Organization of Microtubule Polarity During Neuronal Axon Development in vivo

项目总结 微管的正确组织对神经回路的发育和功能至关重要。MTS表 分子马达运动蛋白和动力蛋白将货物运送到特定细胞位置的轨迹。这个 MTS固有的正端/负端极性指导着汽车的运输。在轴突中，MT与它们的加号排列在一起 末端在细胞体的远端。MT的极性必须在最初的轴突形成过程中以及在轴突形成期间建立 分支事件。对轴突和分支中MT极性的调节机制知之甚少，但 大量证据表明，分子马达对MT的极性至关重要。激动素和动力蛋白有助于 MT的极性，并被假设为通过MT的运输起作用。然而，可视化MT聚合物传输是 具有挑战性，而且只在体外或培养的神经元中完成。 我们的实验室之前表明，Clstn1，一种Kinesin-1适配器，调节感觉神经元中的轴突分支。我们的 初步数据显示，Clstn1也是轴突MT极性所必需的。如何知道Kinesin适配器 主要是调节货物运输，也调节MT的极性未知。我的目标是揭示潜在的 机械装置。已知Clstn1结合并激活KLC，KLC激活KHC和Kinesin的运动活性，以及 抑制KHC尾部与MTS的结合。我假设KLC的Clstn1激活可以防止过多的MT 通过KHC Tail-MT结合进行交联。在缺乏Clstn1的情况下，这种交联会与动力蛋白的能力相反 从轴突中移除方向错误的MTS，导致极化错误的MTS增加。我会用高速列车 活体4D成像方法来验证这些假设。 在目标1中，我将首先测试使用Clstn1 W-酸性结构域肽激活KLC是否足以挽救MT Clstn1-/-突变体的极性。其次，我将使用两种方法来破坏KHC Tail与MTS的绑定，以 减少KHC对MTS的交联，并测试MT极性Clstn1-/-突变体的挽救。最后，我将测试这些角色 KLC蛋白的磷酸化和Clstn1在MT极性的蛋白水解性切割。 在目标2中，我建议与光学和光学实验室合作，使用先进的成像技术 活体MT动态成像的计算仪器。我会给MT聚合物和正负末端贴上标签 使用多个荧光团，并使用光漂白后的荧光恢复来确定MT 运输对体内的极性有贡献。我将测试Clstn1丢失是否会影响MT传输，从而 影响极性。我还将测试Clstn1在轴突期间组织MT极性的功能的假设 通过成像分支点的MT动力学进行分支。这些实验将加深我们对 建立轴突MT极性的细胞和分子机制。

PROJECT SUMMARY Proper organization of microtubules (MTs) is critical for development and function of neural circuits. MTs form the tracks on which the molecular motors kinesin and dynein transport cargo to specific cell locations. The inherent plus end/minus end polarity of MTs directs motor transport. In axons MTs are arranged with their plus ends distal to the cell body. MT polarity must be established during initial axon formation, and also during axon branching events. The mechanisms regulating MT polarity in axons and branches are poorly understood, but a body of evidence suggests that molecular motors are crucial to MT polarity. Kinesin and dynein contribute to MT polarity and are hypothesized to act by transport of MTs. However, visualizing MT polymer transport is challenging and has only been done in vitro or cultured neurons. Our lab showed previously that Clstn1, a kinesin-1 adaptor, regulates axon branching in sensory neurons. Our preliminary data show that Clstn1 is also required for MT polarity in axons. How a kinesin adaptor, known primarily to mediate cargo transport, also regulates MT polarity is unknown. My goal is to reveal the underlying mechanisms. Clstn1 is known to bind and activate KLC, which activates KHC and kinesin motor activity, and inhibits KHC tail binding to MTs. I hypothesize that Clstn1 activation of KLC prevents excessive MT crosslinking by KHC tail-MT binding. In the absence of Clstn1, this crosslinking may oppose dynein’s ability to remove misoriented MTs from the axon, leading to an increase in mispolarized MTs. I will use high-speed in vivo 4D imaging approaches to test these hypotheses. In Aim 1, I will first test if activation of KLC using a Clstn1 W-acidic domain peptide is sufficient to rescue MT polarity in Clstn1-/- mutants. Second, I will use two approaches to disrupt the binding of the KHC tail to MTs to reduce KHC crosslinking to MTs, and test for rescue of MT polarity Clstn1-/- mutants. Finally, I will test the roles of KLC phosphorylation and proteolytic cleavage of Clstn1 in MT polarity. In Aim 2 I propose to use advanced imaging technologies in collaboration with the Laboratory of Optical and Computational Instrumentation to image MT dynamics in vivo. I will label MT polymers and plus/minus ends with multiple fluorophores, and use fluorescence recovery after photobleaching to determine whether MT transport contributes to polarity in vivo. I will test whether Clstn1 loss affects MT transport, and thereby influences polarity. I will also test the hypothesis that Clstn1 functions to organize MT polarity during axon branching by imaging MT dynamics at branch points. These experiments will enhance our understanding of the cellular and molecular mechanisms that establish axon MT polarity.