The non-canonical role of the outer kinetochore protein KNL-1 in axonal development

Abstract

The nervous system is composed of specialized cells; glia and neurons, that form an interconnected network to relay information. Proper transmission of information relies on the two main compartments within neurons, dendrites, that receive information, and axons, that relay the information through specialized domains called synapses. Axon development is a multi-step process that involves, axon outgrowth, guidance, termination and synaptogenesis. In every step of axonal development rearrangements in the microtubule and actin cytoskeleton are essential to mediate the morphological changes that the axon undergoes. The molecular mechanism governing cytoskeletal regulation during axon development is still not fully characterized. Recent findings have highlighted a novel, non-canonical role for the outer kinetochore protein network, KMN (Knl-1, Mis12, Ndc80), in neuronal development. KMN, primarily recognized for its role in tethering chromosomes to spindle microtubules during chromosome segregation in cell division, has emerged as a potential key cytoskeletal regulator in neurons. This work investigates the noncanonical neuronal role of the outer kinetochore signalling and scaffolding protein KNL-1, in brain organization and axon development. In my thesis I show that KNL-1 is essential for axon organization and termination in the nervous system of C. elegans. In the first part I show that KNL-1 is required for the organization of the C. elegans nerve ring axons and ganglia organization in the brain. Specifically, loss of KNL-1 affects the correct placement and fasciculation of the axons within the nerve. Structure-function analysis of KNL-1 showed that this function requires both the signalling and microtubule binding domains of KNL-1. The second part of my work reveals an essential role for KNL-1 in axon termination, a process whereby the axonal growth cone is destabilized and stops its growth upon reaching its target. The effect of KNL-1 in axon termination, requires reorganization of F-actin at the axonal tip and is regulated by microtubule dynamics. In the final part, I have used Correlative Light-Electron Microscopy and a GFPTrap of KNL-1 in C. elegans embryonic neurons to identify the neuronal structures and proteins that KNL-1 associates within the axon. KNL-1 associates with endo-lysosomal structures in the cell body and synaptic vesicles in the axon. Mass spectrometry analysis revealed a synaptic protein as a potential interactor-candidate of KNL-1. This work showed a new potential link of KNL-1’s neuronal activity with synaptic organization and function. Overall, this study provides insights into the mechanism by which the outer kinetochore component KNL-1 functions in brain development, identifies a novel role for this protein in axon termination and reveals neuronal interactors of KNL-1 highlighting a potential role of the protein in synapses