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

NIMA 相关激酶在神经元微管稳定性和运输中的作用

• 批准号: 10464307
• 负责人: Kaiden Michael Power
• 金额: $ 4.3万
• 依托单位: RUTGERS, THE STATE UNIV OF N.J.
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-07-01 至 2024-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10464307
• 关键词: Affect; Afferent Neurons; Animals; Bronchiectasis; Caenorhabditis elegans; Cell physiology; Cells; Cellular biology; Cilia; Clustered Regularly Interspaced Short Palindromic Repeats; Code; Cytoskeleton; Defect; Dendrites; Disease; Electron Microscopy; Flagella; Genetic; Growth; Health; Hippocampus (Brain); Homologous Gene; Human; Image; Impairment; Kinesin; Lead; Learning; Location; Malignant Neoplasms; Mammalian Cell; Measures; Microscopy; Microtubules; Mitochondria; Mitosis; Molecular Motors; Motor; Movement; Mucociliary Clearance; Mus; Mutation; NIMA; Nematoda; Nerve Degeneration; Nervous system structure; Neurodegenerative Disorders; Neuronal Injury; Neurons; Organelles; Pattern; Phenotype; Phosphorylation; Phosphotransferases; Play; Polycystic Kidney Diseases; Post-Translational Protein Processing; Process; Proteins; Purkinje Cells; Regulation; Reporter; Research; Resolution; Role; Signal Transduction; Structure; Tail; Testing; Time; Transgenic Organisms; Travel; Tubulin; Vesicle; Work; base; cell motility; ciliopathy; cilium biogenesis; cilium motility; experimental study; in vivo; innovation; insight; mutant; neuron loss; neuronal transport; phosphoproteomics; prevent; time use; tomography; trafficking

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.

项目摘要 微管蛋白编码通过微管蛋白尾部的谷氨酰化，调节纤毛的结构、运输和功能， 线虫C.优雅的我们确定了CCPP-1脱谷氨酰胺酶缺乏症的遗传抑制因子， 导致人类神经变性，海马神经元微管运输受损 培养，C.优雅的NIMA相关激酶NEKL-4/NEK 10的突变 抑制CCPP-1诱导的纤毛变性。在人类中，NEK 10突变导致支气管扩张， 由于活动纤毛的缺陷导致的呼吸道中粘液纤毛运输的紊乱。NEK 10在某些情况下也很重要。 细胞的纤毛发生和线粒体功能，并预测调节几个纤毛相关的过程 通过磷酸化活性。In C.在线虫中，NEKL-4在所有纤毛神经元中表达，但不 定位于纤毛，表明NEKL-4间接影响CCPP-1对纤毛稳定性的调节， 谷氨酰化该项目旨在了解NEKL-4/NEK 10在神经元中的作用机制。一是 将基于其活性状态和微管稳定性来检验NEKL-4定位是动态的这一假设。 我们将确定NEKL-4激酶活性是否对其功能至关重要，并研究NEKL-4在细胞凋亡中的作用。 纤毛微管超微结构接下来，我们将测试活性NEKL-4蛋白参与 树突状线粒体转运在小鼠中，CCP 1突变导致浦肯野细胞变性和免疫缺陷。 线粒体融合和运输。因此，我们将在ccpp-1中测量线粒体的树突状运输 使用延时成像来观察突变体。我们还将确定nekl-4是否抑制ccpp-1表型， 改变贩运动态。最后，我们将测试NEKL-4与微管- 通过调节感觉神经元中的驱动蛋白马达，基于运输和/或纤毛运输。我们的创新 这种方法利用了C.秀丽隐杆线虫与转基因和 CRISPR生成的内源性荧光报告基因和超分辨率显微镜;这将使我们能够 可视化活体完整动物的神经元运输。我们的研究将提供对基本细胞的深入了解 微管、分子马达和纤毛/鞭毛以及人类纤毛病的生物学， 神经退行性疾病