Master of Science / Department of Biochemistry / Bruce D. Schultz / John M. Tomich / Peptides based on the second transmembrane segment of the glycine receptor (M2GlyR) were made to provide a potential therapeutic treatment for cystic fibrosis (CF) and a latent absorption enhancer for drug delivery. For similarity of presentation, unique synthetic peptide sequences have been given alpha-numeric designations. Results are presented from studies focusing on four peptides. In the first study, the contributions of synthetic peptides p1171, p1172 and p1173 to net transepithelial ion transport were measured as a first step toward the goal of testing whether pore length or electrostatics of pore lining residues will affect anion transport. Peptide p1130 exhibits many attributes that make it an ideal synthetic peptide for CF treatment, but has low permselectivity for anions. Therefore, it is used as a platform for modification. Peptide p1171 is doubly substituted with diaminopropionic acid at positions T13 and T17. Peptide p1172 and p1173 are separately one and two helical turn(s) inserted into the p1130 backbone. Apical exposure of MDCK monolayers to these peptides caused a rapid increase in short circuit current (Isc), an indicator of net ion transport. The increase in Isc caused by p1172 or p1173 was accompanied by increase in transepithelial electrical conductance (gte). The electrophysiological results suggested that these modified peptides can assemble in the apical membrane of epithelial cells to form functional ion-conducting pores. Peptide NC-1059, which provides for ion transport across epithelial cells derived from many sources, was studied further to assess cellular changes that account for increased gte. NC-1059 increased Isc, gte and enhanced permeation of dextrans in a concentration dependent manner. Results from previous and current studies show that NC-1059 modulated the epithelial paracellular pathway by altering the distribution and abundance of junctional proteins. Immunoblotting and immunolabeling with confocal microscopy showed that NC-1059 induces reorganization of actin and causes a reduction in F-actin abundance in epithelial cells. The distributions were changed and cellular abundances were reduced of tight junction proteins occludin and ZO-1 and adherens junction proteins E-cadherin and β-catenin by NC-1059. These effects were largely reversed in 24 hr and fully recovered in 48 hr. Therefore, NC-1059 has the therapeutic potential to increase the efficiency of drug delivery across barrier membranes.

Endocytosis is essential for the pathogenic development of Ustilago maydis. It has been shown that the initiation of pathogenicity relies upon the ability of the cell to recognize pheromone (a1 or a2) released from its mating partner and subsequently to form conjugated hyphae. The actin and microtubule cytoskeleton plays an essential role in all aspects of cell growth. A component of the actin cytoskeleton, the filamentous actin is required for cell-cell fusion, whereas the molecular motors, kinesin and dynein, move along microtubules and provide the long distance transport of many proteins and they are important in cell growth and pathogenicity. In this thesis, we investigated the role of the cytoskeleton in endocytosis and a1 pheromone transport, using a fluorescently labelled derivative of the a1 pheromone. We confirmed that uptake of the a1 pheromone is also receptormediated. In addition, we have shown that pheromone transport towards the cellular vacuole requires the actin and microtubule cytoskeletons. Furthermore, we revealed that the microtubule-dependent motors kinesin-1 and kinesin-3 and dynein were shown to be essential in the delivery of the pheromone to vacuoles. Moreover, a mutation in the early endosomal protein Yup1 gene causes a stop in delivery of the synthetic pheromone to the vacuole. This suggests that it travels with early endosomes. Within the actin cytoskeleton, we analysed the dynamics of actin patches in the presence of the synthetic pheromone and found that the dynamics of the patches increased significantly. Additionally, in the presence of an over-expression of the tail domain of the molecular motor myosin-5, the dynamics of the patches were significantly reduced and their intensity diminished.

<p>Primary cilia are evolutionarily conserved, acetylated microtubule-based organelles that transduce mechanical and chemical signals. Primary cilium assembly is tightly controlled and its deregulation causes a spectrum of human diseases. Formation of primary cilium is a collaborative effort of multiple cellular machineries, including microtubule, actin network and membrane trafficking. How cells coordinate these components to construct the primary cilia remains unclear. In this dissertation research, we utilized a combination of cell biology, biochemistry and light microscopy technologies to tackle the enigma of primary cilia formation, with particular focus on isoform-specific roles of non-muscle myosin II family members. We found that myosin IIB (Myh10) is required for cilium formation. In contrast, myosin IIA (Myh9) suppresses cilium formation. In Myh10 deficient cells, Myh9 inactivation significantly restores cilia formation. Myh10 antagonizes Myh9 and increases actin dynamics, permitting pericentrosomal preciliary complex formation required for cilium assembly. Importantly, Myh10 is upregulated upon serum starvation-induced ciliogenesis and this induction requires Atat1/Mec-17, the microtubule acetyltransferase. Our findings suggest that Atat1/Mec17-mediated microtubule acetylation is coupled to Myh10 induction, whose accumulation overcomes the Myh9-dependent actin cytoskeleton, thereby activating cilium formation. Thus, Atat1/Mec17 and myosin II coordinate microtubules and the actin cytoskeleton to control primary cilium biogenesis.</p> / Dissertation

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.

Extraction and quantitation of myosin light chain two coupled with myograph recordings from Ascaris muscle perfused with calmodulin inhibitors and neurotransmitters in conjunction with their respective agonists and antagonists have been used to establish the regulation of contraction in this muscle. Densitometric tracings of isolectric focusing gels separating the regulatory light chain were used to quantitate phosphorylation in resting, contracted and flaccid muscle. These studies indicated that inhibitory neurostimulation is mediated by a true GABA receptor. Myosin-mediated contraction is responsible for maintaining the level of tension observed in resting actin-mediated muscle. Actin-mediated contraction is responsible for the rapid rise in tension following excitatory stimuli. Both systems function simultaneously and are independant.

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Adherens junctions (AJs) and tight junctions (TJs) are characteristic features of differentiated epithelial cells and are critical for regulation of epithelial barriers and cell polarity. Integrity and remodeling of epithelial junctions depend on their interactions with underlying actomyosin cytoskeleton. Anillin is a multifunctional scaffold able to interact with different cytoskeletal proteins including F-actin and Myosin II. This project aimed to investigate roles of anillin in regulating epithelial AJs and TJs. Using A549 human lung epithelial and DU145 human prostate epithelial cells, we demonstrated the anillin depletion-induced loss of AJs and TJs. This was accompanied by disorganization of perijunctional actomyosin belt and disruption of the adducin-based membrane skeleton that links actin filaments to the plasma membrane and epithelial junctions. Depletion of anillin decreased protein levels of γ-adducin and downregulation of γ-adducin mimicked effects anillin knockdown on AJ and TJ integrity. These findings suggest a novel role for anillin in the assembly of epithelial junctions.

Resonance energy transfer was employed to study the conformational changes of actomyosin during ATP hydrolysis. To calibrate the technique, the parameters for resonance energy transfer were defined. With conformational searching algorithms to predict probe orientation, the distances measured by resonance energy transfer are highly consistent with the atomic models, which verified the accuracy and feasibility of resonance energy transfer for structural studies of proteins and oligonucleotides. To study intramyosin distances, resonance energy transfer probes were attached to skeletal myosin's nucleotide site, subfragment-2, and regulatory light chain to examine nucleotide analog-induced structural transitions. The distances between the three positions were measured in the presence of different nucleotide analogs. No distance change was considered to be statistically significant. The measured distance between the regulatory light chain and nucleotide site was consistent with either the atomic model of skeletal myosin subfragment-1 or an average of the three models claimed for different ATP hydrolysis states, which suggested that the neck region was flexible in solution. To examine the participation of actin in the powerstroke process, resonance energy transfer between different sites on actin and myosin was measured in the presence of nucleotide analogs. The efficiencies of energy transfer between myosin catalytic domain and actin were consistent with the actoS1 docking model. However, the neck region was much closer to the actin filament than predicted by static atomic models. The efficiency of energy transfer between Cys 374 and the regulatory light chain was much greater in the presence of ADP-AlF4, ADP-BeFx, and ADP-vanadate than in the presence of ADP or no nucleotide. These data detect profound differences in the conformations of the weakly and strongly attached crossbridges which appear to result from a conformational selection that occurs during the weak binding of the myosin head to actin. The resonance energy transfer data exclude a number of versions of the swinging lever arm model, and indicate that actin participation is indispensable for conformational changes leading to force generation. The conformational selection during weak binding at the actomyosin interface may precock the myosin head for the ensuing powerstroke.

The dynamics and function of the actin cytoskeleton depends on polymerization and branching of actin filaments, an event that is stimulated by Arp2/3. Arp2/3-dependent branching is closely linked to the pentameric WASH complex which consists of WASH, strumpellin, SWIP, CCDC53 and FAM21. WASH complex is associated mainly with endosomes. It was traditionally localized to retromer-coated domains of early endosomes which enable sorting and recycling of endocytosed material. However, latest scientific data extend the role of WASH complex to other endosomal or even non-endosomal sites. Of all the subunits of the WASH complex, FAM21 is the most prominent hub for protein-protein interactions, thanks to its long unstructured C-terminal domain. In my diploma thesis FAM21 was localized to early and late endosomes and lysosomes of U2OS human cell line. Dictyostelium discoideum was then used as a model organism to investigate FAM21 protein interactions as well as the proteins associated specifically with the C terminal domain of FAM21. Results of the study shed new light on the complex network of FAM21 interactions and question the long-standing theories on the function of WASH complex in cells. Powered by TCPDF (www.tcpdf.org)

(English) Both paxillin and PIP2 are well known components of the cell, although of a distinct origin. Focal adhesion protein paxillin spreads the signals from extracellular matrix via integrins and growth factor receptors to affect cellular motility and migration (Schaller, 2001). PIP2, a major structural component of cytoplasmic membrane, is utilized by phospholipase C to generate second messenger molecules (Hokin and Hokin 1953; Streb et al. 1983). Both molecules were recently shown to be localized in the nucleus. Their original functions have been well established, but together with other research colleagues we are now shedding more light on completely different functions of these biological molecules and moreover, in the different compartments than they were primarily believed to function in. Here, we introduce paxillin as an important factor of the cell nucleus, where it regulates transcription of two important growth-related genes, IGF2 and H19. It does not affect the allelic expression of these imprinted genes, it rather regulates long-range chromosomal interactions between H19 or IGF2 promoter, and the shared distal enhacer on an active allele. In detail, paxillin stimulates the interaction between the enhancer and the IGF2 promoter, activating IGF2 gene transcription, while it restrains...