Modulation of Microtubule Dynamics in Axon Guidance

描述(由申请人提供)：适当的轴突生长和路径寻找对于在发育中的神经系统形成功能神经元电路是必不可少的。不同的引导信号、它们的受体和细胞内信号转导级联的协调最终在协调细胞骨架动力学上收敛，以操纵生长锥导航。Netrins是一个保守的经典引导信号家族，在神经系统发育中的轴突生长和引导中发挥着重要作用。轴突引导信号(如netrin-1)与微管(MT)动力学的耦合对于生长锥(GC)导航至关重要。然而，轴突引导信号是否直接或间接调节MT的动力学尚不清楚。我们的初步数据表明，DCC和DSCAM直接与神经元中最动态的微管蛋白亚型TUBB3相互作用，而Netrin-1诱导了这种相互作用。在发育中的神经系统中，TUBB3是Netrin-1诱导的轴突生长和寻路所必需的。这些结果表明，DCC和DSCAM在Netrin-1介导的轴突吸引中直接耦合MT动力学。有趣的是，unc5也与TUBB3相互作用。在这项研究中，我们将确定Netrin-1是如何通过其受体DCC、DSCAM和UNC5C与动态TUBB3在吸引和排斥信号中偶联来调节微管动力学的。目的：1.解开TUBB3在协调netrin-1吸引信号中的作用。为了进一步研究TUBB3在Netrin/DCC/DSCAM介导的轴突吸引信号中的协调作用，我们将研究：1)Netrin-1调节TUBB3与DCC和DSCAM的相互作用；2)Netrin-1通过动态TUBB3与DCC和DSCAM的相互作用调节MT的动力学；3)TUBB3在Netrin吸引信号中的功能重要性。目的2.确定TUBB3在协调Netrin-1排斥信号中的作用。Ok5与DCC和DSCAM相互作用，这些相互作用介导对netrin-1的排斥反应。我们的初步数据显示，TUBB3与unc5C、DCC和DSCAM相互作用，提示它可能在协调netrin-1排斥信号方面发挥作用。为了进一步阐明这一假说，我们将重点研究：1)Netrin-1对TUBB3与UNC5C、DCC和DSCAM相互作用的调节；2)Netrin-1通过协调动态TUBB3与UNC5C、DCC和DSCAM的相互作用来调节MT的动力学；3)TUBB3在Netrin-1介导的轴突排斥中的功能重要性。目的3.研究Netrin信号与MT动力学之间的机制联系。为了研究Netrin-1如何通过TUBB3直接调节轴突转动中的MT动力学，我们将集中在以下几个方面：1)评估Netrin信号中TUBB3的酪氨酸磷酸化；2)利用活细胞成像追踪Netrin-1介导的吸引过程中GC中MT动力学的调制；3)追踪Netrin-1介导的GC中轴突排斥期间Netrin-1介导的MT动力学。这项研究应该揭示netrin-1是如何通过其受体与轴突转动中动态的TUBB3的差示耦合来直接调节MT的动力学的。

DESCRIPTION (provided by applicant): Proper axon outgrowth and path finding are essential for the formation of functional neuronal circuits in the developing nervous system. Coordination of different guidance cues, their receptors and intracellular signal transduction cascades eventually converges on orchestrating cytoskeleton dynamics to maneuver growth cone navigation. Netrins, a conserved family of canonical guidance cues, play an important role in axon outgrowth and guidance in the developing nervous system. The coupling of axon guidance cues, such as netrin-1, to microtubule (MT) dynamics is crucial for growth cone (GC) navigation. However, whether axon guidance signaling regulates MT dynamics directly or indirectly is unclear. Our preliminary data indicate that DCC and DSCAM interact directly with TUBB3, the most dynamic ?-tubulin isoform in neurons, and netrin-1 induces these interactions. TUBB3 is required for netrin-1- induced axon outgrowth and path finding in the developing nervous system. These results suggest that DCC and DSCAM directly couple MT dynamics in netrin-1-mediated axon attraction. Interestingly, UNC5 also interacts with TUBB3. In this study, we will determine how netrin-1 modulates microtubule dynamics through coupling of its receptors DCC, DSCAM and UNC5C with dynamic TUBB3 in attractive and repulsive signaling. Aim1. Untangle the role of TUBB3 in coordination of netrin-1 attractive signaling. To further investigate the coordinating role of TUBB3 in netrin/DCC/DSCAM-mediated axon attractive signaling, we will examine: 1) Netrin-1 regulation of the interaction of TUBB3 with DCC and DSCAM; 2) Modulation of MT dynamics by netrin-1 via the interaction of dynamic TUBB3 with DCC and DSCAM; 3) The functional importance of TUBB3 in netrin attractive signaling. Aim 2. Determine the role of TUBB3 in coordination of netrin-1 repulsive signaling. UNC5 interacts with DCC and DSCAM and these interactions mediate repulsive responses to netrin-1. Our preliminary data show that TUBB3 interacts with UNC5C, DCC and DSCAM, suggesting it may play a role in coordinating netrin-1 repulsive signaling. To further characterize this hypothesis, we will focus on studying: 1) Netrin-1 regulation of the interaction of TUBB3 with UNC5C, DCC and DSCAM; 2) Modulation of MT dynamics by netrin-1 via coordination of the interaction of dynamic TUBB3 with UNC5C, DCC and DSCAM; 3) The functional importance of TUBB3 in netrin-1-mediated axon repulsion. Aim 3. Investigate the mechanistic link between netrin signaling and MT dynamics. To examine how netrin-1 directly regulates MT dynamics via TUBB3 in axon turning, we will focus on: 1) assessing tyrosine phosphorylation of TUBB3 in netrin signaling; 2) tracking modulation of MT dynamics in the GC during netrin-1-mediated attraction using live cell imaging; 3) tracking netrin-1- mediated MT dynamics in the GC during axon repulsion. This study should reveal how netrin-1 directly modulates MT dynamics via differential coupling of its receptors with dynamic TUBB3 in axon turning.