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.

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.

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.

Hyphal organisms are a commonly used model system for studies of polarised growth. While growing hyphal tips offer a good example of polarised growth, little detail of the process of polarisation can be determined from them. Hyphal branching offers a good example of the development of polarity, however to date it has been largely impractical to study hyphal branching, due to the irregular timing and location along the hypha of natural branch formation. Chemical induction of branches circumnavigates this problem, using a localised concentration of nutrients adjacent to the growing hypha to stimulate controlled branching. Using previous studies of hyphal branching combined with the current understanding of hyphal tip growth, a model of the branching process was established (Jackson et al. 2001). Reception of a branching cue leads to the formation of a radial F-actin array at the new branch site. This, by means of either delivery of cell wall softening enzymes or direct mechanical pressure, leads in turn to the emergence of a visible bump in the hyphal wall. This bump enlarges and then progresses into the branch proper. The bump stage of the branching process is perhaps the least understood, with existing studies giving detail of pre- and post-bump events. The research described in this thesis suggests that bump emergence is a two stage process; an early bump stage, where localised cell wall softening leads to turgor pressure in the cell pushing out the bump, and a late bump, where F-actin is arranged into the developing branch. The addition of an F-actin inhibitor to the induction solution confirmed that the early bump stage is relatively independent of the F-actin cytoskeleton, however this experiment was unable to test F-actin's role in full branch development.

Tip growth is a form of cellular expansion characteristic of fungal hyphae and some types of plant cells. Currently there is no unified model that satisfactorily describes this in hyphal species. Traditionally turgor has been considered an essential driving force behind cell expansion. In recent years this hypothesis has been challenged by evidence that in some species tip growth can occur despite the absence of measurable hydrostatic pressure. There are currently two contentious theories of hyphal extension. These are the turgor-driven model and the amoeboid-movement theory. Though the essential mechanism underlying cell growth differs between these theories, the actin cytoskeleton is considered important in both. It has been suggested that both the turgor-driven and amoeboid-like modes of growth could occur depending on the whether the hyphae are growing invasively or non-invasively respectively (Money, 1990). It has also been proposed that both modes may occur within the same mycelium (Garrill, 2000). Two distinct patterns of actin have been identified in the hyphal tips of oomycetes. This has lead to the hypothesis that two different mechanisms of apical extension may be employed by some hyphal organisms. During the course of this thesis, actin deplete zones have been observed in a significantly higher number of invasive compared to non-invasive hyphae of the oomycete Achlya bisexualis. Furthermore the difference between burst pressures was found to be lower in invasive hyphae compared to non-invasive hyphae suggestive of a weaker cell wall. A lack of significant difference in turgor pressures between the invasive and non-invasive hyphae of this organism suggests that the deplete zone and weaker wall plays a functional role in enabling hyphae to penetrate substrate. Fractal analysis of mycelial colonies shows that the variation in agar concentration and therefore substrate solidity has a significant effect on mycelial morphology. This is most likely due to an effect at the cellular level. The results of the experiments carried out during the course of this thesis provide the basis for future work towards elucidating the mechanisms of hyphal extension.

Regensburg, Univ., Diss., 2007

Stuttgart, Univ., Diss., 2006.

Thesis (Ph. D.)--Ohio State University, 2008.

Universiẗat, Diss., 2001--München.

Thesis (M.Phil.)--Hong Kong University of Science and Technology, 2006. / Includes bibliographical references (leaves 107-121). Also available in electronic version.