Role of a NIMA-related kinase in neuronal microtubule stability and transport

项目摘要 微管蛋白编码通过微管蛋白尾部的谷氨酰化作用，调节纤毛的结构、运输和功能 线虫线虫。我们发现了CCPP-1脱谷氨酸酶缺乏症的基因抑制因子 导致人类神经变性，海马神经元微管转运障碍 培养，线虫的神经元纤毛退化。NIMA相关激酶NEKL-4/NEK10的突变 抑制CCPP-1诱导的纤毛变性。在人类中，NEK10突变会导致支气管扩张， 由运动纤毛缺陷引起的呼吸道粘液纤毛运输障碍。NEK10在某些情况下也很重要 纤毛发生和线粒体功能的细胞，并被预测调节几个纤毛相关的过程 通过磷酸化活性。在线虫中，NEKL-4在所有纤毛神经元中都有表达，但不表达 提示NEKL-4间接影响CCPP-1和CCPP-1对纤毛稳定性的调节 谷氨酰化。本项目旨在了解NEKL-4/NEK10在神经元中的作用机制。首先，我们 将根据NEKL-4的活性状态和微管稳定性来检验NEKL-4定位是动态的假设。 我们将确定NEKL-4激酶活性是否是其功能所必需的，并研究NEKL-4在 纤毛微管超微结构。接下来，我们将检验一种假设，即活性NEKL-4蛋白参与了 树突状线粒体运输。在小鼠中，CCP1突变导致浦肯野细胞退化和缺陷 线粒体的融合和运输。因此，我们将测量CCPP-1中线粒体的树突运输 使用延时成像的突变体。我们还将确定nekl-4是否通过以下途径抑制CCPP-1表型 人口贩运动态的变化。最后，我们将测试NEKL-4协调微管的假设- 通过调节感觉神经元中的运动蛋白马达，基于运输和/或纤毛运输。我们的创新 该方法利用线虫简单、透明的神经系统，结合转基因和 CRISPR生成的内源荧光记者和超分辨率显微镜；这将使我们能够 想象活着的完好无损动物的神经元运输。我们的研究将提供对基本细胞的洞察 微管、分子马达和纤毛/鞭毛的生物学以及人类纤毛疾病和 神经退行性疾病。

Project Summary The Tubulin Code, via glutamylation of tubulin tails, regulates ciliary structure, transport, and function in the nematode C. elegans. We identified genetic suppressors of CCPP-1 deglutamylase deficiency, which causes neurodegeneration in humans, impairment of microtubule-based transport in hippocampal neuron culture, and neuronal ciliary degeneration in C. elegans. Mutation in the NIMA-related kinase NEKL-4/NEK10 suppresses ccpp-1-induced ciliary degeneration. In humans, NEK10 mutation causes bronchiectasis, a disorder of mucociliary transport in the airway due to defective motile cilia. NEK10 is also important in some cells for ciliogenesis and mitochondrial function, and is predicted to regulate several cilia-related processes through phosphorylation activity. In C. elegans, NEKL-4 is expressed in all ciliated neurons but does not localize to cilia, suggesting that NEKL-4 indirectly influences regulation of ciliary stability by CCPP-1 and glutamylation. This project aims to understand the NEKL-4/NEK10 mechanism of action in neurons. First, we will test the hypothesis that NEKL-4 localization is dynamic based on its activity state and microtubule stability. We will determine if NEKL-4 kinase activity is essential for its function and investigate the role of NEKL-4 on ciliary microtubule ultrastructure. Next, we will test the hypothesis that active NEKL-4 protein is involved in dendritic mitochondrial transport. In mice, CCP1 mutation causes Purkinje cell degeneration and defects in mitochondrial fusion and transport. We will therefore measure dendritic trafficking of mitochondria in ccpp-1 mutants using time-lapse imaging. We will also determine if nekl-4 suppresses ccpp-1 phenotypes through alteration of trafficking dynamics. Finally, we will test the hypothesis that NEKL-4 coordinates microtubule- based transport and/or ciliary transport by regulating kinesin motors in sensory neurons. Our innovative approach utilizes the simple, transparent nervous system of C. elegans in combination with transgenic and CRISPR-generated endogenous fluorescent reporters and super-resolution microscopy; this will allow us to visualize neuronal transport in living intact animals. Our studies will provide insight to the fundamental cell biology of microtubules, molecular motors, and cilia/flagella as well as human ciliopathies and neurodegenerative diseases.