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

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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.