Establishment and Preservation of Microtubule Polarity in the Axon

 描述（由申请人提供）：本提案旨在确定细胞质动力蛋白在轴突中微管极性模式的建立和维持中的作用。微管的近一致的正末端远端取向是轴突的标志性特征，并且在整个自然界中发现的神经元中高度保守。这种微管极性对于神经元功能至关重要，因为携带诸如细胞器和囊泡等货物的运动蛋白使用微管极性来引导它们的运动。然而，自1981年发现以来，在阐明轴突微管阵列产生和保存的潜在机制方面几乎没有取得进展。细胞质动力蛋白是一种负端导向的运动蛋白，能够以正端为前导转运微管。该提议的中心假设是动力蛋白驱动短微管进入轴突，其正端导致构建轴突的微管阵列，但也驱动负端远端微管离开轴突，以保持轴突微管极性模式的保真度。等 负端远端微管可在可塑性事件如轴突分支形成期间出现，或作为疾病或损伤相关挑战的结果出现。虽然大多数微管转运 在轴突是顺行的，一个显着的分数是逆行的，这是符合存在这样的清除机制。然而，细胞质动力蛋白的作用已被证明在技术上难以研究，因为以前的方法抑制或耗尽这种马达蛋白是逐渐的，从而引入了其他马达蛋白水平的补偿性变化的潜力。此外，由于技术限制，用于可视化微管转运的传统方法是次优的。在这里，将通过两个具体的假设进行测试， 目的-第一个目的将确定抑制细胞质动力蛋白对轴突微管极性取向的影响;第二个目的将测试细胞质动力蛋白通过将它们运输回细胞体而从轴突清除负端远端微管的假设。关于这两个目标的拟议研究利用大鼠交感神经元的原代培养物进行细胞生物学分析，使用创新的活细胞成像技术和复杂的抑制策略，允许动力蛋白的急性可逆抑制。这些策略规避了长期存在的技术问题，使假设得以验证，同时为观察活神经元中微管转运创造了新的黄金标准。动力蛋白驱动的微管运输的概念是负责建立和维持微管极性模式的轴突有深远的影响，神经元发育的理解和神经疾病和损伤的进展和潜在的治疗。

 DESCRIPTION (provided by applicant): This proposal aims to determine the role of cytoplasmic dynein in the establishment and maintenance of microtubule polarity patterns in the axon. The nearly uniform plus-end distal orientation of microtubules is a hallmark characteristic of axons and is highly conserved in neurons found throughout nature. This microtubule polarity is critical for neuronal function, as motor proteins that carry cargoes such as organelles and vesicles use microtubule polarity to guide their movement. Yet since its discovery in 1981, little progress has been made in elucidating the underlying mechanism responsible for generating and preserving the axonal microtubule array. Cytoplasmic dynein, a minus-end directed motor protein, is capable of transporting microtubules with their plus- ends leading. The central hypothesis of this proposal is that dynein drives short microtubules into the axon with their plus-ends leading to build the microtubule array of the axon, but also drives minus-end distal microtubules out of the axon, to preserve fidelity of the axon's microtubule polarity pattern. Such minus-end distal microtubules may arise during plastic events such as axonal branch formation, or as a result of disease or injury-related challenges. While the majority of microtubule transport in the axon is anterograde, a notable fraction is retrograde, which is consistent with the existence of such a clearing mechanism. However, the role of cytoplasmic dynein has proven technically difficult to investigate because previous methods for inhibiting or depleting this moto protein did so gradually, thus introducing the potential for compensatory changes in the levels of other motor proteins. Additionally, traditional methods for visualizing microtubule transport were sub- optimal due to technical limitations. Here, the hypothesis will be tested through two specific aims - the first aim will determine the effects on axonal microtubule polarity orientation of inhibiting cytoplasmic dynein; the second aim will test the hypothesis that cytoplasmic dynein clears minus-end distal microtubules from the axon by transporting them back to the cell body. Proposed studies on these two aims utilize primary cultures of rat sympathetic neurons for cell biological analyses using innovative live-cell imaging techniques and sophisticated inhibition strategies that allow acute, reversible inhibition of dynein. These strategies circumvent longstanding technical issues, allowing the hypothesis to be tested, while simultaneously creating a new gold standard for observing microtubule transport in living neurons. The notion of dynein-driven transport of microtubules as being responsible for the establishment and maintenance of microtubule polarity patterns in the axon has profound implications for the understanding of neuronal development and the progression and potential treatments of neurological diseases and injuries.