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.

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.

Thesis (Ph. D.)--University of Alabama at Birmingham, 2007. / Title from first page of PDF file (viewed Feb. 6, 2008). Includes bibliographical references (p. 81-92).

Thesis (Ph. D.)--Cornell University, 1989. / Vita. Includes bibliographical references.

Thesis (doctoral)--Umeå Universitet, 1985. / Added t.p. with thesis statement inserted. Includes bibliographical references (p. 35-41).

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

No description available.

No description available.

No description available.

Microtubules (MTs) are important components of the eukaryotic cytoskeleton, with critical functions in intracellular trafficking, establishing and maintaining cell morphology, and segregating chromosomes during mitosis. MTs are hollow, cylindrical polymers composed of αβ-tubulin heterodimers. The longitudinal assembly of αβ-tubulin subunits generates protofilaments, and multiple protofilaments (typically 13 in vivo) interact laterally to form the wall of the MT. In vitro, the polymerization of MTs proceeds in two steps: nucleation and elongation. During the nucleation phase, several αβ-tubulin subunits associate to form a seed, from which further MT elongation then occurs. However, at the relatively low αβ-tubulin concentrations found in vivo, the spontaneous assembly of MTs is not favoured, due largely to the slow kinetics of MT nucleation. The nucleation of MTs in vivo requires the γ-tubulin complex (γ-TuC), a ring-like complex composed of γ-tubulin and γ-tubulin complex proteins (GCPs). Two copies of γ- tubulin associate with one copy each of GCP2 and GCP3 to produce the γ-tubulin small complex (γ-TuSC). Multiple γ-TuSCs, along with the additional GCPs 4,5, and 6, assemble to form the larger γ-tubulin ring complex (γ-TuRC). The γ-TuRC contains a ring of 13 γ-tubulins, which acts as a template for the nucleation of MTs. Typically, the γ-TuC nucleates MTs only when localized to specific subcellular sites, referred to as microtubule organizing centres (MTOCs). However, the precise mechanism by which the γ-TuC is activated at MTOCs remains unknown. In fission yeast, the proteins Mto1 and Mto2 form a complex (Mto1/2) required for the nucleation and organization of cytoplasmic MTs. Mto1/2 determines sites of MT nucleation by recruiting the γ-TuC to several different MTOCs. Different sequences in the Mto1 C-terminus independently confer γ-TuC localization to spindle pole bodies, MTs, and the cell equator. Here, I show that the Mto1 N-terminus is necessary for localization to the nuclear envelope (NE). By simultaneously removing the N- and C-terminal localization domains, I generated the "Mto1-bonsai" mutant, which fails to localize to any conventional MTOCs. In mto1-bonsai cells, MTs are still nucleated in the cytoplasm in an Mto1- dependent manner, but nucleation is spatially random. This reveals that targeting of the γ- TuC to conventional MTOCs is not necessary for MT nucleation, and suggests that Mto1/2 has a direct role in activating MT nucleation by the γ-TuC. Live-cell confocal microscopy allows us to detect individual MT nucleation events, in which newly nucleated MTs are associated with single γ-TuCs as well as Mto1/2-bonsai complexes. Fluorescence quantification reveals that these nucleating complexes contain approximately 13 molecules of both Mto1-bonsai and Mto2, matching the 13 copies of γ-tubulin anticipated for a single γ-TuC. We propose that Mto1/2 may contribute to γ-TuC activation by promoting γ-TuSC assembly and/or inducing conformational changes in the γ-TuC upon binding. I also expressed and purified recombinant Mto1/2-bonsai complex, using a baculovirus/insect cell system. This recombinant Mto1/2-bonsai self-assembles into higher-order complexes, comparable in size to the complexes analyzed in vivo by fluorescence microscopy.