Production of infectious retrovirus particles is a complex and poorly-understood process with multiple steps that are often linked to one another. Our aim in this study was to gain better understanding of the path the murine leukemia virus (MLV) structural protein Gag follows to assemble into immature capsid structures, the process of which is central to retroviral assembly and release. Extensive studies of human immunodeficiency virus type 1 (HIV-1) assembly have led to the development of a model proposing that the assembly of immature HIV-1 capsids proceeds sequentially through multiple intermediates, in association with an RNA granule containing some well-conserved cellular factors, such as ATP-binding cassette subfamily E member 1 (ABCE1) and DEAD-box helicase 6 (DDX6). In this work, we provided evidence suggesting that MLV Gag associates with endogenous ABCE1 in human cells expressing assembly-competent MLV, and can be found in at least three high-molecular weight complexes with sedimentation properties highly resembling the HIV-1 assembly intermediates. Furthermore, we assessed the Gag proteins of select assembly-defective MLV mutants in terms of their expression levels, ability to form viral particles, involvement in intracellular complexes, membrane association, and ABCE1 interaction. Our findings were not only consistent with a model of MLV assembly through host-mediated intermediates, but also provided novel information about the effects of various MLV Gag mutations that are associated with defects in particle production.

Cells contain an elaborate cytoskeleton which plays a major role in a variety of cellular functions including: maintenance of cell shape and dimension, providing mechanical strength, cell motility, cytokinesis during mitosis and meiosis and intracellular transport. The cell cytoskeleton is made up of three types of protein filaments: the microtubules, the intermediate filaments and the actin cytoskeleton. These components interact with each other to allow the cell to function correctly. When functioning incorrectly, disruptions to many cellular pathway have been observed with mutations in various cytoskeletal proteins causing an assortment of human disease phenotypes. Characterization of these filament systems in different cell types is essential to the understanding of basic cellular processes and disease causation. The studies in this thesis are concerned with examining specific cytoskeletal tropomyosin-defined actin filament systems in skeletal muscle. The diversity of the actin filament system relies, in part, on the family of actin binding proteins, the tropomyosins (Tms). There are in excess of forty Tm isoforms found in mammals which are derived from four genes: α, β, γ and δTm. The role of the musclespecific Tms in striated muscle is well understood, with sarcomeric Tm isoforms functioning as part of the thin filament where it regulates actin-myosin interactions and hence muscle contraction. However, relatively little known about the roles of the many cytoskeletal Tm isoforms. Cytoskeletal Tms have been shown to compartmentalise to form functionally distinct filaments in a range of cell types including neurons (Bryce et al., 2003), fibroblasts (Percival et al., 2000) and epithelial cells (Dalby-Payne et al., 2003). Recently it has been shown that cytoskeletal Tm, Tm5NM1 defines a cytoskeletal structure in skeletal muscle called the Z-line associated cytoskeleton (Z-LAC) (Kee et al., 2004).The disruption of this structure by over-expression of an exogenous Tm in transgenic mice results in a muscular dystrophy phenotype, indicating that the Z-LAC plays an important role in maintenance of muscle structure (Kee et al., 2004). In this study, specific cytoskeletal Tms are further investigated in the context of skeletal muscle. Here, we examine the expression, localisation and potential function of cytoskeletal Tm isoforms, focussing on Tm4 (derived from the δ- gene) and Tm5NM1 (derived from the γ-gene). By western blotting and immuno-staining mouse skeletal muscle, we show that cytoskeletal Tms are expressed in a range of muscles and define separate populations of filaments. These filaments are found in association with a number of muscle structures including the myotendinous junction, neuromuscular junction, the sarcolemma, the t-tubules and the sarcoplasmic reticulum. Of particular interest, Tm4 and Tm5NM1 define cytoskeletal elements in association with the saroplasmic reticulum and T-tubules, respectively, with a separation of less than 90 nm between distinct filamentous populations. The segregation of Tm isoforms indicates a role for Tms in the specification of actin filament function at these cellular regions. Examination of muscle during development, regeneration and disease revealed that Tm4 defines a novel cytoskeletal filament system that is orientated perpendicular to the sarcomeric apparatus. Tm4 is up-regulated in both muscular dystrophy and nemaline myopathy and also during induced regeneration and focal repair in mouse muscle. Transition of the Tm4-defined filaments from a predominsnatly longitudinal to a predominantly Z-LAC orientation is observed during the course of muscle regeneration. This study shows that Tm4 is a marker of regeneration and repair, in response to disease, injury and stress in skeletal muscle. Analysis of Tm5NM1 over-expressing (Tm5/52) and null (9d89) mice revealed that compensation between Tm genes does not occur in skeletal muscle. We found that the levels of cytoskeletal Tms derived from the δ-gene are not altered to compensate for the loss or gain of Tm5NM1 and that the localisation of Tm4 is unchanged in skeletal muscle of these mice. Also, excess Tm5NM1 is sorted correctly, localising to the ZLAC. This data correlates with evidence from previous investigations which indicates that Tm isoforms are not redundant and are functionally distinct (Gunning et al., 2005). Transgenic and null mice have also allowed the further elucidation of cytoskeletal Tm function in skeletal muscle. Analyses of these mice suggest a role for Tm5NM1 in glucose regulation in both skeletal muscle and adipose tissue. Tm5NM1 is found to colocalise with members of the glucose transport p fibres and analysis of both transgenic and null mice has shown an alteration to glucose uptake in adipose tissue. Taken together these data indicate that Tm5NM1 may play a role in the translocation of the glucose transport molecule GLUT4. In addition to this Tm5NM1 may play a role in adipose tissue regulation, since over-expressing mice found to have increased white adipose tissue and an up-regulation of a transcriptional regulator of fat-cell formation, PPAR-γ. / Doctor of Philosophy (PhD)

Thesis (M.S.)--The University of North Carolina at Greensboro, 2008. / Directed by Paul Steimle; submitted to the Dept. of Biology. Title from PDF t.p. (viewed Mar. 19, 2010). Includes bibliographical references (p. 30-31).

Thesis (Ph. D.)--University of Chicago, Dept. of Neurobiology, Pharmacology and Physiology, August 2001. / Includes bibliographical references. Also available on the Internet.

Glioblastoma multiforme (GBM), a grade IV malignant astrocytic tumor according to WHO classification, is one of the most common and malignant brain tumor. Temozolomide (TMZ) is the current standard treatment for GBM. Nevertheless, resistance to chemotherapy in GBM is common and therefore a major obstacle to successful treatment. Adducin 3 (ADD3), a cytoskeletal protein, has been found to be associated with chemoresistance in osteosarcoma, but its potential role in glioblastoma is unclear. A TMZ-resistant model was established by chronically exposing the glioma cells (D54 cell line) to an increasing dose of TMZ. A resistant subclone (D54-R) was successfully generated. ADD3 expression level was found to be upregulated in the D54-R when compared to the parental D54 cells (D54-C). CD133 is a putative cancer stem cell marker. Its expression level was found also to be higher in D54-R when compared to D54-C cells. Among the D54-R cells, a subgroup of cells was found to express ADD3 intensely. The proportion of these spherical cells was higher in D54-R than D54-C. Moreover, these cells were spherical in morphology and expressed putative cancer stem cell markers: CD133, NANOG and OCT-3/-4. Therefore, ADD3 is associated with cancer stem cells in human glioma. The upregulation of ADD3 expression is associated with TMZ-resistance in GBM. / published_or_final_version / Surgery / Master / Master of Research in Medicine

Thesis (Ph. D.)--Wake Forest University. Dept. of Physics, 2003. / Vita. Includes bibliographical references (leaves 108-114).

Thesis (M.S.)--The University of North Carolina at Greensboro, 2009. / Directed by Paul Steimle; submitted to the Dept. of Biology. Title from PDF t.p. (viewed May 11, 2010). Includes bibliographical references (p. 37-39).

Thesis (Ph. D.)--Ohio State University, 2005. / Title from first page of PDF file. Includes bibliographical references (p. 196-235).

FtsZ, the primary bacterial cytoskeleton protein, drives cytokinesis in the vast majority of archae and eubacteria by forming single-stranded filaments that coat the cell membrane and scaffold the peptidoglycan synthesis machinery. While the biochemistry and kinetics of FtsZ filaments are well studied, the structure details of the filaments remain elusive. Across species, FtsZ monomers are highly homologous, with all but two published monomer crystal structures assuming the same "Relaxed" conformation. However, a second "Tense" conformation has been identified in Staphylococcus aureus FtsZ monomers that is presumed to correspond to the monomer form present in active filaments. As of this writing, Tense monomers have only been directly observed in S. aureus, and while it is widely thought that they correspond to the active monomer form, this has not been confirmed. This dissertation presents a series of structural studies of Escherichia coli FtsZ filaments, primarily using the magic angle spinning solid-state NMR (MAS NMR) technique dynamic nuclear polarization (DNP). DNP uses cryogenic temperatures and high-powered microwave radiation to dramatically increase the NMR signal, making signal-to-noise limited systems, such as the ones presented here, much more efficient. I employ a differential isotopic labelling scheme to selective observe nuclei present at the inter-monomer interface using ZF-TEDOR to recouple the heteronuclear dipolar coupling. When combined with homology modelling and chemical shift prediction, this strategy allows for the generation of distance restraints across the interface and direct model comparison in the absence of full resonance assignments. The size of the EcFtsZ monomer (~300 structured residues), in comparison to the size of the intermonomer interface (30--80 residues depending on the model) makes it very difficult to generate enough signal-to-noise to do multi-dimensional NMR studies and obtain unambiguously assigned spectra. However, by combining 1D NMR with various sparse 13C labelling schemes, I was able to observe inter-monomer 13C--15N contacts and measure a set of 12 distances between 3.0--6.0 Å. Using this set of restraints, along with chemical shift predictions for three potential interface models---one corresponding to the Tense monomer state and two corresponding to different Relaxed states---I performed several different structural analyses on the ZF-TEDOR data, including residue counting, identifying peaks best described by a single model, and chemical shift difference analysis. This study provides multiple sets of evidence that active EcFtsZ filaments are primarily composed of Tense monomers. This is the first such direct structural evidence of the presence of Tense monomers in FtsZ filaments, and the first direct observation of Tense monomers in EcFtsZ. Additionally, I present a previously published study where we characterize chemical reduction of a nitroxide biradical, TOTAPOL, used in DNP experiments, specifically probing the stability in whole-cell pellets and lysates. DNP experiments use paramagnetic species to dramatically increase NMR signals. Although there is considerable excitement about using nitroxide-based DNP for detecting the NMR spectra of proteins in whole cells, nitroxide radicals are reduced in minutes in bacterial cell pellets. We show that addition of the covalent cysteine blocker N-ethylmaleimide to whole cells significantly slows the rate of reduction, suggesting that cysteine thiol radicals are important to in vivo radical reduction. The use of cell lysates rather than whole cells also slows TOTAPOL reduction, which suggests a possible role for the periplasm and oxidative phosphorylation metabolites in radical degradation. Reduced TOTAPOL in lysates can also be efficiently reoxidized with potassium ferricyanide. These results point to a practical and robust set of strategies for DNP of cellular preparations.

Willin is a novel member of the Four-point-one Ezrin Radixin Moesin (FERM) protein superfamily, containing an N-terminal FERM domain most like the Ezrin-Radixin-Moesin (ERM) family but also the closely related protein Merlin. Willin was initially discovered as a yeast two-hybrid binding partner of neurofascin155, and this interaction has now been confirmed by both co-localisation studies and the use of two different biochemical methods. Like neurofascin155, Willin also localises to detergent resistant membranes, and like the ERM family, it is able to bind to phospholipids. The expression of Willin appears to be toxic as the production of cell-lines stably expressing Willin proved to be not possible and this appears to be because it induces apoptosis in cultured cells. This is a proliferation control function consistent with the suggestion that Willin is the human homologue of the Drosophila tumour suppressor ‘Expanded’. Three antibodies to Willin were also characterised and a novel splice variant, Willin2, subcloned into a GFP-tagged plasmid for comparison with the original form.