Shao Qiangqiang, Yang Tao, Huang Huai, Alarmanazi Farrah, Liu Guofa
Department of Biological Sciences, University of Toledo, Toledo, Ohio 43606.
Department of Biological Sciences, University of Toledo, Toledo, Ohio 43606
J Neurosci. 2017 Jun 7;37(23):5620-5633. doi: 10.1523/JNEUROSCI.2617-16.2017. Epub 2017 May 8.
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 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 and caused defects in axon projection of DRG toward the spinal cord 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. Proper regulation of microtubule (MT) dynamics in the growth cone plays an important role in axon guidance. However, whether guidance cues modulate MT dynamics directly or indirectly is unclear. Here, we report that dissociation of UNC5C and polymerized TUBB3, the highly dynamic β-tubulin isoform in neurons, is essential for netrin-1/UNC5C-promoted axon repulsion. These results not only provide a working model of direct modulation of MTs by guidance cues in growth cone navigation but also help us to understand molecular mechanisms underlying developmental brain disorders associated with TUBB3 mutations.
微管（MT）动力学的调节是轴突生长和路径寻找过程中生长锥（GC）细胞骨架重塑的关键事件。我们之前的研究表明，脑内神经元特异性MT亚基聚合TUBB3与网蛋白受体DCC和DSCAM的直接相互作用是网蛋白-1介导的轴突生长、分支和吸引所必需的。在此，我们表明聚合TUBB3与网蛋白-1排斥受体UNC5C的解偶联参与了网蛋白-1介导的轴突排斥。TUBB3与UNC5C直接相互作用，并在来自两性P2小鼠幼崽小脑外颗粒层的原代神经元GC外周区域与UNC5C部分共定位。网蛋白-1减少了这种相互作用以及UNC5C和TUBB3在GC中的共定位。沉降分析结果表明，UNC5C与MTs中的聚合TUBB3相互作用，而网蛋白-1降低了这种相互作用。敲低TUBB3或UNC5C均阻断了网蛋白-1促进的轴突排斥，并导致背根神经节（DRG）轴突向脊髓投射的缺陷。此外，在原代外颗粒层细胞中对用EGFP标记的末端结合蛋白3（EB3-GFP）进行活细胞成像显示，网蛋白-1在排斥过程中差异地增加了GC中的MT动力学，GC远端的MT生长比近端更多，敲低UNC5C或TUBB3消除了网蛋白-1的作用。总之，这些数据表明UNC5C与聚合TUBB3的分离在网蛋白-1/UNC5C介导的轴突排斥中起重要作用。生长锥中微管（MT）动力学的适当调节在轴突导向中起重要作用。然而，导向线索是直接还是间接调节MT动力学尚不清楚。在此，我们报道UNC5C与聚合TUBB3（神经元中高度动态的β-微管蛋白异构体）的解离对于网蛋白-1/UNC5C促进的轴突排斥至关重要。这些结果不仅提供了生长锥导航中导向线索对MT进行直接调节的工作模型，还帮助我们理解与TUBB3突变相关的发育性脑疾病的分子机制。
