Cytoskeletal and signalling mechanisms of axon collateral branching

Abstract

Axon collateral branching is a ubiquitous mechanism during neurodevelopment. More recently, it has been implicated in regeneration in the adult nervous system. Cytoskeleton remodelling is an essential step in facilitating collateral branch formation. Actin structures such as actin patches play essential roles during the initiation of collateral branches from the axon. However, the regulation of actin patches and early molecular mechanism initiating branch formation is poorly understood. FMN2, actin nucleation and elongation protein, has been implicated in various neurodevelopmental disorders. I investigated the role of FMN2 in regulating patch dynamics. This thesis revealed that FMN2 localises to actin patches and increases the lifetime of an actin patch. Furthermore, it was shown that FMN2 localises at the base of early protrusion and is not needed when the branch matures and elongates in length. I use optogenetic and calcium-based techniques to study axon collateral branching. Calcium is an essential secondary messenger that has been implicated in multiple metabolic pathways, including collateral branching. I show that spontaneous calcium transients along the axons do not increase filopodial initiation. NGF-TrkA signalling has been shown to regulate axon collateral branching. Therefore, I attempt to establish an optogenetic system where I can spatiotemporally regulate the formation of a branch. This thesis showed that optical activation of TrkA signalling significantly enhances protrusion density and filopodial lengths. This thesis presents a unique toolset to study the regulation of collateral branching alongside looking at the role of FMN2 in mediating cytoskeleton remodelling during interstitial branching.