Establishment and Preservation of Microtubule Polarity in the Axon

 描述(申请人提供)：这项建议旨在确定细胞质动力蛋白在建立和维持轴突微管极性模式中的作用。微管近乎均匀的正端远端取向是轴突的一个特征，在自然界中发现的神经元中高度保守。这种微管的极性对神经元的功能至关重要，因为携带细胞器和囊泡等货物的马达蛋白使用微管的极性来指导它们的运动。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进展甚微。胞质动力蛋白是一种负端导向的运动蛋白，能够运输具有正端引导的微管。这一提议的中心假设是，动力蛋白驱动短的微管进入轴突，其正端导致建立轴突的微管阵列，但也将负端的远端微管赶出轴突，以保持轴突的微管极性模式的保真度。是这样的 负端远端微管可能出现在整形过程中，如轴突形成，或由于疾病或损伤相关的挑战。而大多数微管运输 轴突是顺行的，有相当一部分是逆行的，这与这种清除机制的存在是一致的。然而，细胞质动力蛋白的作用已被证明在技术上很难研究，因为以前抑制或耗尽这种Moto蛋白的方法是逐渐进行的，从而引入了其他发动机蛋白水平的代偿性变化的可能性。此外，由于技术限制，传统的微管运输可视化方法并不理想。在这里，这一假设将通过两个具体的 AIMS-第一个目标将确定抑制细胞质动力蛋白对轴突微管极性方向的影响；第二个目标将检验细胞质动力蛋白通过将负端微管运输回细胞体来清除轴突末端负端微管的假说。针对这两个目标的拟议研究利用原代培养的大鼠交感神经元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，允许对动力蛋白进行急性、可逆的抑制。这些策略绕过了长期存在的技术问题，允许对这一假设进行检验，同时创建了观察活神经元中微管运输的新黄金标准。动力蛋白驱动的微管运输负责轴突内微管极性模式的建立和维持，这一概念对于理解神经元的发育以及神经系统疾病和损伤的进展和潜在的治疗具有深远的意义。

 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.