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Rising temperatures and growing populations are putting increased pressure on food sources, with pathogens moving polewards in search of more favourable conditions. Stopping crop losses due to disease would provide enough additional food to solve world hunger, with spare left over. My PhD has been centred on discovering more about how plants are able to defend themselves to new or unknown potential pathogens through the use of the model organism, Arabidopsis thaliana. I have helped to develop a novel method of stimulating the basal immune response in order to probe different components of this system. PEN3, a key defence protein that is transported to the site of infection, was observed over the initial 20 minutes of an immune response and its accumulation was quantified. PEN3 transport requires the actin cytoskeleton, which remodels within 30s of artificial peg contact, therefore I developed an image analysis algorithm to segment and quantify various aspects of the cytoskeletal network. This algorithm detected significant differences in the network of loss-of-function mutants of arp2-1 and formin4/7/8 compared to the wildtype, yet the PEN3 distributions were functionally identical. These findings, alongside simulations of PEN3 accumulation with various delivery zone sizes led to the hypothesis of at least one upstream signalling molecule designating PEN3 secretion into the membrane. The novel assay, network extraction tool and PEN3 simulations are discussed in detail, including their limitations and potential improvements as well as other systems they could be applied to.

Filopodia are finger-like cytoplasmic protrusions that contain filamentous actin and extend beyond the leading edge of migrating cells. Filopodia are thought to facilitate both sensing of signaling molecules and the movement of cells towards them. Profuse filopodia formation is seen in metastatic cancer cells and in diseases causing intellectual disability. Conversely, in the central nervous system, growth cone filopodial architecture fails to reform after injury, limiting healing. Despite the major impact that filopodia formation has on embryonic development and pathology, our understanding of the molecular mechanisms underlying filopodia formation remain incomplete and controversial. Using a collection of antibodies isolated by phage display antibody phenotypic screening on an *in vitro* system of filopodia-like structures (FLS) I aimed to discover new molecular mechanisms underlying filopodia formation. To identify the antigens of the phage display antibodies I combined Western blotting, immunoprecipitation and immunostaining approaches with different proteomic techniques and transient expression. The antibody panel was strikingly split into two groups: (1) Antibodies that lengthen FLS and do not recognise specific proteins in their eluates or (2) Antibodies that shorten FLS and show specific candidate proteins. Immunoprecipitation revealed a strong association with actin itself either directly or indirectly for the long phenotype subset of the antibodies. I used multiple mass spectrometry proteomic strategies including quantitative (tandem mass tag liquid chromatography mass spectrometry (TMT LC-MS/MS)) and qualitative (LC-MS/MS) for both immunoprecipitated proteins and excised bands. I developed immunostaining protocols and determined the localisation of the target antigens by immunostaining in FLS as well as in Xl177, U20S, HEK 293 cell lines, retinal ganglion cell growth cones and mouse intestinal organoids. To verify the candidate proteins, I combined Western blotting against purified proteins, sedimentation assays with purified actin, immunostaining and transient expression of proteins of interest, confirming the identity of two target antigens. I also sought to develop an *in vitro* system for filopodia triggered by SARS-CoV2 nucleocapsid protein, with limited success, but promising results for further study. This work shows that novel actin regulatory mechanisms can be identified based on antibody reactivity in the FLS system and gives new candidate molecules and mechanisms underlying filopodia formation and other actin assemblies during development and in different pathologies.

The role of the RNA-binding protein FUS in axonal organisation and disease In this thesis, I discuss the role of the RNA-binding protein Fused in sarcoma (FUS) in axonal organisation and disease. FUS aggregates in forms of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), and ALS-associated mutations change its phase transitions and subcellular localisation. This is known to compromise axonal local protein synthesis (LPS), but it is not well-understood which critical LPS-dependent processes are affected. This is important, as these processes represent potential therapeutic targets. In my work, I built on insights from the field of developmental neurobiology to formulate hypotheses on the physiological consequences of altered FUS phase behaviour. Specifically, I focussed on axonal survival signalling and cytoskeletal remodelling. I tested these hypotheses using the Xenopus laevis retinal ganglion cell (RGC) model system, which is an established model system for combining in vitro and in vivo studies into LPS-dependent axonal properties. First, I studied the effect of FUS mutation on LPS of axonal survival-associated proteins, as changes in the local activity of these could be linked directly to axonal degeneration. I focussed on the essential axonal survival factor nicotinamide mononucleotide adenylyltransferase 2 (NMNAT2), as its mRNA is a potential FUS binding target. I hypothesised that axonal NMNAT2 levels are sustained by LPS, as this protein is known to have a very short half-life. However, by combining in situ hybridisation and quantitative PCR approaches, I found that nmnat2 mRNA does not localise to RGC axons, falsifying this hypothesis. Therefore, I shifted the focus of my research to axonal cytoskeletal reorganisation, as this is known to be regulated by LPS, and ALS is associated with mutations in cytoskeleton-linked genes. I next hypothesised that reorganisation of the axonal cytoskeleton is compromised by mutant FUS, as this process is strongly LPS-dependent, and so studied the dynamic axon terminal. I used two previously reported ALS/FTD X. laevis models: the juvenile ALS-associated nuclear localisation signal mutant FUS(P525L) and an artificial FTD-mimic of hypomethylated FUS, FUS(16R). Mutant FUS compromised the actin cytoskeleton of the growth cone in vitro, resulting in considerable changes in growth cone mechanoproperties. This occurred without detectable reductions in axonal microtubule density or RNA transport function, indicating this defect may be specific to the dynamic actin cytoskeleton. However, axonal cytoskeletal organisation is dependent on local cues in vivo, and ALS is considered a non-cell-autonomous disease. Therefore, I subsequently studied whether this actin change correlates with in vivo phenotypes, hypothesising that mutant FUS would compromise actin-dependent axon branching in vivo. Mutant FUS indeed reduced axon complexity in vivo, and FUS(P525L) additionally induced axon looping, pointing to the occurrence of signalling defects. This loss of branching complexity has implications for synaptic connectivity, changes in which are a key feature of neurological disorders. Therefore, altered actin remodelling could be a central phenotype on which multiple pathomechanisms associated with ALS/FTD converge. These results have wider conceptual implications for our understanding of the nature of ALS/FTD. In particular, the present work demonstrates that mutant FUS-associated changes to axonal organisation can occur early in development already, which supports the emerging concept that neurodevelopmental and neurodegenerative diseases may have overlapping molecular players and may therefore share aspects of pathomechanisms. I conclude this thesis by discussing how mutant FUS-induced developmental phenotypes may be connected to onset of symptoms in adolescence or adulthood, and which future research directions emerge from this perspective.

Background: Migration and homing are essential to B-lymphocyte mediated immunity, and are driven by rapid, directed, and appropriate reorganisation of the actin cytoskeleton. Important observations have linked the cytoskeletal-rearrangements made by leukaemic B-lymphocytes of chronic lymphocytic leukaemia (CLL) to disease pathology. In particular, cytoskeletal alterations mediated by B-cell receptor (BCR) engagement or chemokine-binding are recognised to be central to the migration of CLL cells to lymphoid tissues, where they engage in the complex cellular and molecular interactions that underlie their survival, proliferation, and drug resistance. Further emphasising this importance, has been the observation that highly effective small molecule inhibitors that target key components of the BCR signalling machinery, such as Bruton's tyrosine kinase (BTK), disrupt the migratory behaviour of CLL cells, and that this may, at least in part, underlie their clinical effect. Detailed characterisation of the highly dynamic cytoskeletal alterations in CLL may, therefore, inform novel therapeutic interventions, particularly for subsets with unmet clinical needs, such as those with mutations affecting the tumour protein P53 (TP53), ataxia telangiectasia mutated (ATM), or Notch receptor 1 (NOTCH1) genes, which are all more frequent in IGHV-unmutated disease. This work describes the development of techniques to characterise and quantify morphological responses to inhibitors, aiming to produce a quantitative description of cytoskeletal changes relating to specific signalling pathways, and to suggest rational drug combinations in the disorder. Methods: Primary CLL cells were cultured at high density with autologous T-lymphocytes and monocytes in the presence of specific signal inhibitors. The morphological responses of leukaemic cells were determined using a range of microscopic techniques, including scanning electron microscopy, and immunocytofluorescent detection of cytoskeletal and plasma membrane components. Cytoskeletal alterations were evaluated via computer-aided morphometric analyses of cell shape parameter, homotypic cellular interactions, and migration, generating a precise description of changes to the polymerised F-actin cytoskeleton and cell behaviour. Matrigel™ matrix models were combined with transmission electron microscopy to study cellular morphology within a 3D tumour microenvironment (TME)-like setting. Immunogold labelling of specific proteins within neoplastic lymphocytes was performed to allow visualisation of protein localisation changes in response to signal inhibition at the ultrastructure level. Results: This study tested inhibitors targeting different signalling pathways as a 'proof of principle' evaluation to determine whether the morphological and behavioural responses induced could be effectively distinguished from one another and quantitatively described. Inhibition of BTK by ibrutinib resulted in uniform populations of globular cells with retained polarity and, consequently, increased amoeboid motility. BTK blockage is recognised to impair integrin-mediated retention of leukaemic cells within tissue niches, leading to the observed peripheral blood lymphocytosis seen in CLL patients receiving ibrutinib. Reduced integrin-mediated motility was associated with impaired homotypic cellular interactions within IGHV-mutated cases specifically, indicating that this subgroup may have a greater dependency on elongated-type migration for permitting pro-survival cellular contact than their IGHV-unmutated counterparts. Disruption of Rho-associated protein kinase 1 (ROCK1) activity by Y-27632 lead to impaired actomyosin-mediated retraction of cytoskeletal processes. Loss of the ROCK1-induced cytoskeletal asymmetry required for effective cell migration resulted in reduced CLL cellular interactions; however, CXCL12-driven motility was attenuated in IGHV¬-mutated cases alone. The Abelson kinase 1 (ABL1) inhibitor imatinib caused CLL cells to acquire a globular phenotype with frequent microvilli, similar to that of B-lymphocytes isolated directly from the peripheral blood. Transient cellular interactions were markedly reduced by imatinib, whereas elongated-type motility, being a largely ABL1-independent process, was unaltered. The morphological and behavioural responses of CLL cells were compared to those observed in mantle cell lymphoma (MCL) cell lines. These cells lines, which were utilised as a surrogate model for BTK inhibitor sensitivity in CLL, demonstrated that the establishment of anterior-posterior morphology, mediated by the activity of ROCK1 and ABL1, is essential for effective trafficking of B-lymphocytes to protective niches, regardless of ibrutinib sensitivity. Blockage of NOCTH1 signalling by gamma-secretase inhibitors (GSIs) PF-03084014 and R04929097 resulted in varying morphological responses, possibly indicating differences in NOTCH1 activation between CLL cases. Despite chemotaxis being identified as a key NOTCH1-regulated process, CLL and NOTCH1-mutated MCL cells demonstrated enhanced directional transmigration with GSI treatments. MCL cell lines were utilised to model the effects of GSI sensitivity in CLL. In contrast, NOTCH1-unmutated MCL cells displayed unaltered migration with PF-03084014 pre-treatment, consistent with reports of low GSI sensitivity in MCL cells exhibiting unmutated NOTCH1, and reduced chemotaxis with R04929097. The developed 3D ex vivo culture system preserved CLL cell viability, migration, and dynamic cellular interactions, as demonstrated by flow cytometry and time-lapse live-cell imaging. Interrogation with transmission electron microscopy enabled high-resolution visualisation of cell morphology within a TME-like setting; however, further optimisation of immunogold labelling of effector proteins is required. Conclusion: Using novel imaged-based morphometric analyses, distinct signal inhibitor-induced cytoskeletal adaptations were identified in CLL B-lymphocytes. This approach may be applied to prognostically-defined subgroups or resistant cases to provide in-depth characterisation of morphological responses to novel therapeutic agents and to assess treatment responses within the TME. These observations, when combined with transcriptional data, may allow more effective combinational targeting of behavioural signatures unique to the patient and, thus, improve treatment outcomes in the disease.

The actin cytoskeleton is a complex and dynamic network of protein filaments involved in a great number of cellular processes essential for cellular homeostasis and survival. The dynamic nature of the cytoskeleton requires careful control of actin stabilisation and severing. Cofilin is an important promoter of actin turnover, promoting the severing and disassembly of actin filaments, resulting in increased availability of actin and driving assembly of new actin structures . A major reported negative regulator of cofilin is p21 activated kinase 1 (PAK1). These dynamic structures and the importance of actin turnover result in targets for manipulation by bacterial pathogens, such as Salmonella Typhimurium’s ability to enter non-phagocytic cells, and Shigella flexneri to assemble actin ‘comet tails’ for intracellular propulsion and bacterial dissemination. This work sought to investigate the role that cofilin and PAK1, as well as its upstream and downstream regulators has in actin remodelling, and how this may be usurped by bacterial enteric pathogens, with a particular focus on Shigella flexneri comet tail formation. Shigella LPS O-antigen null mutants have been previously shown to have poor comet tail formation, however the mechanism behind this defect remains controversial. Previous studies have focused on the relationship between LPS length and the functionality of IcsA, the protein primarily responsible for intracellular motility. Here we demonstrate that LPS length does not affect IcsA’s ability to bind the vital actin related proteins necessary to produce actin comet tails, leading to investigation of the wider actin dynamics and the role they play in Shigella infection post-invasion. This work establishes key roles for both PAK1 and cofilin in bacterial manipulation of the actin cytoskeleton, demonstrating that the activation of both cofilin and PAK is required for Salmonella invasion and Shigella comet tail invasion. I provide evidence that the artificial activation of PAK and cofilin via ATP and calcium signalling results in the restoration of several attenuated bacterial mutant strains ability to manipulate the actin cytoskeleton. I provide evidence that Salmonella is able to limit its invasion, by the inhibition of cofilin post-invasion via the bacterial effector SopB, preventing the overloading of host cells and prolonging infection. I propose that Shigella activates cofilin post-invasion in response to caspase-1 activation by the bacterial effector protein IpaB, maintaining the dynamic cytoskeleton required for comet tail formation. This work also provides a potential mechanism for the failure of R-LPS Shigella mutants to form actin comet tails by indicating that the loss of O-antigen results in the secretion impairment of IpaB, resulting in its association to the bacterial membrane. Together these finding outline a novel function for IpaB’s activation of caspase-1 in the promotion of comet tail formation and provides further insight into the relationship between inflammation and actin reorganisation.

A critical step in the pathogenesis of enteric bacteria such as Salmonella is entering the host cell. To achieve this, Salmonella inject effector proteins into the cytosol of the cell and manipulate the actin cytoskeleton, driving pathogen entry. Two of the delivered effectors, namely SopE and SopE2, activate host Rho GTPases and subsequently the Arp2/3 complex, which results in actin reorganisation. Whereas the role of SopE in promoting bacterial uptake is well-established, the precise function of SopE2 is not known. SopE2 was previously shown to activate Cdc42, which leads to the mobilisation of N-WASP and the resulting Arp2/3 complex-mediated actin assembly. This work establishes that the activity of SopE2 restricts excessive Salmonella uptake via the generation of actin structures that exhibit anti-phagocytic properties. The loss of SopE2 or N-WASP is associated with unrestricted pathogen entry, highlighting the importance of those proteins for limiting bacterial invasion. The C-terminal VCA domain of N-WASP is revealed as the key region for the formation of the invasion-limiting actin networks. Those SopE2- and N-WASP-generated actin structures also obstruct the translocation of effectors from the invading bacteria. The recruitment of different subunit isoforms by the Arp2/3 complex can also modulate Salmonella uptake, however the precise role of the VCA in that event remains elusive. Finally, the loss of N-WASP is associated with a reduction in the cellular content of filamentous actin and a decrease in focal adhesions made by the cell, which may also play a role in boosting pathogen uptake. Together these results outline a novel mechanism utilised by Salmonella to control its own invasion, which most likely sustains the viability of the host and thus maintains the replicative niche. This work also highlights the dual role of the actin network for promoting and inhibiting both effector translocation and the uptake of the pathogen.

Egg activation is a series of highly coordinated processes that prepare the mature oocyte for embryogenesis. Typically associated with fertilisation, egg activation results in the resumption of the cell cycle, expression of maternal mRNAs and cross-linking of the vitelline membrane. While some aspects of egg activation, such as initiation factors in mammals and environmental cues in sea animals, have been well-documented, the mechanics of egg activation in many animals are still not well understood. This is especially true for animals where fertilisation and egg activation are unlinked. In order to elucidate how egg activation is regulated independently of fertilisation, I use Drosophila melanogaster as a model system. This insect provides extensive genetic tools, ease of manipulation for experimentation and is amenable for imaging. Through visualisation of calcium, Processing bodies and meiotic spindles, I show that osmotic pressure acts as an initiation cue for the calcium wave and downstream processes, including the resumption of cell cycle and the dispersion of the translational repression sites. I further show that aquaporin channels, together with external sodium ions, play a role in coordinating swelling of the oocyte in response to the osmotic pressure. I proceed to identify the requirement of internal calcium sources together with a dynamic actin cytoskeleton for a calcium wave to occur. Through co-visualisation of calcium and actin, I provide the first evidence that the calcium wave is followed by a wavefront of non-cortical F-actin at egg activation, which requires the calcium wave. Genetic analysis supports a model where changes in osmotic pressure trigger the calcium wave via stretch sensitive calcium channels in the oocyte membrane and the calcium wave is relayed by nearby channels via the actin cytoskeleton. My work concludes that the mechanism of egg activation in Drosophila is more similar to plants, compared to most vertebrates.

The actin cytoskeleton plays a vital role in various biological processes such as cell migration, morphogenesis, and intracellular trafficking. The polymerization of actin filaments at membranes provides the force for generating dynamic actin structures such as protrusions and invaginations that drive these processes. In filopodia, which are finger-like protrusions comprised of bundled actin filaments, actin regulatory proteins are believed to assemble a distal 'tip complex' which stimulates actin nucleation at the membrane. However how these regulators collectively behave in a macromolecular complex still remains poorly understood. To understand the macromolecular nature of these complexes, I investigated the dynamic properties and spatial organization of actin regulatory factors, using an in vitro reconstitution assay for filopodia-like structures (FLS) utilizing artificial lipid bilayers and Xenopus laevis egg extracts. FRAP analysis of seven actin regulatory factors (Toca-1, N-WASP, GTPase-binding domain, Ena, VASP, Diaph3, Fascin) revealed that the FLS tip complex has both dynamic and stable properties, with different proteins displaying distinct dynamics. Further analyses on the membrane-binding protein Toca-1 showed that its dynamic turnover is controlled by interactions with actin and exchange of molecules with solution. Single-molecule localization microscopy resolved the nanoscale organization of Toca-1, showing its arrangement into flat plaque-like and narrowly elevated tubular substructures. Plaque-like structures showed similarities to phase-transition patterns, while tubule-like structures closely resembled those previously found to decorate membrane tubules in vitro, which are thought to be involved in endocytic membrane remodeling. Endocytic accessory proteins such as SNX9 and Dynamin2 were also found to localize to FLS tips. This work provides new insights into the dynamics and organization of protein ensembles at actin nucleation sites, and proposes a novel link between endocytosis and filopodia formation, which is relevant to understanding how cells decide when and where to assemble actin at the membrane.

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.

Actin cytoskeletal regulation is of critical importance for a number of diverse cellular functions, including cell motility, endocytosis, cell division and transcription. Tight regulation of actin is critical for many aspects of cancer biology and in particular invasion and metastasis. ADF/cofilins are among the most important actin regulatory proteins. Mammals have three highly conserved members, ADF, CFL1 and CFL2, which regulate actin dynamics by severing and depolymerizing actin filaments. Despite a huge literature on the roles of ADF/cofilins in actin treadmilling and cell migration in vitro and in cancer cell behavior during invasion, very little is known about their collective roles in tissue homeostasis. By employing genetic knock-outs of ADF, in conjunction with conditional depletion of CFL1 using a Cre-LoxP system under the control of the keratin 14 promoter, we were able to study the effects of ADF/CFL1 loss in vivo in the mouse epidermis. Furthermore, by generating ADF-null squamous cell carcinoma (SCC) cell lines and by transiently downregulating CFL1 with RNAi, we were able to investigate further the cellular responses after ADF/CFL1 depletion in vitro. Co-depletion of ADF and CFL1 from the mouse epidermis triggered loss of tissue homeostasis characterized by abnormal thickening of the tissue, actin filament accumulation and nuclear deformation. Loss of ADF/CFL1 in cultured malignant keratinocytes also led to aberrant cell morphology accompanied by unrestrained accumulation of actin stress fibers tethered to enlarged focal adhesions. Enhanced SRF/MAL-mediated transcription fuels this uncontrolled actin polymerization which is also mediated by Arp3. Furthermore, these actin filaments are decorated with phospho-myosin light chain, which indicates their contractile nature. As a consequence, the increased intracellular acto-myosin tension results in nuclear deformation, which is promoted by the deregulated actin filaments tethered to the nuclear envelope via the linker of nucleoskeleton and cytoskeleton (LINC) complex. Overall, we describe new conceptual insight into the cellular functions of ADF/cofilins. We show that their activities are essential for the dynamic regulation of contractile actin filaments that, if left unchecked, lead to loss of cellular homeostasis and cell death promoted by loss of nuclear integrity. Additionally, the critical roles of nuclear actin and actin-associated proteins have recently started being appreciated. Thus, for the first time we set out to investigate new functions of cofilins in the nucleus using proteomics, and identify new cofilin binding partners that implicate them in novel cellular pathways, expanding our knowledge on these small actin-binding proteins.

The brain is a highly complex structure; neurons extend axons which follow precise paths to make connections with their targets. This extension is guided by a specialised and highly motile structure at the axon tip -the growth cone- which integrates guidance cues to steer the axon through the environment. Aberrant pathfinding is likely to result in developmental impairments causing disruption to brain functions underlying emotion learning and memory. Furthermore, pre-existing connections are constantly remodelled, the ability to do so declines with age, and can have huge impacts on quality of life and well-being. Examining how changes in growth cone behaviour triggered by external cues occurs is crucial for understanding processes in both development and disease. Controlled reorganisation of growth cone cytoskeletal components, such as actin filaments, generate membrane protrusions forming lamellipodia and filopodia. Filopodium formation is commonly associated with sensing the mechanical and chemical environment of the cell. Despite our understanding of the guidance choices that can be made, how filopodia transmit information at a molecular level leading to profound changes in morphology, motility and directionality remains largely unknown. Various actin-binding proteins regulate the number, stability and branching of filopodia. They may therefore have a key role in priming or abrogating the ability of the growth cone to respond to a given guidance cue. I have shown that the actin binding proteins drebrin and cofilin, whilst displaying opposing molecular activities on actin filaments, work synergistically in a temporally regulated manner. A fluorescent membrane marker combined with tagged cofilin and drebrin enabled accurate correlation of cofilin and drebrin dynamics with growth cone morphology and filopodial turnover in live neurons. In contrast to previous in vitro experiments, cofilin was found to enhance the effect of drebrin to promote filopodia formation in intact neurons, and that growth cone spread was significantly constrained when cofilin was knocked down. Importantly, this adds to our understanding of how the two actin binding proteins contribute to directed motility in neuronal growth cone filopodia during guidance. Furthermore, following acute treatment with low concentrations of the repulsive guidance cue semaphorin-3A, neuronal growth cones expressing cofilin displayed increased morphological complexity and filopodial stability. This suggests that traditional collapse signals may serve as pause signals allowing neurons to increase the surface area to sense the environment adequately and enable precise wiring decisions. Remodeling of the cytoskeleton is perturbed in a number of degenerative diseases including Alzheimer's, Huntington's, and Amyotrophic Lateral Sclerosis. These conditions are associated with widespread synaptic loss, resulting in memory loss, cognitive impairment, and movement disorders which leads to severe deterioration in quality of life for those afflicted in addition to wider negative socioeconomic impacts. How widespread synaptic loss occurs is poorly understood. One common characteristic is neuronal stress which can be initiated through different conditions such as neuroinflammation, energetic stress, glutamate excitotoxicity, and accumulation of misfolded proteins, all of which have been associated with perturbation of the actin cytoskeleton and the initiation of the cofilin-actin rod stress response. Dysfunction of the cytoskeleton can lead to the disruption of synaptic activity by blocking the delivery of elements such as organelles and proteins required for maintenance of the synapse. Modulating this stress response offers an approach to protecting the integrity of normal synaptic function. Actin interacting protein-1 is a conserved actin binding protein that enhances the filament disassembly activity of cofilin. I have discovered that AIP-1 has a potent ability to prevent the formation of cofilin rods which are thought to contribute to the neuronal dysfunction in several neurodegenerative disorders, even when they are treated with amyloid-β or subjected to metabolic stress. This is the first study to demonstrate a molecular mechanism for preventing rod formation in the presence of a neuronal stressor and has the potential to protect against rod formation by other stressors associated with disease such as inflammation and excitotoxicity. AIP-1 offers the exciting possibility of a means to reverse cofilin rod formation and the subsequent cytoskeletal pathology associated with dementia and has potential for therapeutic exploitation in human disease. Furthermore, it is the first study to demonstrate that AIP-1 localises to areas of rapid actin remodeling in neuronal growth cones. Exploiting the action of AIP-1 therefore represents an exciting and novel therapeutic avenue to tackle neurodegeneration.

The increasing demand on healthcare from pregnancies complicated by gestational diabetes (GDM) and obesity is caused in large part by fetal macrosomia (FM). Alterations to the vasculature of the placenta leading to changes to nutrient flux may be more frequent when GDM and obesity occur concomitantly. However, the impact of obesity as an independent comorbidity is poorly understood. The current study sought to characterise structural and functional changes in placenta from pregnancies complicated by GDM and/or obesity and examine the involvement of miRs in this phenomenon, as the phenotype of vascular smooth muscle (VSM) has been documented to be influenced by microRNA (miR) expression. Patients were stratified according to the presence or absence of GDM and/or obesity, which resulted in four groups. Morphometric analysis of CD31 immuno-stained placentas showed that pregnancies complicated by GDM or obesity both had a higher mean sum ratio of the area of the lumen compared to the endothelium. No relationship was found with FM. The ratio increased with maternal body mass index (BMI) in all pregnancies. Immunohistochemistry with a panel of VSM markers suggested an altered phenotype of VSM in pregnancies complicated by GDM and/or obesity. RT-QPCR and immunoblotting showed a higher expression of smooth muscle myosin (SM-MHC), h-caldesmon (HC) and alpha smooth muscle actin (ASMA) in pregnancies complicated by obesity, consistent with a greater contractile capacity. This was most marked when obesity occurred without GDM.Studies were conducted on two miRs, miR-145, which is associated with VSM in many vascular tissues, and the snoRNA-derived species miR-664a-3p, which microarray studies had shown to be higher in placentas from pregnancies complicated by GDM. Dicer and dyskerin, components of the snoRNA-derived miR biogenesis pathway, were increased and reduced respectively in GDM placenta. However, studies in cultured placental villous explants suggested that neither miR species was regulated by glucose, insulin or IGF-I. Placental mesenchymal cells are the developmental precursors of VSM. In primary culture, these cells expressed both miRs. To determine the function of miR-664a-3p, a nucleofection protocol was developed in a fetal mesenchymal cell line, WI38, and applied to first-trimester placental mesenchymal cells. Preliminary proteomic analysis after nucleofection-mediated knockdown of miR-664a-3p suggested a series of novel candidate target proteins for this uncharacterised miR species. Blood vessel structure and VSM phenotype are both altered in pregnancies complicated by GDM and/or obesity. The significance of apparently higher level of contractile proteins with wider vessel lumens in obesity requires further investigation. Translational regulation by miRs including miR-145 and miR-664a-3p is implicated in these alterations. In future, targeted therapies that alter miR levels in the placenta may be useful in control of fetal overgrowth such as FM.

Axons are up-to-a-meter-long cable-like cellular processes of neurons. The proper function of nervous systems requires that axons grow and wire up correctly during development or regeneration. The uniquely challenging architecture of axons has to be sustained for an organism's lifetime, and renders them key lesion sites during healthy ageing, in injury and neurodegenerative diseases. Notably, axon degeneration is considered as the cause rather than consequence for neuron decay in the context of various neurodegenerative diseases. The structural backbones of axons are formed by parallel bundles of microtubules (MTs) which also provide the highways for life-sustaining long-distance transport between cell bodies and the growth cones or synaptic endings. To better understand axon development, regeneration, maintenance and degeneration during ageing, my PhD project has focused on mechanisms underpinning the regulation of MT bundles in axons. For this, I have capitalised on fast and genetically and experimentally amenable research possible in Drosophila neurons, both in primary culture and in vivo. I have used systematic combinatorial genetics and pharmacological approaches to unravel mechanisms and roles of actin as well as the cortical collapse factor Efa6 in MT regulation during axon formation and maintenance. I was able to gain a number of novel mechanisms contributing to the de novo alignment and maintenance of ordered MT bundles. First, it has been proposed that Spectraplakins (large actin-microtubule linkers) guide the extension of polymerising MTs along cortical F-actin, thus directly laying axonal MTs out into parallel bundles. Here, I have used manipulations of actin networks as well as hybrid constructs of Shot where the actin binding domain was replaced by actin associating domains of other molecules. My data strongly suggest that Shot's ABD domain has unique properties that can sense specific properties of F-actin networks, and this is important for its ability to appropriately regulate MT behaviours. Second, using combinations of actin and Shot manipulations, I found that Shot displays not only these actin-dependent guidance functions, but it displays novel actin-independent function in MT bundle maintenance for which I present a working hypothesis. Third, I found a novel and Shot-independent role of axonal actin in maintaining MTs and promoting axon growth, and my results suggest that these functions involve promotion of MT polymerisation. MT maintenance is therefore mediated through two complementary mechanisms involving Shot on the one hand and actin on the other, and simultaneous removal of Shot and actin leads to entire loss of axons. Finally, I have unravelled novel axonal functions of the cortical collapse factor Efa6 which serves as a check point in MT bundle maintenance by eliminating "off track" MTs that have escaped the axonal bundle organisation. In the absence of this factor, a gradual increase of disorganised, criss-crossed MTs occurs as a matter of days. These new mechanisms strongly suggest that different MT-regulatory mechanisms act in parallel in axons and complement each other in one common mechanism of MT bundle formation and maintenance. I propose here a local homeostasis model of axonal MT bundle maintenance which provides new ways to think about problems of ageing as well as a range of different neurodegenerative diseases.

Meckel-Gruber syndrome (MKS) is a recessive genetic disease that is uniformly lethal in affected children due to resultant developmental defects in the kidney and brain. 13 MKS genes have been identified, and further candidate genes have been linked to this disease, all encoding unrelated proteins. Their role is believed to be in generation and compartmentalisation of the primary cilium, a microtubule-based organelle that functions in signal transduction of developmentally-crucial pathways. However, recent evidence indicates that these proteins are also likely involved in regulation of the actin cytoskeleton. Furthermore, research is beginning to uncover roles of other ciliopathy proteins in regulation of additional subcellular structures, such as the microtubule cytoskeleton, focal adhesions and the Golgi. To begin to understand the roles of the MKS proteins beyond the cilium, I examined a number of cellular features of patient fibroblasts carrying mutations in TMEM216 (MKS2) and TMEM67 (MKS3). In this thesis, I describe the temporal appearance and nature of prominent actin bundles observed in these cells, and analyse the dependency of these on the Rho/ROCK signalling pathway. Furthermore, I identify novel alterations to the microtubule cytoskeleton and organisation of the Golgi complex in MKS patient cells, and subsequently establish a temporal order of these phenotypes, demonstrating microtubule defects as the first to occur in these cells. Finally, I connect these phenotypic defects to Rho/ROCK signalling. In contrast to the prevailing view in the ciliopathy field, I believe that a diffusion barrier at the transition zone is not the primary role of MKS proteins. Instead I propose, supported by these data, that MKS protein complexes play a dual role as effectors of Rho signalling in addition to performing a structural role with particular importance in tethering the cytoskeleton to membranes. I therefore conclude that these, and other ciliopathy protein complexes, may act as important signal transduction and structural components at multiple locations throughout the cell.

My thesis, written in an alternative format, consists of three manuscripts. The first one is published in Journal of Cell Science and is entitled "GAS2-like proteins mediate communication between microtubules and actin through interaction with end-binding (EB) proteins." This article describes the mechanisms of how members of the GAS2 family of proteins mediate the crosstalk between actin and microtubules (MTs). We show that in particular GAS2-like 1 (G2L1) and GAS2-like 2 (G2L2) coordinate this cross-communication, as their exogenous expression leads to the stabilisation of MTs and guidance along actin stress fibres. We found that the association of GAS2-like members with MTs is mediated through their binding to EB proteins. The second article is a follow up story of the first article, in which we further elucidate the role of GAS2-like proteins during cell division. We show that G2L1 localises to the mitotic spindle and cleavage furrow during cell division. G2L1 knockdown leads to reduced cell division rates, multinucleation and nuclear deformation. As for MT guidance along actin filaments, we demonstrate that the binding of G2L1 to EB proteins plays an important role in cell division. Although overexpression of G2L1 had no effect, the expression of a mutant that blocks the association with EB proteins phenocopies the knockdown effect of G2L1 on cell division. Actin and MTs undergo major reorganisation during cell division. This reorganisation involves the fast remodelling of focal adhesions (FAs) but the mechanisms of this remodelling were not clear. In the third paper we demonstrate that the majority of FAs disassemble shortly before cell division and reassemble in newly formed daughter cells during cytokinesis. Interestingly, our data suggest that the regulation of FA disassembly during cell division differs from the disassembly processes during cell migration. While in migrating cells FAs can be stabilised by the expression of constitutively active vinculin (vinT12, known to circumvent the requirement forces for FA stability), this was not case for FAs during cell division. Further experiments using inhibitors suggested that calpain-driven cleavage of FA components but not endocytosis play a key role in FA disassembly during cell division. Altogether, the three manuscripts provide insight into important molecular aspects involved in the regulation of cell cytoskeletal networks and cell adhesion during cell division.

Accumulating evidence highlights the importance of studying the migration of cells within the context of their natural environment as manipulating the substrate on which a cell is migrating can have a dramatic impact on the mode/mechanisms employed by cells during migration. Central to this phenomenon is the requirement of adhesion to the ECM in order to gain traction during migration. Integrins constitute the main family of cell receptors involved in mediating cell-ECM interactions during motility. Whilst traditionally two-dimensional cell culture studies have placed emphasis on the importance of these receptors for spreading and migration, it has become evident that within more confined environments these receptors, at least for some cell types, are less crucial. In this research we utilise Drosophila embryonic hemocytes as an in vivo model for cell migration. We show that whilst hemocytes migrate within confined environments in vivo, these cells depend on integrins for powering both developmental and inflammatory migrations. Given the close association between these receptors and the actin cytoskeleton we were surprised to discover that removal of the main β integrin subunit, Myospheroid, did not affect cell spreading in vivo and had only a small impact on lamellipodial structure and dynamics. Furthermore we discovered that, in contrast to other cell types previously analysed, removal of this integrin subunit in hemocytes was not accompanied by an increase in the rate of actin retrograde flow within the protrusions, which we believe could reflect abrogation of a positive feedback between Rho, ROCK and Myosin II contraction. Instead, we discover a key role for integrins in regulating the microtubule cytoskeleton, in the maintenance of a polarised microtubule bundle, termed a ‘microtubule-arm’. Although the molecular mechanisms by which this stabilisation is coordinated have yet to be identified, this provides important insight into the co-regulation of adhesion and microtubule cytoskeleton important for the migratory behaviour of these cells. Cell migration reflects the complex and integrated regulation of the actin cytoskeleton by diverse families of actin regulatory proteins. Using hemocytes as a model system, we also explore the regulatory interactions between two main actin regulatory proteins, Diaphanous and Enabled, in vivo. Whilst the function of these proteins in the formation of filopodial protrusions is overlapping, recent research has highlighted the ability of these proteins to regulate the activity of one another. We find that co-expression of Enabled in hemocytes is able to rescue the morphological and migratory defects resulting from overexpression of active Diaphanous. Thus, data here presents Enabled as a negative regulator of Diaphanous, which may play an important role in the migration of hemocytes in vivo.

Understanding the internal organization of cells from the molecular up to organelle level is a current challenge for biology if we are to better comprehend the mechanisms by which cellular processes occur. A prevailing view of the cell interior is that biochemical reactions and molecular movements are dominated by random diffusion. However, in addition to being compartmented into organelles, cells may well be organized at a finer level. Proteins may localise to specific areas within sub-cellular compartments, by associating with each other, the cytoskeleton and organelle membranes. Thus giving rise to distinct microcompartments. Considerable in vitro evidence exists for such interactions between enzymes and larger cellular components. This strengthens the idea that cells may be microcompartmented. However, little in vivo evidence supports this hypothesis, especially in plants. As a test-case for the concept of microcompartmentation this project investigated the sub-cellular distribution of the glycolytic enzyme fructose-bisphosphate aldolase in Arabidopsis thaliana; the ultimate aim being to establish whether it is microcompartmented in vivo and, further, to test the potential function(s) of such microcompartmentation.

Enterohaemorrhagic Escherichia coli (EHEC) have recently emerged as significant zoonotic pathogens. O157:H7 is one of the most common EHEC serotypes associated with human disease, which is transmitted faeco-orally from a bovine reservoir. EHEC O157:H7 preferentially colonises the bovine terminal rectum (BTR). Injection of virulence factors by type-III secretion is necessary for colonisation of cattle and results in re-modelling of the host cytoskeleton. Flagella machinery is evolutionarily related to the Type III secretion apparatus and O157 strains lacking H7 flagella show reduced adherence to the BTR. Vaccination with FliC, the main component of H7 flagella, has the potential to protect cattle against E. coli O157:H7 infection. The focus of this work was to investigate the molecular basis for H7 flagella binding to the BTR, in order to understand the basis for FliCH7 being an immuno-protective antigen. H7 flagella were shown to adhere across the surface and penetrate into BTR epithelial cells. Both the FliC shaft and the FliD cap components of flagella filaments showed the capacity to adhere to BTR epithelial cells. Preliminary studies indicate that the current FliCH7 vaccination of cattle results in FliD-specific antibodies where oral challenge with O157:H7 does not. FliD is more conserved than FliCH7, which contains a predicted 88aa structural insertion, but variation occurs along the full length of the FliD protein. There was no evidence for post-translational modification of FliCH7. A number of actin binding proteins were identified as potential FliC and FliD binding partners from BTR epithelial cell lysates. From this, a panel of purified galectin-4, cofilin-1 and βγ-actin was used to compare binding of flagella from different pathogens. H7 flagella bound more to cofilin-1 than βγ-actin, whereas phase-1 and phase-2 flagella from Salmonella Typhimurium bound more to βγ-actin, than to cofilin-1. Size-exclusion chromatography indicated that cofilin-1 alters H7 flagella filament polymerisation dynamics. αβ-ctin polymerisation and depolymerisation experiments indicate that H7, phase-1 and phase-2 flagella interactions with actin affect actin dynamics.

This thesis deals with the organisation and function of actin and septins in the model filamentous fungus, Neurospora crassa. Firstly, study demonstrates the utility of the Lifeact peptide probe for the investigation of actin dynamics in N. crassa. Lifeact fused to fluorescent proteins allowed live-cell imaging of actin patches, cables and rings without interfering with cellular functions. Actin cables and patches localised to sites of active growth during the establishment and maintenance of cell polarity in germ tubes and conidial anastomosis tubes (CATs). Recurrent phases of formation and retrograde movement of complex arrays of actin cables were observed at growing tips of germ tubes and CATs. Two populations of actin patches exhibiting slow and fast movement were distinguished, and rapid (1.2 μm/s) saltatory transport of patches along cables was observed. Actin cables accumulated and subsequently condensed into actin rings associated with septum formation. F-actin organisation was markedly different between the tip regions of mature hyphae and germ tubes. Only mature hyphae displayed a sub-apical collar of actin patches and a concentration of F-actin within the core of the Spitzenkörper. Proper organisation of actin cables required the class-V myosin, MYO-5, and the frequency of rapid transport of actin patches was reduced in its absence, suggesting that MYO-5 participates in actin patch translocation. Deletion of myo-5 caused gross morphological and polarity defects, demonstrating the importance of this motor for normal cell function. GFP-tagged MYO-5 localised as a crescent at germ tube tips and to the core of the Spitzenkörper in mature hyphae. Secondly, analysis of septin null mutants demonstrated that septins limit the emergence of germ tubes and are important for septation and conidiation in N. crassa. Septins showed different patterns of localisation at hyphal tips, with GFP-CDC-10 and CDC- 11-GFP organised as a collar with lower signal intensity at the tip apex, CDC-3-GFP and CDC-12-GFP constituted as a cap at the tip apex and GFP-SPN-1 forming an extended collar. Septins formed a range of different higher-order structures in N. crassa – rings, loops, fibres, bar-like structures, and caps – which can co-exist within the same cell. Purification of the septin complex and mass spectrometry of isolated proteins revealed that the septin complex consists predominantly of CDC-3, CDC-10, CDC-11 and CDC-12. Immunoprecipitation of SPN-1 revealed that this septin interacts with the core septin complex.

Epithelia act as protective barriers and it is therefore essential that wounded epithelia are rapidly repaired to maintain barrier function. Cells surrounding epithelial wounds become motile following wounding, which involves generating dynamic actin structures that drive closure of the wound. These actin structures include filopodia which are important in the final stage of epithelial closure in which the opposing epithelial edges are joined together. The molecular mechanisms that trigger wound edge cells to become motile are not well understood. Using Drosophila wound healing and the morphogenetic process dorsal closure as models, we find that phosphatidylinositol 3,4,5-triphosphate (PIP3) regulates epithelial closure by promoting the formation of filopodia at epithelial edges. PIP3 accumulates at epithelial edges and genetically depleting PIP3 results in reduced filopodia and defects in epithelial closure. We demonstrate that the GTPase Rac and guanine nucleotide exchange factor Myoblast City function downstream of PIP3 to promote filopodia formation. We also demonstrated that the scaffolding protein Par3/Bazooka and the lipid phosphatase PTEN are responsible for restricting the localisation of PIP3 and consequently the downstream signals to the epithelial leading edge, so acting to determine the location of filopodia formation. This project reveals a novel mechanism by which actin protrusions, required for epithelial closure, are formed in response to epithelial damage. Additionally, we have identified an additional role for PIP3 in regulating the extrusion of cells from epithelial sheets in the Drosophila embryo. This finding implicates PIP3 in the regulation of tissue homoeostasis, and could contribute to our understanding of tumour initiation as unregulated tissue growth can result in the formation of tumours.

The I-BAR proteins are a family of actin regulators which include IRSp53 and Missing-In-Metastasis (MIM). These proteins possess an N-terminal I-BAR domain which associates with both the actin cytoskeleton and membrane phospholipids and is able to induce membrane curvature. Previous cell culture and in vitro studies have implicated I-BAR proteins in the regulation of actin protrusion formation, however their roles within the organism are poorly understood. We have used Drosophila melanogaster as a model system in which to study I-BAR protein function at the cellular and organismal level. Drosophila possess two I-BAR proteins, one homologous to IRSp53 and one homologous to MIM. Using full- length and truncated splice variants generated for both dMIM and dIRSp53, we have performed structure/function analysis to determine the role of specific domains in the localisation and actin modifying abilities of the proteins in both cell culture and in vivo. We found that dMIM overexpression typically promotes a lamellipodial morphology, with dMIM localising to the edges of extending lamellipodia. dIRSp53 expression induced a more filopodial phenotype in cell culture, which was not as notable in vivo, however expression of a dIRSp53 splice variant with a WH2 domain resulted instead in a predominantly lamellipodial morphology. Similar to dMIM, dIRSp53 localises to the tip of extending protrusions, albeit more transiently. We found that multiple domains contribute to the localisation and activity of dIRSp53 and dMIM. Following overexpression analysis, complementary loss-of-function analysis was performed in vivo using Drosophila mutants lacking dMIM, dIRSp53 or both genes together. Surprisingly these mutants were viable and morphologically normal. Absence of these genes individually or together did not greatly affect cell migration or actin dynamics in haemocytes or epithelial cells undergoing dorsal closure. However, a role for I- BAR proteins in axonal filopodia formation within primary neuronal cultures was apparent, as was a notable role in neuromuscular junction morphology. We have also identified potential redundancy between Drosophila MIM and the Drosophila F-BAR protein Cip4 in actin bundle regulation within embryonic haemocytes, with an additional novel role for Cip4 alone in haemocyte lamellipodial maintenance. Our results suggest that the Drosophila I-BAR proteins contribute to actin cytoskeleton regulation in vitro and in vivo, particularly within the CNS, and with novel shared functions with other BAR domain family proteins contributing to their regulation of actin cytoskeletal organisation and function.

Yersinia pestis is a human pathogen and the causative agent of bubonic plague, responsible for causing three massive pandemics, resulting in hundreds of millions of deaths in the 14th century alone. Yersinia’s virulence stems from its ability to overcome host immune defences by the injection of six Yersinia outer proteins (Yops) into the host cells via its Type III secretion system. One of these Yops, YopO specifically disables the actin polymerization machinery, leading to the crippling of phagocytosis. YopO consists of a GDI domain which sequesters Rac and Rho, and a kinase domain, the activity of which is dependent on host actin. Little is known about the targets of the kinase domain and the mechanism of function of YopO remains incomplete. In this work, YopO was crystallized in complex with actin, revealing that YopO binds to actin on subdomain 4, away from the 'hotspot’ between subdomains 1 and 3 which is involved in binding most actin-binding proteins. The structure reveals how recruitment of YopO-bound actin monomers stalls actin polymerization by steric hindrance. The structure also demonstrates how YopO uses actin for self-activation and suggests that actin is being used by YopO as bait for recruitment into actin machineries. Using SILAC mass spectrometry, actin cytoskeletal machineries within macrophages that recruit YopO are identified and these include, amongst others: VASP family proteins, gelsolin family proteins, formins and WASP. Of these, VASP, EVL, diaphanous1, WASP and gelsolin have been identified to be phosphorylated by YopO and were validated by in vitro phosphorylation. This work demonstrates that YopO uses actin as a scaffold for selection of kinase substrates, enabling targeted phosphorylation of the actin machinery and provides insight into the regulation of the actin cytoskeleton by phosphorylation under non-pathogenic conditions.

Cell migration is essential for embryonic development, it occurs in adult organisms during processes like wound healing and its misregulation contributes to pathological conditions such as metastasis. Despite this, most studies of cell migration have been undertaken in vitro. Ena/VASP proteins, believed to be actin anti-capping proteins, have been studied extensively in fibroblasts in vitro, and using Drosophila macrophages (hemocytes) within the developing embryo, the role of the Drosophila homologue of Mena, Ena, is investigated in vivo. Consistent with data from fibroblasts in vitro, Ena localised to regions of actin dynamics within migratory hemocytes, where this protein stimulated lamellipodial dynamics and positively regulated filopodial number and length. However, whilst overexpression of Ena/VASP proteins in fibroblasts reduced migration speeds, Ena overexpression in hemocytes dramatically increased migration speeds in three different assays. This positive regulation of migration speed closely resembled the increased motility of breast cancer cells that overexpress Mena and evidence presented here, suggests that this key difference may be explained by spatial constraints that are imposed upon cells within three dimensional environments. Indeed, such constraints prevented ruffling, a more detrimental form of retraction, in hemocytes in vivo. Furthermore, fibroblasts overexpressing Mena in vitro form membrane ruffles more frequently. Therefore Ena/VASP proteins drive migration by enhancing lamellipodial protrusion, but in certain environments these protrusions are lost as ruffles slowing migration. The method by which Ena regulates lamellipodial protrusion and migration speeds was then investigated: Ena increased Fascin-mediated actin bundling and the number of Fascin rich-actin bundles that coalesced. Analysis of individual actin bundles revealed that coalescence increased protrusion rate and that both protrusion rate and coalescence, increased cell migration speeds. This suggests that Ena facilitates an increase in cell migration by promoting the coalescence of Fascin bundles, and positions Ena as a key regulator of migration speeds in vivo.

Chondroplasia, the development of cartilage-like characteristics in tendinous tissue, is a form of degeneration found in tendons including those of the rotator cuff. The molecular mechanism of its development is currently unknown. An examination of the features of the torn rotator cuff and the cartilage literature led to the identification of several potential drivers of chondroplasia including cell shape change/actin cytoskeleton and hypoxia. Lovastatin caused actin cytoskeleton disruption and promoted cartilage matrix deposition in the ATDC5 model. It was the most effective member of a panel of cytoskeletal inhibitors, increasing expression of the chondrocytic markers Sox5 and Sox9 and decreasing expression of COL1A1 and COL3A1 in primary human tenocytes. Its effects were dose dependent, reversible by mevalonate addition and long term treatment induced de novo expression of collagen II. Short term hypoxia upregulated VEGF-A and chondrocytic marker gene DEC1 expression but not other chondrocyte markers. Combination treatment with hypoxia did not enhance the effects of lovastatin. These data suggest that modulation of pathways that regulate the actin cytoskeleton and cell shape may alter tenocyte phenotype.

Myxomatous mitral valve degeneration (MMVD) is the single most common cardiac disease of the dog, and is analogous to Mitral Valve Prolapse in humans. Very little is known about the aetiopathogenesis of this disease or the changes in valvular interstitial cell populations in diseased valves. The aim of this study was to identify morphological, cellular and molecular changes associated with MMVD. Mitral valve leaflets from both normal and varying grades (Whitney’s 1-4) of diseased dogs were subject to image analysis, immunophenotyping, proteomics and RT-PCR. Image analysis - leaflet thickening due to accumulation of glycosaminoglycan was significant in this disease. MMVD is associated with loss of connective tissue, reduction in cell numbers but no change in cell shape in the overtly myxomatous area. Near the surface, increase in valvular interstitial cells (VIC) towards the damaged endothelium in concert with destruction of collagen and building up of ground substance was manifested during the disease process. Immunophenotyping - activated myofibroblasts were increased and fibroblast-like VICs were reduced without any change in desmin and myosin expression in MMVD compared to clinical normal dogs. In addition, other cell types like macrophage, adipocyte, chondrocyte, mast cell, and stem cell were identified and their possible role in MMVD is discussed. Proteomics - a protein expression profile was established, with 64 proteins being positively identified from dog’s mitral valve using 1-D SDS PAGE LC/MS. Amongst them 44 proteins were differentially expressed comparing normal and severely diseased. Two actin binding proteins, tropomyosin alpha and myosin light chain-2 were found to be differentially expressed in the normal but down regulated in the diseased. RT-PCR was used to assess the expression of 8 genes of interest. Their expression was compared with 3 different housekeeping genes.

Endocytosis and cell migration are cellular processes requiring transient localised remodelling of the cell cortex. Several lines of evidence suggest a key regulatory role in these activities for members of the Ras family of small GTPases. I have generated Dictyostelium cells lacking one member of this family, RasS, and the mutant cells are perturbed in both endocytosis and cell migration. Mutant amoebae are impaired in phagocytosis and strongly impaired in fluid-phase endocytosis. Conversely, the rasS- cells show an enhanced rate of migration, moving 3 times faster than wild-type controls. The mutant cells have abnormal morphology; they are highly polarised, carry many elongated microspikes and show a concomitant decrease in formation of pinocytotic crowns on the cell surface. 3D analysis of the movement of the mutant cells indicates their rapid movement is due to differences in pseudopodium behaviour. There are fewer pseudopodia per cell, but they have an increased volume and turn over more rapidly. The rasS- phenotype correlates with significant changes in the expression and organisation of components of the actin cytoskeleton. Under specific conditions, the mutant cells show a significant elevation in the expression levels of the actin-binding proteins, talin and coronin. The mutant cells also have abnormal levels of the polymerised form of actin. The polymerisation of actin which occurs in response to chemoattractant stimulation is also distinct in the mutant cells, which show a stronger response than parental cells. The level of the PI 3-kinase product, PI(3,4)P2, is normal in the mutants. The rasS- cells have a lowered cellular level of an unidentified lipid. The axenic mutations, which are known to impinge upon cell motility and endocytosis, can act as genetic modifiers of the rasS- phenotype.