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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.

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.

More than 30 missense mutations in the ACTA2 gene, which encodes α–smooth muscle (α–SM) actin, cause thoracic aortic aneurysms and dissections (TAAD). Aortic cell samples obtained from patients harboring the R256H mutation, the third most common of this group, reveal a lack of α–SM organization. However, the biochemical mechanisms contributing to this disorganization have yet to be elucidated. Biochemical analysis of these mutations is critical to understanding the mechanisms underlying this disease; however, this goal proves difficult due to the inability to obtain diseased aortic tissue, the difficulty in purifying pure mutant actin from smooth muscle tissue, and the absence of a sufficient animal model. The yeast Saccharomyces cerevisiae has a single actin-encoding gene, ACT1, that shares 86% homology with human α–SM actin, making it a viable model system for analysis of TAAD mutations. Regulation of actin function by actin binding proteins is conserved between yeast and humans and plays a key role in cytoskeletal assembly and disassembly events. Two such actin binding proteins are cofilin and Aip1p, which work together to facilitate filament disassembly. Cofilin severs actin filaments, an action enhanced by actin interacting protein 1 (Aip1p) through mechanisms that are not well understood. Normal regulation of filament disassembly is important for cell proliferation and migration. In vivo data obtained by lab colleagues reveal that, in the absence of Aip1p, cells have abnormal cytoskeleton and organelle morphologies and poor growth. This thesis summarizes the investigation into the regulation of R256H mutant actin by Aip1p. S. cerevisiae was used to express R256H actin as the sole actin in the cell, and the effect of the mutation was assessed in vitro. The mutant actin exhibited decreased thermal stability indicative of an effect on monomer integrity. Filament stability was also affected as evidenced by aberrant polymerization kinetics, and high critical concentration and phosphate release rates. While the mutant actin was more sensitive to cofilin-mediated severing, it was less sensitive to disassembly in the presence of Aip1p with incomplete oligomer breakdown at varying Aip1p concentrations. Collectively, these data suggest an alteration in the filament in the presence of the R256H mutation that interferes with proper actin–Aip1p interaction. The biochemical effects observed with this mutant suggest how its presence leads to α–SM cytoskeletal disorganization and TAAD.

Antimicrotubule drugs are used in the treatment of childhood neuroblastoma and acute lymphoblastic leukaemia (ALL). Resistance to these agents can be a major clinical problem and mechanisms mediating resistance are not fully understood. Previous studies have reported an association between the actin cytoskeleton and resistance to antimicrotubule drugs. Thus, the aim of this study was to investigate the role of the actin regulating proteins, LIM kinases (LIMK1 and LIMK2) in drug resistance. In addition, the role of ?? actin, a major actin isoform, in drug resistance was also examined. Chapter 1 reviewed the known mechanisms of antimicrotubule drug resistance and the interaction between the microtubules and actin cytoskeleton. The methodologies used in this study are described in chapter 2. LIMKs are known to regulate the actin cytoskeleton via phosphorylation of cofilin. Real Time RT PCR and western blotting was used in chapter 3 and showed that expression of LIMKs and their downstream target cofilin was altered in antimicrotubule resistant neuroblastoma and leukaemia cells. Moreover, altered LIMK expression was detected in in vivo derived vincristine resistant ALL xenografts and ALL clinical samples, further demonstrating that alterations in LIMKs and cofilin are associated with antimicrotubule drug resistance. Importantly, in chapter 4, gene silencing and drug treated clonogenic assays were performed to elucidate the functional role of LIMK1 and LIMK2 in drug response. Silencing of LIMK1 and/or LIMK2 increased sensitivity of neuroblastoma cells to microtubule targeting drugs and DNA damaging agents, suggesting that LIMKs may be useful targets to improve the efficacy of anticancer drugs. ??-Actin has been associated with drug resistance and chapter 5 used gene silencing and drug treated clonogenic assays to show that decreased ?? actin expression conferred resistance to anitmicrotubule drugs but not to DNA damaging agents. Microscopy and tubulin polymerisation assays showed that reduced ??-actin protects microtubules from paclitaxel induced polymerisation. This data supports a functional role for ?? actin in antimicrotubule drug action. In conclusion, this study showed that LIMKs and ?? actin mediate the action of antimicrotubule drugs and other anticancer agents, demonstrating that the actin cytoskeleton may serve as a useful drug target to improve the efficacy of anticancer drugs.

The major outer sheath protein (Msp) is the most prominent surface antigen of the periodontal pathogen Treponema denticola. It mediates adhesion to extracellular matrix and dysregulation of cytoskeletal homeostasis of host cells. Disassembly of actin filaments and the coincident subcortical de novo synthesis of actin filaments in fibroblasts upon exposure to Msp were investigated with a barbed-end fluorescent labeling method. The functional impact of actin cytoskeleton disorganization was determined with a scratch wound migration assay in fibroblast monolayers and a videomicroscopy migration assay in neutrophils. Msp pretreatment had a significant inhibitory effect on the migration of the fibroblasts across a collagen substratum and inhibited the neutrophil chemotactic migration towards a chemoattractant. In a study originally aimed to find the biologically active domains of Msp that may perturb actin, short peptides were selected from the deduced and predicted surface exposed regions of Msp and investigated for their role in actin dynamics and cell motility. A novel BSA-conjugated peptide (P34BSA) was found serendipitously to induce stress fiber formation and stability in fibroblasts. This activity was found to be mediated by Rho activation and cofilin phosphorylation, which are important tandem signaling pathways in the regulation of a variety of actin-dependent cellular functions. P34BSA was internalized by the cells. Yet, a mechanistic study using low-temperature treatments and depletion of cholesterol with methyl-β-cyclodextrin (MβCD) revealed that P34BSA most likely induces actin stress fiber formation extracellularly through a Rho-dependent signaling pathway. P34BSA induced Rho activation via binding of guanosine nucleotide exchange factor p114RhoGEF to RhoA, one of many exchange factors that have been shown to play a role in activating Rho signaling. Pretreatment with P34BSA partially protected the fibroblasts against the actin-disrupting effects of cytochalasin D and latrunculin B, and the cells maintained most of their actin filaments. P34BSA treatment caused retardation of fibroblast migration on a collagen substratum. It also inhibited the chemotactic movement of neutrophils towards the chemoattractant fMLP. P34 may represent a novel amino acid sequence of a bacterial virulence protein that, when conjugated to BSA, can be used as a chemical reagent to investigate RhoA signaling pathways in host cells.

The major outer sheath protein (Msp) is the most prominent surface antigen of the periodontal pathogen Treponema denticola. It mediates adhesion to extracellular matrix and dysregulation of cytoskeletal homeostasis of host cells. Disassembly of actin filaments and the coincident subcortical de novo synthesis of actin filaments in fibroblasts upon exposure to Msp were investigated with a barbed-end fluorescent labeling method. The functional impact of actin cytoskeleton disorganization was determined with a scratch wound migration assay in fibroblast monolayers and a videomicroscopy migration assay in neutrophils. Msp pretreatment had a significant inhibitory effect on the migration of the fibroblasts across a collagen substratum and inhibited the neutrophil chemotactic migration towards a chemoattractant. In a study originally aimed to find the biologically active domains of Msp that may perturb actin, short peptides were selected from the deduced and predicted surface exposed regions of Msp and investigated for their role in actin dynamics and cell motility. A novel BSA-conjugated peptide (P34BSA) was found serendipitously to induce stress fiber formation and stability in fibroblasts. This activity was found to be mediated by Rho activation and cofilin phosphorylation, which are important tandem signaling pathways in the regulation of a variety of actin-dependent cellular functions. P34BSA was internalized by the cells. Yet, a mechanistic study using low-temperature treatments and depletion of cholesterol with methyl-β-cyclodextrin (MβCD) revealed that P34BSA most likely induces actin stress fiber formation extracellularly through a Rho-dependent signaling pathway. P34BSA induced Rho activation via binding of guanosine nucleotide exchange factor p114RhoGEF to RhoA, one of many exchange factors that have been shown to play a role in activating Rho signaling. Pretreatment with P34BSA partially protected the fibroblasts against the actin-disrupting effects of cytochalasin D and latrunculin B, and the cells maintained most of their actin filaments. P34BSA treatment caused retardation of fibroblast migration on a collagen substratum. It also inhibited the chemotactic movement of neutrophils towards the chemoattractant fMLP. P34 may represent a novel amino acid sequence of a bacterial virulence protein that, when conjugated to BSA, can be used as a chemical reagent to investigate RhoA signaling pathways in host cells.

Cancer metastasis is a multi–step process that initiates with a tumor cell obtaining the ability to migrate. A multitude of changes occur in such a cell including changes to cell adhesion molecules such as integrins. In cancer cells, integrins are known to be involved in migration, invasion and metastasis. Investigation by our group of the α6 integrin led to the discovery of a cleaved form of the integrin lacking the ligand binding domain, called α6p. While it is known that the integrin is cleaved by urokinase plasminogen activator (uPA) little is known about how this process is regulated. There is a need to better understand the players involved in regulation of α6 cleavage as inhibiting this event from occurring may contribute to prolonged or increased patient survival or ultimately a cure.The existence of the integrin–actin complex has been known for many years. In this study actin was identified as a potential regulator of α6 cleavage. Using a diverse set of tumor cell lines (DU145, PC3 and MDA–MB–231) and a number of actin modifing compounds (latrunculin A, jasplakinolide and siRNA) it is reported here that disassembling actin filaments leads to an increase in α6p production. Although the increase in cleavage product did not always correlate with an increase in uPA receptor, an increase in uPAR was observed when actin was complexed by small molecule inhibitors. Taken together the results demonstrate a potential role for actin filaments to protect α6 integrin from uPA–uPAR induced cleavage via a multi–protein complex.

The development of fibrosis involves disruption of connective tissue homeostasis that may include inhibition of collagen remodeling pathways such as phagocytosis, as well as the differentiation of myofibroblasts, pro-fibrotic cells. Myofibroblast differentiation is dependent on actin assembly, which can alter cell shape and is required for collagen phagocytosis and remodeling. Cyclosporin A (CsA) is a commonly used drug for prevention of organ transplant rejection that causes marked fibrosis in periodontal tissues by inhibiting collagen phagocytosis. As gelsolin is a Ca2+-dependent actin severing protein that mediates collagen phagocytosis, I determined whether gelsolin is a CsA target. Compared to vehicle-treated controls, CsA-treatment of wild-type mice increased collagen accumulation by 60% in periodontal tissues; equivalent increases were seen in vehicle-treated gelsolin-null mice. From a series of in vitro experiments, I conclude that CsA-induced accumulation of collagen in the periodontal ECM involves disruption of the actin severing properties of gelsolin. This disruption inhibits the binding step of collagen phagocytosis and promotes fibrosis. During the development of pressure-induced cardiac hypertrophy, collagen accumulates in the interstitium, due to myofibroblasts which express alpha-smooth muscle actin (SMA). As focal adhesion complexes are putative mechanosensing organelles, I examined the role of focal adhesion kinase (FAK) and its interaction with gelsolin, in the regulation of SMA expression. After application of mechanical force to cultured fibroblasts through collagen-coated magnetite beads attached to beta1 integrins, FAK and gelsolin were recruited to beads and there was increased nuclear translocation of MRTF-A, a transcriptional co-activator of SMA. These data suggested a novel pathway in which mechanosensing by FAK regulates actin assembly through gelsolin; actin assembly in turn controls SMA expression through MRTF-A. I also examined a potential role for the actin nucleators, mammalian Diaphanous-related formins (mDia), in the mechanosensing pathway that leads to force-induced expression of SMA. siRNA knockdown of mDia inhibited actin assembly at force-induced focal adhesions. In anchored collagen gels to model myofibroblast-mediated contraction of the matrix, mDia knockdown reduced contraction by 50%. Collectively, these experiments indicate that the regulation of actin assembly plays an important role in the development of force-induced transcriptional activation of SMA, myofibroblast differentiation and collagen phagocytosis.