The generation of a functional nervous system requires that neuronal cells and axons navigate precisely to their appropriate targets. The Eph Receptor Tyrosine Kinases (RTKs) and their ephrin ligands have emerged as one of the important guidance cues for neuronal and axon navigation. However, the molecular mechanisms of how Eph RTKs regulate these processes are still incomplete. The purpose of this work was to contribute to the understanding of how Eph receptors regulate axon guidance by identifying and characterizing components of the Caenorhabditis elegans Eph RTK (VAB-1) signaling pathway. To achieve this objective I utilized a hyper active form of the VAB-1 Eph RTK (MYR-VAB-1) that caused penetrant axon guidance defects in the PLM mechanosensory neurons, and screened for suppressors of the MYR-VAB-1 phenotype. Through a candidate gene approach, I identified the adaptor NCK-1 as a downstream effector of VAB-1. Molecular and genetic analysis revealed that the nck-1 gene encodes for two isoforms (NCK-1A and NCK-1B) that share similar expression patterns in parts of the nervous system, but also have independent expression patterns in other tissues. Genetic rescue experiments showed that both NCK-1 isoforms can function in axon guidance, but each isoform also has specific functions. In vitro binding assays showed that NCK-1 binds to VAB-1 in a kinase dependent manner. In addition to NCK-1, WSP-1/N-WASP was also identified as an effector of VAB-1 signaling. Phenotypic analysis showed that nck-1 and wsp-1 mutants had PLM axon over extension defects similar to vab-1 animals. Furthermore, VAB-1, NCK-1 and WSP-1 formed a complex in vitro. Intriguingly, protein binding assays showed that NCK-1 can also bind to the actin regulator UNC-34/Ena, but genetic experiments suggest that unc-34 is an inhibitor of nck-1 function. Through various genetic and biochemical experiments, I provide evidence that VAB-1 can disrupt the NCK-1/UNC-34 complex, and negatively regulate UNC-34. Taken together, my work provides a model of how VAB-1 RTK signaling can inhibit axon extension. I propose that activated VAB-1 can prevent axon extension by inhibiting growth cone filopodia formation. This is accomplished by inhibiting UNC-34/Ena activity, and simultaneously activating Arp2/3 through a VAB-1/NCK-1/WSP-1 complex. / Thesis (Ph.D, Biology) -- Queen's University, 2011-07-28 16:20:31.957

Regulation of the actin cytoskeleton is key to cellular function and underlies processes including cell migration, mitosis and endocytosis. Motile cells send out dynamic actin protrusions that enable them to sense and interact with their environment, as well as generating physical forces. Linking of the actin cytoskeleton to the cell membrane is essential for the formation of these protrusions. The proteins that are thought to fulfil such a role have a membrane interacting domain (such as the PH domain in lamellipodin, or I-BAR protein in IRSp53) and a domain which interacts with actin regulatory proteins (such as the SH3 domain of IRSp53, which binds Ena and VASP). I investigated the contribution of the F-BAR protein Toca-1 in linking actin polymerisation to membranes, by characterising a new protein-protein interaction and the interaction of Toca-1 with giant unilamellar vesicles. FBP17, a homologue of Toca-1, can oligomerise to form 2D flat lattices and 3D tubules on membranes. Proteins of the Toca-1 family have previously been implicated in actin polymerisation in cell-free systems and during endocytosis. However, there is emerging evidence that Toca-1 family proteins could also be involved in the formation of outward facing protrusions, lamellipodia and filopodia. In an in vitro system that recapitulates the formation of filopodia-like structures (FLS) on supported lipid bilayers, Toca-1 is recruited early, suggesting a Toca-1 scaffolding mechanism could precede the recruitment of other actin regulators. One prediction of this model is that Toca-1 would bind proteins previously implicated in filopodia formation, such as formins. I found that extracts depleted of Toca-1 binding partners no longer forms filopodia-like structures and subsequently optimised pull-down assays to identify Toca-1 binding partners by mass-spectrometry. I identified four formins, Diaph1, Diaph3, FHOD1 and INF2, and as well as the actin elongation factors and filopodia proteins, Ena and VASP. I further characterised these interactions and found that Toca-1 binds Ena and VASP via its SH3 domain. The interaction is direct and is strongly reduced if the proline-rich region in Ena is deleted. VASP was still able to bind without its proline rich region, suggesting there could be additional binding sites. I discovered that the binding of Ena and VASP was dependent on the clustering state of Toca-1, whilst the binding of the previously identified Toca-1 binding partner N-WASP was not. This further supports the importance of Toca-1 oligomerisation in actin polymerisation. I tested these interactions in the FLS system and found that increasing Toca-1 concentration leads to increased recruitment of N-WASP, as well as the novel binding partner Ena to the structures, whereas an increase in VASP was not observed. SH3-domain mediated interactions are required for Toca-1 recruitment to FLS, suggesting that its membrane and protein binding activities act cooperatively. I showed that unlike N-WASP, which promotes the formation of branched actin, Ena and VASP are not required for actin polymerisation on supported lipid bilayers, suggesting that they are redundant with other factors in the elongation step of FLS formation. Ena and VASP are known to be important for the formation of neuronal filopodia and so I began to further test the role of these interactions in a cellular context using a neuronal cell culture system. As well as recruiting protein binding partners, F-BAR family proteins are implicated in stabilising lipid microdomains and can induce the clustering of phosphoinositides. I investigated the role of Toca-1 in actin polymerisation on PI(4,5)P2-rich giant unilamellar vesicles (GUVs). Actin-rich tails formed on the GUVs only when excess Toca-1 was supplemented into the extracts, and I propose that this is due to lipid organisation by Toca-1. In summary, my work suggests a model in which Toca-1 clusters, stabilises the membrane lipids and recruits regulators of actin polymerisation, such as Ena. This mechanism could be used to link actin polymerisation to the membrane in cellular protrusions, such as filopodia.