Uncoupling of UNC5C with Polymerized TUBB3 in Microtubules Mediates Netrin-1 Repulsion

Abstract

Modulation of microtubule (MT) dynamics is a key event of cytoskeleton remodeling in the growth cone (GC) during axon outgrowth and pathfinding. Our previous studies have shown that the direct interaction of netrin receptor DCC and DSCAM with polymerized TUBB3, a neuron-specific MT subunit in the brain, is required for netrin-1-mediated axon outgrowth, branching, and attraction. Here, we show that uncoupling of polymerized TUBB3 with netrin-1-repulsive receptor UNC5C is involved in netrin-1-mediated axonal repulsion. TUBB3 directly interacted with UNC5C and partially colocalized with UNC5C in the peripheral area of the GC of primary neurons from the cerebellar external granule layer of P2 mouse pups of both sexes. Netrin-1 reduced this interaction as well as the colocalization of UNC5C and TUBB3 in the GC. Results from the in vitro cosedimentation assay indicated that UNC5C interacted with polymerized TUBB3 in MTs and netrin-1 decreased this interaction. Knockdown of either TUBB3 or UNC5C blocked netrin-1-promoted axon repulsion in vitro and caused defects in axon projection of DRG toward the spinal cord in vivo. Furthermore, live-cell imaging of end-binding protein 3 tagged with EGFP (EB3-GFP) in primary external granule layer cells showed that netrin-1 differentially increased MT dynamics in the GC with more MT growth in the distal than the proximal region of the GC during repulsion, and knockdown of either UNC5C or TUBB3 abolished the netrin-1 effect. Together, these data indicate that the disengagement of UNC5C with polymerized TUBB3 plays an essential role in netrin-1/UNC5C-mediated axon repulsion.

miR-92 Suppresses Robo1 Translation to Modulate Slit Sensitivity in Commissural Axon Guidance

“…In ovo electroporation into the embryonic chicken neural tube was performed at HH10-12 and the DSC was collected at HH22-26 after electroporation. The immunoblotting procedure on cell lysates from HEK293 cells, primary cortical neurons and the DSC of chicken embryos was similar to those previously described (Qu et al, 2013a;Shao et al, 2017).…”

“…The procedures of primary neuron cultures were performed as previously described (Huang et al, 2015(Huang et al, , 2018Shao et al, 2017). For primary cortical neuron culture, cerebral cortices were collected from embryonic day 14.5 (E14.5) mouse embryos.…”

UNC-6/Netrin and its Receptors UNC-5 and UNC-40/DCC Control Growth Cone Polarity, Microtubule Accumulation, and Protrusion

“…UNC-6 and UNC-5 might inhibit growth cone protrusion by preventing F-actin formation in the ventral/lateral regions of the growth cone to restrict protrusion to the dorsal leading edge, and by preventing accumulation of MT + ends in the growth cone. In cultured growth cones, MTs are involved in both DCC and UNC5C-mediated axon outgrowth, and DCC and UNC5C physically associate with MTs in a Netrin-dependent manner in cultured cells (Qu et al 2013; Shao et al 2017; Huang et al 2018). Our results suggest a link between UNC-6/Netrin signaling and VD growth cone MTs in vivo .…”

“…This is consistent with results from cultured growth cones showing that MT stabilization results in growth cone turning in the direction of stabilization, and MT destabilization results in growth cone turning away from MT destabilization (Buck and Zheng 2002). Also, MTs are involved in both DCC and UNC5C-mediated axon guidance (Qu et al 2013; Shao et al 2017; Huang et al 2018), and physically associate with UNC5C, which is decoupled by Netrin and associated with growth away from Netrin (Shao et al 2017). We have no evidence that UNC-5 or UNC-40 physically associate with MTs, but these data are consistent with MTs having a pro-protrusive role (i.e.…”

“…Thus, motility of the growth cone is achieved through proper regulation and coordination between microtubules and the actin cytoskeleton (Dent and Kalil 2001; Buck and Zheng 2002; Zhou et al 2002; Zhou and Cohan 2004). MTs have been implicated in Unc5 signaling (Shao et al 2017; Huang et al 2018), but the role of MTs in UNC-5-mediated VD growth cone outgrowth in vivo remains unclear.…”