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Function of kinetochore proteins in post-mitotic neurons

有丝分裂后神经元着丝粒蛋白的功能

基本信息

批准号：
10026166
负责人：
Melissa Rolls
金额：
$ 43.37万
依托单位：
PENNSYLVANIA STATE UNIVERSITY, THE
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-09-01 至 2023-08-31
项目状态：
已结题

项目摘要

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.

项目摘要微管既是重要的结构元件，也是长距离运输的通道。他们是在神经元中比其他细胞更稳定，而且，令人惊讶的是，这种稳定性需要有丝分裂中的动粒。KMN网络蛋白将动粒连接到动态微管在有丝分裂和最近被证明影响细胞形状在有丝分裂后的小鼠，蠕虫和苍蝇神经元该提案的初步数据表明，它们的功能是抑制微管动力学，神经元在调节神经元微管动力学中，染色体乘客复合物（CPC）和纺锤体组装检查点（SAC）蛋白。如何以及在何处将研究这些有丝分裂蛋白在成熟神经元中控制微管稳定性的作用。目的1着丝粒蛋白通过什么途径控制神经元微管加末端数目？当有丝分裂后果蝇神经元中动粒蛋白的水平降低时，更多的微管加在树突中观察到末端，但在轴突中未观察到末端。上调的微管断裂或成核可以解释增加了正端数。由切断产生的负端被Patronin识别，所以将使用大量生长的Patronin标记的负末端来区分这两种可能性。遗传相互作用的分析也将用于确定动粒蛋白是否抑制成核或切断。在神经元中，增加的成核与神经保护有关，而切断则先于因此，重要的是要了解哪些是由动粒蛋白调节的。目标2. KMN蛋白在神经元中的位置和方式？试点研究表明，不同的KMN网络蛋白定位于神经元细胞体中的点。这些斑点的身份将确定系留地点。最有可能的候选者是高尔基复合体和溶酶体，因为这些细胞器主要位于细胞体中。目标3。动粒蛋白能感知微管和末端到达细胞体吗？这场比赛的最终目标是该计划旨在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。月初在有丝分裂中，KMN网络与CPC和SAC蛋白合作，以感知微管到达有丝分裂细胞。动粒在微管到达后，CPC和SAC改变定位。在神经元中，这些蛋白质共同检测从树突生长到细胞体中的微管数量。为了验证这一假设从树突进入的微管数量会增加和减少，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。该初始探索它们在神经元中的作用方式和位置将为理解它们的功能提供坚实的基础。对有丝分裂后生命的贡献。