Mechanisms that control neuronal microtubule polarity

项目摘要 神经元微管与神经元健康密切相关，但许多基本原则， 控制神经元微管组织仍然是个谜。例如，微管可以成核 在轴突和树突中，但将成核位点定位在远离细胞体的调节剂没有 被识别。轴突损伤和应激使整个神经元的成核上调， 重要的是了解它是如何控制的。如果控制神经元微管的基本机制 组织不阐明，将很难理解微管之间的关系， 调节蛋白和神经变性。遗传性痉挛性截瘫是遗传学上最简单的 神经退行性疾病的形式，以及编码微管切断蛋白的基因突变 40%的病例是由痉挛引起的然而，痉挛蛋白在成熟神经元中的作用方式和位置尚不清楚。 为什么它的减少会使神经元对退化敏感，也不清楚。本提案中 果蝇被用作一个模型系统，在其中有效地识别调节神经元的基本机制， 微管提供了一个框架，为未来的工作再生和退化。 目标1.一种意想不到的途径控制树突中的微管成核。使用筛分 方法，Wnt信号蛋白作为定位微管成核位点的关键调节因子出现在细胞中。 树突这一目标的初步数据表明，Wnt信号蛋白可能激活细胞核的形成。 核内体这些初步的发现将得到加强配对一个新的成核测定与 Wnt信号传导蛋白和内体的定位。 目标2.切断蛋白质在将负末端成核转化为负末端生长中的作用。这是 最近的研究表明，负末端生长在枝晶中，这对极性控制和枝晶生长非常重要。 再生初步数据表明，痉挛蛋白在产生负端生长中起作用。使用新 与遗传工具相结合的可视化成核和切断的测定，微管如何以及在哪里形成， 将研究在树突中切断。 目标3.通过检查点系统控制分裂成核。微管成核有可能 通过产生随机方向的微管来破坏极性。详细的实时成像， 微管加末端决定了检查点系统解聚了在微管中成核的微管。 “错误”的方向。促进"正确"方向微管生长所需的两个参与者， 鉴定它们的功能，定位和相互作用将被调查。 结论：我们将阐明控制树突的基本但知之甚少的机制 微管组织填补了我们对神经元细胞骨架理解的关键空白， 理解神经元再生和退化的背景。

Project Summary Neuronal microtubules are intimately interconnected with neuronal health, yet many basic principles that control neuronal microtubule organization remain mysterious. For example, microtubules can be nucleated throughout axons and dendrites, but regulators that position nucleation sites far from the cell body have not been identified. Nucleation is upregulated throughout the neuron by axon injury and stress so it is particularly important to understand how it is controlled. If basic mechanisms that control neuronal microtubule organization are not elucidated, it will be very difficult to understand the relationships between the microtubule regulatory proteins and neurodegeneration. Hereditary spastic paraplegia is genetically one of the simplest forms of neurodegenerative disease, and mutations in the gene that encodes the microtubule severing protein spastin cause 40% of cases. However, how and where spastin functions in mature neurons has not been pinned down, and why its reduction sensitizes neurons to degeneration is also not clear. In this proposal Drosophila is used as a model system in which to efficiently identify basic mechanisms that regulate neuronal microtubules to provide a framework for future work on regeneration and degeneration. Aim 1. An unexpected pathway controls microtubule nucleation in dendrites. Using a screening approach, Wnt signaling proteins emerged as key regulators that position microtubule nucleation sites in dendrites. Preliminary data in this aim indicates Wnt signaling proteins likely activate nucleation on endosomes. These preliminary findings will be strengthened by pairing a new nucleation assay with localization of Wnt signaling proteins and endosomes. Aim 2. The role of severing proteins in converting minus end nucleation to minus end growth. It was recently shown that minus ends grow in dendrites and this is important for polarity control and dendrite regeneration. Preliminary data indicates spastin plays a role in generating growing minus ends. Using new assays to visualize nucleation and severing in combination with genetic tools, how and where microtubules are severed in dendrites will be investigated. Aim 3. Control of disruptive nucleation by a checkpoint system. Microtubule nucleation has the potential to disrupt polarity by generating microtubules in random orientations. Detailed live imaging of growing microtubule plus ends determined that a checkpoint system depolymerizes microtubules nucleated in the “wrong” orientation. Two players required for promoting growth of “right” orientation microtubules have been identified. Their function, localization and interactions with one another will be investigated. Conclusion: We will elucidate fundamental but poorly understood mechanisms that control dendrite microtubule organization to fill in key gaps in our understanding of the neuronal cytoskeleton that are essential context for understanding neuronal regeneration and degeneration.