to an interaction with APC, which itself binds to EB1 49 . In vitro experiments have shown that Kinesin-2 can guide microtubule plus ends along adjacent microtubule when Kinesin-2 is artificially tethered to the plus end 86,87 , and this guidance mechanism appears to be true at dendritic branchpoints in vivo where Kinesin-2 is necessary for microtubules to turn towards the soma 47,49 . APC-2 is believed to recruit APC to branchpoints where it can function in plus end turning 47 , but APC-2 also accumulates with the soma of class I neurons 47,49 .…”

“…In both neuronal classes, microtubules in axons are predominantly plus-endout throughout development, but microtubule polarity in dendrites progressively becomes more minus-end-out as the neurons develop, such that by late larval stages the majority of dynamic microtubules are minus-end-out 44,49 . Microtubule polarity within da neurons can be disrupted under various mutant or RNAi knockdown conditions 10,12,41,42,[45][46][47][48] , but we still don't have a full understanding of how microtubule polarity is normally established and maintained.…”

Spastin, atlastin, and ER relocalization are involved in axon but not dendrite regeneration

“…Because EB1 acts as a dynamic platform at growing microtubule ends that recruits other proteins (Akhmanova and Steinmetz, 2008), the presence of large amounts of EB1-GFP could reduce recruitment of other plus end–binding proteins. Indeed, in Drosophila , neurons EB1 binds Apc, which in turn brings kinesin-2 to growing dendritic microtubules to help maintain minus-end-out polarity (Mattie et al ., 2010; Weiner et al ., 2016), and high levels of EB1-GFP result in mixed polarity (Mattie et al ., 2010). Because of this, we express EB1-GFP at low levels, but it is still possible that there is a subtle defect in microtubule growth or organization.…”