Mechanisms Of Drebrin-mediated F-actin Network Modification

Filamentous actin (F-actin) is an essential dynamic and modular cytoskeletal polymer serving diverse functions in cell motility, morphogenesis and signalling. Responsive architecture modifications of single filaments and their arrangement into higher-order networks of multiple filaments underlie function, driven by the association of F-actin binding proteins (ABPs). Drebrin is a key vertebrate ABP with diverse functions in multiple cell types and is implicated in pathological processes including opioid addiction, epilepsy, Down Syndrome, Alzheimer’s disease and normal aging. Drebrin has best known functions in neurons. In adult neurons, drebrin is localised at the dendritic post-synapse and is important in synaptic plasticity underlying learning and memory. In developing neurons, drebrin is a key modulator of developmental migration, projection outgrowth and connective pathfinding via its localisation and action at the leading-edge growth cone. Drebrin achieves these functions through binding F-actin and coincident single filament and downstream network modification, yet the mechanism of binding is unknown, or how binding translates into modifications of F-actin or higher-order networks. There is some evidence drebrin modifies the architecture of single F-actin filaments directly and modifies their stability. Drebrin F-actin association has been hypothesised to further regulate higher-order networks in cells by displacing other organiser ABPs either via rendering an incompatible F-actin architecture or by directly outcompeting for ABP-binding surfaces on F-actin. The core goal of this project is to reveal how drebrin interacts with F-actin and how this drives its modification both at the single filament and network level. We will first use cutting-edge cryo-electron microscopy (cryo-EM) to detail the interaction of drebrin with F-actin at near-atomic resolution. This objective will also reveal how drebrin modifies filament architecture and competes with ABPs. We will test our models by assessing the F-actin association-dependent ability of structure-based designed drebrin mutants to induce cellular filopodia. Filopodia are an important cell projection in motility and signal sensing formed and maintained by F-actin reorganisation. Secondly, we will determine how drebrin induces filopodia in cells. Nano-scale F-actin architecture reorganisation in drebrin-induced filopodia will be described using in situ cryo-electron tomography (cryo-ET). The possible roles of ABPs in drebrin-mediated filopodial induction will then be analysed by knockdown or overexpression of various ABPs. Furthermore, to analyse if they are displaced by drebrin, their relocalisation after drebrin overexpression will be assessed using immunofluorescence microscopy. Finally, we will determine how drebrin influences F-actin architectures in the neuronal growth cone, where it is concentrated at particular subregions; the transition zone (T-zone) and the base of filopodia. Using cryo-ET, the nano-scale F-actin architectures of these subregions where drebrin is concentrated will be revealed, comparing basal conditions with drebrin overexpression or removal. The potential relocalisation of ABPs from their normal localisations after drebrin overexpression or knockdown will also be assessed using immunofluorescence microscopy. This proposal will unveil the mechanisms of a fundamental regulator of the vertebrate F-actin cytoskeleton with important roles in neurons and numerous other cell types. In addition to advancing our basic understanding of fundamental biology, given drebrin’s implication in a variety of pathologies, this research promises auxiliary biomedical impact. The utilisation and development of cutting edge cryo-EM/cryo-ET techniques in this research will have supplemental impact in advancing the competitiveness of the UK scientific workforce.