Function of kinetochore proteins in post-mitotic neurons

项目摘要 微管既是重要的结构元件，又是长距离传输的通道。他们是 神经元比其他细胞更稳定，令人惊讶的是，与 有丝分裂中的动粒是这种稳定性所必需的。KMN网络蛋白将动粒连接到动态 有丝分裂中的微管，最近发现影响有丝分裂后的小鼠、蠕虫和苍蝇的细胞形状 神经元。该方案中的初步数据表明，它们具有抑制微管动力学的功能 神经元。在神经元微管动力学的调节中，也发现了类似的作用。 染色体乘客复合体(CPC)和纺锤体组装检查点(SAC)蛋白。如何以及在哪里 这些有丝分裂蛋白作用于成熟神经元以控制微管的稳定性将被研究。 目的1动粒蛋白通过什么途径控制神经元中的微管和末端数目？ 当有丝分裂后果蝇神经元中动粒蛋白水平降低时，更多的微管+ 在树突中观察到末端，但在轴突中观察不到末端。上调的微管断裂或成核可以解释 对于加端数量的增加。Patronin识别由切断产生的减端，因此 越来越多的Patronin标记的负端将被用来区分这两种可能性。 对遗传相互作用的分析也将被用来确定动粒蛋白是否抑制 成核或断核。在神经元中，核化增加与神经保护有关，而切断则先于 因此，了解哪些是由动粒蛋白调控的是很重要的。 目的2.KMN蛋白在神经元中定位于何处以及如何定位？初步研究表明，有三项 不同的KMN网络蛋白定位于神经元胞体中的斑点。这些点号标识 系绳位置将确定。最有可能的候选者是高尔基复合体和溶酶体，如 细胞器主要分布在细胞体中。 目的3.动粒蛋白是否感知微管+末端到达细胞体？这个项目的最终目标是 建议测试动粒蛋白在神经元和有丝分裂细胞中是否具有类似的功能。在早期 有丝分裂KMN网络与CPC和SAC蛋白合作，感知微管到达 动毛虫。微管到达后，CPC和SAC的定位发生改变。在神经元中，这些蛋白质可以 共同检测从树突长入细胞体的微管数量。为了测试这一点 假设从树突进入的微管数量会增加和减少，而 监测动粒蛋白在细胞体中的定位。 动粒蛋白代表了神经元微管行为的主要新调节因子。这个首字母 探索它们在神经元中如何以及在哪里发挥作用，将为理解它们的 有丝分裂后对生命的贡献。

Project Summary Microtubules are both critical structural elements and the tracks for long-range transport. They are much more stable in neurons than other cells, and, surprisingly, the regulatory machinery associated with kinetochores in mitosis is required for this stability. KMN network proteins link the kinetochore to dynamic microtubules in mitosis and were recently shown to impact cell shape in post-mitotic mouse, worm and fly neurons. Preliminary data in this proposal indicates they function to suppress microtubule dynamics in neurons. A similar role in regulation of neuronal microtubule dynamics was found for the regulatory Chromosome Passenger Complex (CPC) and Spindle Assembly Checkpoint (SAC) proteins. How and where these mitotic proteins act in mature neurons to control microtubule stability will be investigated. Aim 1 What pathway is used by kinetochore proteins to control microtubule plus end number in neurons? When levels of kinetochore proteins are reduced in post-mitotic Drosophila neurons, more microtubule plus ends are observed in dendrites, but not axons. Upregulated microtubule severing or nucleation could account for the increase in plus end number. Minus ends generated by severing are recognized by Patronin, so the number of growing Patronin-tagged minus ends will be used to distinguish between these two possibilities. Analysis of genetic interactions will also be used to determine whether kinetochore proteins suppress nucleation or severing. In neurons increased nucleation is linked to neuroprotection while severing precedes degeneration, so is it important to understand which is regulated by kinetochore proteins. Aim 2. Where and how are KMN proteins localized in neurons? Pilot studies have demonstrated that three different KMN network proteins localize to puncta in the neuronal cell body. The identity of these punctate tether sites will be determined. Most likely candidates are the Golgi complex and lysosomes as these organelles are localized predominantly in the cell body. Aim 3. Do kinetochore proteins sense microtubule plus end arrival in the cell body? The final goal in this proposal is to test whether kinetochore proteins function analogously in neurons and mitotic cells. In early mitosis the KMN network cooperates with CPC and SAC proteins to sense microtubule arrival at the kinetochore. After microtubule arrival the CPC and SAC change localization. In neurons these proteins could work together to detect number of microtubules growing into the cell body from dendrites. To test this hypothesis the number of microtubules entering from dendrites will be increased and decreased while monitoring kinetochore protein localization in the cell body. Kinetochore proteins represent major new regulators of neuronal microtubule behavior. This initial exploration of how and where they function in neurons will provide a strong foundation for understanding their contribution to life after mitosis.