Dissertations / Theses on the topic 'Cytoskeletal proteins'
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Liu, Gang. "Cytoskeletal proteins of Dictyostelium." Thesis, University of Oxford, 1990. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.292634.
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Baisden, Joseph M. "AFAP-110 is a cSrc activator." Morgantown, W. Va. : [West Virginia University Libraries], 2003. http://etd.wvu.edu/templates/showETD.cfm?recnum=2766.
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Thesis (Ph. D.)--West Virginia University, 2003.Title from document title page. Document formatted into pages; contains v, 149 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references.
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Alwash, Ban Hussein Kadhim. "S100 proteins control cytoskeletal dynamics in cancer." Thesis, University of Leicester, 2018. http://hdl.handle.net/2381/42867.
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The S100 family of calcium binding proteins exhibits a unique pattern of cell type specific expression. These proteins are found in the cytoplasm and/or nucleus of a variety of cells, and involved in the control of a wide range of cellular processes such as cell cycle progression and differentiation. S100A4 and S100A6 are members of the S100 protein family that interact with several molecular targets including the heavy chain of non-muscle myosin IIA (NM IIA) and annexin II, respectively. NM IIA is a major actin-associated motor protein, which is involved in cell motility and cytokinesis. Assembly/disassembly of myosin filaments is primarily controlled by myosin light chain phosphorylation. However, small calcium-binding proteins of the S100 family also play an active role in the dynamics of actin-myosin filaments, leading to an increase in the dissemination of tumour cells. Accordingly, the main aim of this work was to study the molecular mechanism underlying S100A4/A6 function in epithelial mesenchymal transition (EMT) and provide in vivo data highlighting their role in the regulation of myosin dynamics. Intriguingly, we employed a novel transition electron microscopy approach to study the function of non-muscle IIA isoforms and their interactions with S100A4/A6 in A431/ZEB2 cells undergoing an EMT. Our data confirmed that both 6S and 10S myosin isoforms do exist in cells and directly interact with S100A4/A6 in vivo. Depletion of S100A4 resulted in the disappearance of the peaks corresponding to monomeric myosin indicating that S100A4 is required for balancing monomer-polymer equilibrium in cells. In blot overlay, both S100A4 and S100A6 showed similar binding site on myosin fragment 4 (C-terminus). However, a new S100A6 binding site was mapped on myosin heavy chain represented in M53 fragment which is a part of rod domain. In addition to the solubility of myosin in high ionic buffer, S100A4 and S100A6 are able to solubilise the myosin which was measured by the turbidity assay. Moreover, a decrease in ATPase activity of actomyosin complex in cells undergoing EMT was observed in the presence of S100A4/A6. In conclusion: This study shows that S100A4/A6 protein interacts with NM IIA. There is no redundancy and both proteins promote myosin dynamics, cell migration and invasion. S100A4 and S100A6 are up-regulated by ZEB2 and is implicated in the dynamic regulation of myosin filaments by switching the balance towards monomeric myosin.
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Snyder, Heidi Ghent. "Fiber type-specific desmin content in human single muscle fibers /." Diss., CLICK HERE for online access, 2006. http://contentdm.lib.byu.edu/ETD/image/etd1253.pdf.
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McCarthy, David James. "Analysis of the novel Lyn-associated cytoskeletal modular protein, LACM." University of Western Australia. School of Medicine and Pharmacology, 2009. http://theses.library.uwa.edu.au/adt-WU2009.0180.
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A yeast-two hybrid screen with Lyn identified a novel 130 kDa multidomain protein with a 36% identity to Actin Filament Associated Protein (AFAP) 110 and similar domains, including PH domains, potential sites of tyrosine and serine/threonine phosphorylation, a leucine-zipper domain, a potential actin binding site and multimerization site. AFAP110 has been shown to have a role in modulating actin filament integrity and induce lamellipodia formation, and is known to interact with Src family kinases. The aim of this thesis was to characterize this novel protein named Lyn-Associated Cytoskeletal Modulator (LACM) and determine any molecular interactions in order to attempt to elucidate a role for the protein in cell signaling through Lyn. LACM is encoded by a gene consisting of 18 exons and is located on human chromosome 5q33.1 and mouse chromosome 18 E1. LACM protein is expressed through a number of cell types including the R11 erythroid cell line, and mouse tissues including brain, lung, heart and embryos. LACM was shown to multimerize, and subcellular localization of the protein was observed to concentrate around the cell membrane at sites of filamentous actin in filopodia, lamellipodia and stress fibres. The carboxy-terminus of LACM was observed to localize the protein to sites at the cell membrane and through the cytoplasm. Removal of this terminal region resulted in all LACM protein localizing to the nucleus in punctuate spots. LACM protein was observed in heart muscle and potentially has a role at sites of nerve junctions on cardiac myocytes. LACM was shown to interact with the SH3 domain of Lyn at a polyproline motif on LACM. LACM was observed to co-localize and co-immunoprecipitate with Lyn and was tyrosine phosphorylated by the kinase domain of Lyn. Interestingly, the consititutively active Lyn and LACM caused transfected cells to
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MacDonald, Louisa M. "Characterisation of the benzimidazole-binding site on the cytoskeletal protein tubulin." Thesis, MacDonald, Louisa M. (2003) Characterisation of the benzimidazole-binding site on the cytoskeletal protein tubulin. PhD thesis, Murdoch University, 2003. https://researchrepository.murdoch.edu.au/id/eprint/173/.
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The binding kinetics of several benzimidazole compounds were determined with recombinant tubulin monomers and heterodimers from benzimidazole-sensitive and -insensitive organisms. This study utilised the naturally occurring high efficacy of the benzimdazoles for the parasitic protozoa Giardia duodenalis and Encephalitozoon intestinalis. The benzimidazoles are not active against the protozoan Cryptosporidium parvum or mammalian hosts, including humans. The affinity of several benzimidazole derivatives for monomeric and heterodimeric beta-tubulin was clearly demonstrated, thus supporting previous studies of drug-resistant nematode and fungal populations. A homology model of protozoan alpha beta-tubulin, produced using the three-dimensionalstructure of mammalian alpha beta-tubulin, identified a strongly hydrophobic domain only on the beta-tubulin protein of sensitive protozoa. This domain is proposed to be the benzimidazole-binding domain and the amino acid residues within it include three key residues which are substituted between benzimidazole-sensitive and -insensitive organisms. These residues are Ile-189, Val-199, and Phe-200 that all have non-polar, hydrophobic side groups and are proposed to bind with the R5 side chain of several benzimidazole derivatives. In addition to this, the benzimidazole derivatives were able to bind irreversibly with assembling microtubules from sensitive parasites. The incorporation of benzimidazole-bound alpha beta-heterodimers into assembling microtubules was shown to arrest polymerisation in vitro although the addition of benzimidazole compounds to assembled microtubules did not result in depolymerisation. Taken together, these results suggest that the mechanism of action of these compounds is through disruption of the dynamic equilibrium that balances the cycle of microtubule polymerisation and disintegration within these protozoa. Further, this effect is brought about by preferential binding of the benzimidazoles to a hydrophobic region on the beta-tubulin protein.
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MacDonald, Louisa M. "Characterisation of the benzimidazole-binding site on the cytoskeletal protein tubulin." Murdoch University, 2003. http://wwwlib.murdoch.edu.au/adt/browse/view/adt-MU20050107.94048.
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The binding kinetics of several benzimidazole compounds were determined with recombinant tubulin monomers and heterodimers from benzimidazole-sensitive and -insensitive organisms. This study utilised the naturally occurring high efficacy of the benzimdazoles for the parasitic protozoa Giardia duodenalis and Encephalitozoon intestinalis. The benzimidazoles are not active against the protozoan Cryptosporidium parvum or mammalian hosts, including humans. The affinity of several benzimidazole derivatives for monomeric and heterodimeric â-tubulin was clearly demonstrated, thus supporting previous studies of drug-resistant nematode and fungal populations. A homology model of protozoan áâ-tubulin, produced using the three-dimensionalstructure of mammalian áâ-tubulin, identified a strongly hydrophobic domain only on the â-tubulin protein of sensitive protozoa. This domain is proposed to be the benzimidazole-binding domain and the amino acid residues within it include three key residues which are substituted between benzimidazole-sensitive and insensitive organisms. These residues are Ile-189, Val-199, and Phe-200 that all have non-polar, hydrophobic side groups and are proposed to bind with the R5 side chain of several benzimidazole derivatives. In addition to this, the benzimidazole derivatives were able to bind irreversibly with assembling microtubules from sensitive parasites. The incorporation of benzimidazole-bound áâ-heterodimers into assembling microtubules was shown to arrest polymerisation in vitro although the addition of benzimidazole compounds to assembled microtubules did not result in depolymerisation. Taken together, these results suggest that the mechanism of action of these compounds is through disruption of the dynamic equilibrium that balances the cycle of microtubule polymerisation and disintegration within these protozoa. Further, this effect is brought about by preferential binding of the benzimidazoles to a hydrophobic region on the â- tubulin protein.
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Ritchie, Sian. "Identification of cytoskeletal proteins as substrates for Ca'2'+ dependent protein kinase." Thesis, University of Reading, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.240317.
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Höng, J. "Investigating the structures of evolutionarily conserved cytoskeletal proteins." Thesis, University of Cambridge, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.604097.
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The parasitic protist Giardia intestinalis, considered to be one of the most ancient eukaryotic organisms, contains a typical eukaryotic cytoskeleton composed of microtubules (αβ-tubulin), microfilaments (actin) and intermediate filaments. Proteins associated with the microtubules include kinesins and additional members of the tubulin superfamily. δ-Tubulin is a member of the tubulin superfamily. The methylotropic yeast P. pastoris was identified as the most suitable host for recombinant expression of G. intestinalis δ-tubulin. High-resolution crystal structures of G. intestinalis wild-type kinesin-2 GiKIN2a motor domain with docked neck linker and its hydrolysis deficient mutant GiKIN2aT104N, expressed in and purified from E. coli, were solved in complex with ADP and Mg2+ at 1.6 Å and 1.8 Å resolutions, respectively. They represent the first high-resolution structures of a kinesin-2 motor domain. They confirm that the structural fold of the kinesin motor domain is remarkably conserved and also provide insights into the nucleotide coordination within its active site. Furthermore, in vivo experiments confirmed the role of G. intestinalis kinesin-2 in intraflagellar transport. Previously, structural and functional homologues of eukaryotic cytoskeletal proteins have been identified in prokaryotes. In magnetotactic bacteria, magnetosomes are held in place by cytoskeletal filaments formed by a protein, MamK, that is predicted to be actin-like. Magnetospirillum magnetotacticum MamK was expressed, purified and crystallised with apparent space group P321 and P422. However, these crystals appear to be perfectly twinned and thus determination of MamK structure has not yet been possible. Electron microscopy analysis of filamentous sheets, assembled from MamK in the presence of AMPPCP nucleotide, showed a longitudinal repeat of 53 Å, characteristic for actin-like protofilaments.
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Holmes, Fiona Elizabeth. "A study of cytoskeletal proteins in the neuron." Thesis, University of Kent, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.242927.
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Hayes, Nandini V. L. "A study on cytoskeletal proteins in the synapse." Thesis, University of Kent, 1992. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.314471.
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Bolton, Sarah J. "Studies on the cytoskeletal proteins vinculin and talin." Thesis, University of Leicester, 1995. http://hdl.handle.net/2381/35153.
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The role of vinculin in cell adhesion was investigated by isolating NIH3T3 and Balb/c 3T3 cells containing a plasmid vector expressing a vinculin antisense RNA under the control of the MMTV promoter. Stable cell lines of NIH3T3 cells were cultured with dexamethasone but none showed any reduction in vinculin protein levels. Two Balb/c 3T3 cell lines, when cultured with dexamethasone displayed a marked reduction in vinculin levels and displayed an altered cell shape from a spread to a more spindle-shaped morphology. These phenotypic changes were reversible upon removal of the dexamethasone. The reduced adhesion corresponded with a reduction in the growth rate of the clones. The cells were also unable to spread on fibronectin and the ususal increase in the phosphotyrosine content of two signalling proteins, paxillin and pp125FAK, normally associated with cell adhesion, was not observed. The results establish that vinculin is a key component of integrin-mediated cell adhesion. To explore the structure-functional relationship of talin, six anti-talin monoclonal antibodies were microinjected into human fibroblasts and the effect upon the actin cytoskeleton was assessed. Two of the antibodies, TA205 and TD77 resulted in the disintegration of actin stress fibres, and migration of CEF was also severely impaired following microinjection of either antibody. The epitopes recognised by TA205 and TD77 had been mapped to talin residues 102-497 and 2269-2541 respectively. Microinjection of CEF with a polypeptide containing residues 102-497 demonstrated that they were mainly associated with the detergent-soluble cytoplasmic portion of the cell and were also able to disrupt stress fibre and focal adhesion integrity. Residues 2269-2541 were associated with the detergent-insoluble cytoskeletal fraction of the cell but did not affect stress fibre or focal adhesion integrity. Attempts to identify proteins that interacted with residues 102-497 of talin were unsuccessful but an actin-binding site was identified within residues 2269-2541. The results indicate the presence of domains within the N- and C-terminus of talin that are essential to the involvement of talin in the formation of focal adhesions.
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Park, Jin Gyoon. "Function of Cytoskeletal Proteins in GLUT4 Vesicle Transport in Adipocytes: Dissertation." eScholarship@UMMS, 2003. http://escholarship.umassmed.edu/gsbs_diss/273.
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Insulin stimulates glucose uptake in adipose and muscle cells via translocation of the intracellular vesicles containing GLUT4. It was largely unknown whether and/or how the signaling molecules such as PI 3-kinase and Akt regulate the mechanical movements of the GLUT4-containing vesicles. Hence, this study was performed to test the hypothesis that actin and microtubules function in translocating GLUT4 vesicles. Treatments of insulin as well as endothelin-1 (ET-1), an insulin-mimicking peptide which does not act through PI 3-kinase, induced polymerization of actin without affecting the microtubular network. By mass spectrometry, the tyrosine kinase PYK2 was identified to be tyrosine phosphorylated specifically by ET-1 but not by insulin. Expression of the carboxyl-terminal fragment (CRNK) PYK2, but not wild type nor kinase-deficient PYK2 mutants, inhibited ET-1-stimulated actin polymerization while expression of all three PYK2 constructs had no effect on insulin-stimulated actin polymerization. More importantly, expression of CRNK, but not wild type nor kinase-deficient PYK2 constructs, blocked ET-1- but not insulin-stimulated GLUT4 translocation to the plasma membrane. These suggest that ET-1 and insulin stimulate actin polymerization via distinct signaling pathways, and that the actin polymerization is required for GLUT4 vesicle translocation.
In order to test the possible involvement of microtubule in GLUT4 vesicle translocation, time lapse imaging of 3T3-L1 adipocytes expressing GLUT4-YFP and tubulin-CFP was performed. GLUT4-YFP vesicles move long-range bi-directionally on microtubules, which suggests the presence of molecular motors on the vesicles. Moreover, insulin increased the number of vesicle movements on microtubules without changing the velocities. Interestingly, the stimulatory action of insulin appears to be independent of PI 3-kinase activation. Conventional kinesin was identified as a highly expressed kinesin isotype in adipocytes. Notably, expression of dominant negative mutants but not wild type kinesin inhibited insulin-stimulated long-range GLUT4 vesicle movements and GLUT4 translocation to the plasma membrane in live and fixed cells, respectively. These data indicate that insulin signaling induces the movement of GLUT4 vesicles on microtubule which is mediated by conventional kinesin. Overall, the data presented here provide evidence supporting the hypothesis that actin and microtubule cytoskeletons are required for insulin to mobilize GLUT4 vesicles in adipocytes.
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Auxier, Benjamin. "Evolutionary history of the septin cytoskeletal proteins in opisthokonts." Thesis, University of British Columbia, 2017. http://hdl.handle.net/2429/63550.
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Septins are cytoskeletal proteins important in morphogenesis, cell division and establishing and maintaining cell polarity. Over the course of more than a billion years as Animals and Fungi originated and diversified, their septin genes duplicated and diverged, giving rise to paralogs that encode modular proteins. The septin monomers assemble into heteropolymeric higher order structures that affect cell form by creating physical barriers to diffusion or serving as scaffolds organizing groups of diverse proteins. Here we take advantage of newly sequenced genomes to track the history of septin gene expansions and losses within the phylogeny of Animals and Fungi, including their close protist relatives. By sampling broadly across Opisthokonts, we identified the likely presence of early-diverging animal lineages within Groups 4 and 2A and discovered a novel group of fungal septins not found in Ascomycetes or Basidiomycetes. We hypothesized that previously identified sequence conservation is linked to interface interactions. Using protein homology folding, we mapped interacting residues across Opisthokonts and found that all previously identified motifs were involved in interface interactions, and contained almost all interacting residues. As septin subunit interactions are likely driven by residue identity, we categorized the interacting residues and found specific interface residues associated with each septin Group. We suggest that these residues may explain patterns of septin subunit binding affinity. Notably, we found that Group 3 septins show little conservation within the polybasic region that forms the first alpha helix, found in the NC interface of other septin Groups. This may explain the capping role of Group 3 septins in the yeast septin octamer. With increased sampling, this work identified increased diversity of Opisthokont septins. These proteins show patterns of sequence conservation that are largely driven by conserved interface interactions, in addition to binding of GTP. This work highlights the likely duplications that predate the Opisthokont ancestor, and the structural constraints that shaped the evolution of these multi-purpose septins.
Additionally, I attempted to validate and optimize an Agrobacterium-mediated transformation protocol for the chytrid fungus Blastocladiella. While I was unable to conclusively repeat previously published experiments, my work highlighted the difficulties in transformation of these distinct fungi.Science, Faculty of
Botany, Department of
Graduate
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Gilmore, Andrew Peter. "Molecular studies on the cytoskeletal proteins vinculin and talin." Thesis, University of Leicester, 1993. http://hdl.handle.net/2381/35231.
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Vinculin and talin are components of adherens type cell/ECM junctions (adhesion plaques) where they are thought to form part of the linkage between the cytoplasmic domain of the beta-subunit of integrins and the actin cytoskeleton. Vinculin has been shown to interact with the adhesion plaque proteins talin, paxillin and beta-actinin, and talin has been shown to interact with vinculin, integrins beta1 and beta3, and actin. This study has examined the domain structure of vinculin and talin with regard to the interaction between them. Saturation binding analysis using purified chicken vinculin and talin demonstrated that the interaction was biphasic, with two dissociation constants of 3x10.;-8Mand 5.5x10.;-7M. The lower affinity interaction indicated that multiple bindingsites existed, with three moles of vinculin binding per mole of talin. The high affinity interaction was substoichiometric, and this remains to be explained. To localise the talin binding domain in vinculin, contiguous regions of the molecule were expressed as fusion proteins in E. coli and tested for binding iodinated talin in solid phase assays. Binding was localised to the N-terminal 258 amino acids of vinculin, and no further truncation of this region left detectable talin binding activity. This region was found to bind talin as effectively as intact vinculin, and no evidence was obtained to suggest that short linear sequences within this region could account for the interaction. Two independently isolated vinculin cDNA clones, one of which lacked 123bp of coding sequence, had suggested that alternative splicing of the vinculin mRNA within the region encoding the talin binding domain could be a mechanism for regulating talin binding. Though this region was found to be contained on a single exon within the chicken vinculin gene, no evidence was found to authenticate the existence of such a spliced message using a reverse transcriptase/PCR protocol to map vinculin transcripts. Vinculin binding sites were mapped in talin by expressing a series of overlapping talin fusion proteins and investigating their ability to bind vinculin in solid phase assays. This identified three non-overlapping regions of talin within its 190kDa domain which bound vinculin with dissociation constants around 10-7M. Two of these were within residues 498-950, whilst the most C-terminal mapped to between residues 1554-2268. Further experiments on the most C-terminal of these sites suggested that a 40 amino acid sequence was able to bind vinculin.
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Whiteman, Ineka T. "Cytoskeletal proteins and early neurodegenerative mechanisms in Alzheimer's disease." Thesis, The University of Sydney, 2011. https://hdl.handle.net/2123/28860.
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Alzheimer’s Disease (AD) is a devastating neurodegenerative disorder that is histopathologically characterized by several hallmark lesions, including extracellular plaques comprised of amyloid beta (AB) peptides, neuropil threads and intra-neuronal neurofibrillary tangles, both of which are comprised of hyperphosphorylated microtubule-associated (MAP) protein tau. Neuropil threads are one of the earliest lesions observed in AD brain and the extent of their presence correlates with cognitive decline and disease progression. In addition, rod-like aggregates of actin and actin depolymerizing factor (ADF)/cofilin (‘AC’ or ‘cofilin’ rods) have also been described throughout the neuropil of AD brains.
Sporadic AD accounts for over 90% of all AD cases and although aging has been identified as the most significant risk factor for developing this form of the disease, the pathogenic mechanisms involved in initiation of sporadic AD remain poorly understood. Decreased mitochondrial function and increased oxidative stress are common features of the aging brain and a growing body of evidence suggests these mitochondrial changes may play a central role in the pathogenesis of sporadic AD. The key question therefore is can mitochondrial dysfunction induce histopathological features of AD and if so, by what mechanisms? Answers to these important questions may be pivotal in the development of more effective AD therapeutics.
This thesis investigated the effects of mitochondrial dysfunction on the interrelationships between two AD-related cytoskeletal systems, MAP/tau and AC-actin. Employing primary neuron culture models, organotypic brain slice cultures and a range of immuno—labeling and microscopy techniques, we show that mitochondrial dysfunction rapidly induces rod-Iike aggregations of activated AC throughout neurites that subsequently recruit AD-relevant epitopes of phosphorylated MAP/tau. The resulting cytoskeletal complexes closely resemble neuropil threads observed in human AD brain. Mechanistically, the relationship between these inclusions was explored through use of actin modifying
drugs, knockdown of ADF/cofilin in primary neuron culture and knockout of tau in transgenic mouse brain slices. Overall, the results suggest that during neuronal stress, AC rods form rapidly and serve as a nucleation seed for subsequent recruitment of phosphorylated MAP/tau. Moreover, this initial recruitment is specific to MAP/tau phosphorylated in the functional microtubuIe-binding domain which is of significance, since this is one of the first phosphosites identified during the early pathogenesis of AD tau pathology. Furthermore, treatments with synthetic or naturally-secreted preparations of AB peptides induced the same effects in primary neuron and brain slice cultures, thus suggesting that the major histopathologies of AD may all be reconciled in one common pathway.
The studies reported here provide evidence suggesting that mitochondrial dysfunction is central to the pathogenesis of two AD-related cytoskeletal pathologies: MAP/tau neuropil threads and AC rods. Moreover, the results presented here show for the first time that these two neuritic inclusions are closely interrelated and together implicate a disrupted cytoskeletal network that may account for the widespread axonal transport deficits and axonal degeneration characteristic of this disease. To that extent, we propose that association of MAP/tau and AC-actin proteins constitutes one of the earliest events in the pathogenesis of sporadic AD.
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Fawell, E. H. "Studies on the microvillus cytoskeleton." Thesis, University of Leeds, 1985. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.355700.
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Che, Alexis Pun Kit. "Studies of band 3 rotational mobility in normal and ovalocytic human red blood cell membranes by transient dichroism." Thesis, University of Essex, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.241212.
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Woodward, Robert. "Molecular and immunological characterization of cytoskeletal proteins in Trypanosoma brucei." Thesis, University of Kent, 1991. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.292369.
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Rindisbacher, Lorenz. "Studies on cytoskeletal and cell surface proteins of African trypanosomes /." [S.l.] : [s.n.], 1991. http://www.ub.unibe.ch/content/bibliotheken_sammlungen/sondersammlungen/dissen_bestellformular/index_ger.html.
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Vlahovich, Nicole. "The role of cytoskeletal tropomyosins in skeletal muscle and muscle disease." Thesis, View thesis, 2007. http://handle.uws.edu.au:8081/1959.7/32176.
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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.
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Gupta, Sneha. "Understanding Regulation of the Cytoskeleton during Cell Cycle Transitions through Examination of Crosstalk between Homologous Fission Yeast Pathways, Septation Initiation Network and Morphogenesis ORB6 Network: A Dissertation." eScholarship@UMMS, 2013. http://escholarship.umassmed.edu/gsbs_diss/693.
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The fission yeast Schizosaccharomyces pombe has become a powerful model system for studying cytokinesis, a process of cytoplasmic division by which one cell divides into two identical daughter cells. Like mammalian cells, S. pombe divides through the use of an actomyosin contractile ring, which is composed of a set of highly conserved cytoskeletal proteins. Cytokinesis in S. pombe is primarily regulated by the SIN pathway, which is activated in late mitosis and is required for actomyosin contractile ring and septum assembly, and also plays a role in spindle checkpoint inactivation, and telophase nuclear positioning. The various functions of the SIN are carried out by the terminal kinase in the pathway called Sid2. The lack of information in the downstream targets of Sid2 has limited our understanding of the different functions of the SIN. We recently showed that, in addition to its other functions, the SIN promotes cytokinesis through inhibition the MOR signaling pathway, which normally drives cell separation and initiation of polarized growth following completion of cytokinesis (Ray et al, 2010). The molecular details of this inhibition and the physiological significance of inhibiting MOR during cytokinesis was unclear. The results presented in Chapter II describe our approach to identify Sid2 substrates, particularly focusing on Nak1 and Sog2 that function in the MOR signaling cascade. We identified and characterized Sid2 phosphorylation sites on the Nak1 and Sog2 proteins. Chapter III explores how post translational modification of MOR proteins by Sid2 regulates polarized growth during cytokinesis. This includes delineating the effect of Sid2 mediated phosphorylation of Nak1 and Sog2 on protein-protein interactions in the MOR pathway as well as on the regulation of their localization during late mitosis. Finally, results in Chapter IV demonstrate that failure to inhibit MOR signaling is lethal because cells initiate septum degradation/cell separation before completing cytokinesis thereby emphasizing the importance of cross-regulation between the two pathways to prevent initiation of the interphase polarity program during cytokinesis.
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Vlahovich, Nicole. "The role of cytoskeletal tropomyosins in skeletal muscle and muscle disease." View thesis, 2007. http://handle.uws.edu.au:8081/1959.7/32176.
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Thesis (Ph.D.)--University of Western Sydney, 2007.A thesis presented to the University of Western Sydney, College of Health and Science, School of Natural Sciences, in fulfilment of the requirements for the degree of Doctor of Philosophy. Includes bibliographies.
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Roberts-Pilgrim, Anna M. Phillips Charlotte L. "Glomerulosclerosis in the Col1a2-deficient mouse model homotrimer pathogenesis and MMP expression /." Diss., Columbia, Mo. : University of Missouri--Columbia, 2009. http://hdl.handle.net/10355/6155.
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Title from PDF of title page (University of Missouri--Columbia, viewed on Feb. 20, 2010). The entire thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file; a non-technical public abstract appears in the public.pdf file. Dissertation advisor: Charlotte L. Phillips. Vita. Includes bibliographical references.
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Teoh, Désiree Ann. "The role of cytoskeletal proteins in Giardia lamblia-induced epithelial injury." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1999. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape7/PQDD_0025/MQ48048.pdf.
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Hughes, Richard Anthony. "Motor and cytoskeletal proteins and their interactions in the inner ear." Thesis, King's College London (University of London), 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.420601.
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Willetts, Alison. "The role of cytoskeletal proteins in the mechanism of insulin release." Thesis, Aston University, 1988. http://publications.aston.ac.uk/12565/.
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This thesis is concerned with the role of /3-cell cytoskeletal proteins in the mechanism of insulin release from islets of experimental animals, the Aston obese diabetic hyperglycaemic (ob/ob) mouse and their lean littermates and the cultural insulin secreting /?-cell lines, HIT-TT5 and RINm5F. Investigations were carried out into the glucose induced insulin response of the lean and obese mouse islets and HIT-TI5 cells and the D-glyceraldehyde response of RINm5F cells using a static incubation system. Colchicine was found to inhibit insulin release from both lean and obese mouse islets more significantly than cultured TTT-TI5 and RINm5F cells. (Colchicine pre-treatment also inhibited the second phase of insulin release from perifused lean mouse islets and HIT-TI5 cells). Cytocha-lasin B, used to investigate the role of the microfilamentous system in the mechanism of insulin release enhanced insulin release from both lean and obese mouse islets to a significantly greater degree than that from cultured HIT-TI5 and RINm5F cells. Pre-treatment of isolated lean and obese mouse islets and cultured /?-cells with a combination of colchicine and cytochalasin B significantly reduced the insulin response of the HIT-TI5 and RINm5F cells compared with the control values suggesting that intact microtubules are more important for the sustained release of insulin than the microfilamentous system. However, the response was not so clearly defined with the lean and obese mouse islets. Tubulin was separated from the extracts of lean mouse islets and the HIT-TI5 and RINm5F cells and actin was separated from all of the cell types including the obese mouse islets by SDS- polyacrylamide electrophoresis. A tubulin radioimmunoassay and a colchicine binding assay were developed to measure the tubulin content of lean and obese mouse islets, and the shift between the proportions of tubulin dimers and polymerized tubulin under stimulatory and non-stimulatory conditions. The assay methods developed were not prone to be accurate, sensitive and precise but gave some indication of the shift from unpolymerised to polymerised tubulin during glucose stimulated insulin release. These studies show that microtubules do play a fundamental role in the mechanism of insulin release from both islets and cultured HIT-TI5 and RINm5F cells.
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Cai, Xinming Schaller Michael D. "Cytoskeletal signaling proteins complex regulatory mechanisms and roles in innate immunity /." Chapel Hill, N.C. : University of North Carolina at Chapel Hill, 2007. http://dc.lib.unc.edu/u?/etd,2155.
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Thesis (Ph. D.)--University of North Carolina at Chapel Hill, 2007.Title from electronic title page (viewed Feb. 26, 2009). "... in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department or Cell and Developmental Biology, School of Medicine." Discipline: Cell and Developmental Biology; Department/School: Medicine.
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Guljamow, Arthur. "Characterization of two eukaryotic cytoskeletal proteins horizontally transferred to a cyanobacterium." Doctoral thesis, Humboldt-Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, 2012. http://dx.doi.org/10.18452/16481.
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Das Cyanobakterium Microcystis aeruginosa PCC 7806 enthält zwei Proteine unbekannter Funktion, welche eine hohe Sequenzähnlichkeit mit Bausteinen des eukaryotischen Aktinzytoskeletts haben. Eines dieser Proteine ist Aktin selbst, das andere ist das Aktinbindeprotein Profilin. Die vorliegende Arbeit enthält eine detaillierte Charakterisierung beider Proteine sowie Vergleiche mit ihren eukaryotischen Verwandten. So inhibiert, im Gegensatz zu Eukaryoten, cyanobakterielles Aktin nicht das Enzym DNaseI. Es bildet jedoch Polymere, die hier mit Phalloidin visualisiert wurden. Konfokale Mikroskopie offenbart klare Unterschiede in den Polymeren, da die cyanobakteriellen eine Länge von 10 µm nicht überschreiten und breiter sind als die zylindrischen, ca. 100 µm langen Filamente eukaryotischen Aktins. Röntgen-Kleinwinkelstreuungsdaten zeigen, dass cyanobakterielle Aktinpolymere in ihrer Form am ehesten einem Band ähneln. Es bestehen auch Unterschiede hinsichtlich des Profilins: während es in Eukaryoten ausschließlich Aktinmonomere bindet, assoziiert cyanobakterielles Profilin mit Aktinfilamenten und vermittelt die Entstehung flächiger Heteropolymere. GFP-Fusionsstudien zeigen, dass die Koexpression von Aktin und Profilin die Bildung eines Hohlraumkompartiments in E.coli nach sich zieht. Ähnliche Gebilde wurden bereits in Microcystis gezeigt und könnten auf die beobachteten Heteropolymere zurückzuführen sein. Diese Arbeit verdeutlicht, dass beide Proteine in einer natürlichen Bakterienpopulation etabliert sind und dort Merkmale tragen, die ihre eukaryotischen Vorläufer nicht zeigen. Folglich könnte die Anpassung an die räumlichen Begrenzungen einer Bakterienzelle, welcher die für die Regulierung der Polymerisation notwendigen Aktinbindeproteine fehlen, die Triebkraft für eine Koevolution von cyanobakteriellem Aktin und Profilin gewesen sein. Dieser Prozess gipfelte möglicherweise in der Entstehung eines neuartigen intrazellulären Gebildes von potentiell struktureller Bedeutung.The cyanobacterium Microcystis aeruginosa PCC 7806 harbors two proteins with unknown functions that were transferred horizontally from eukaryotes and show a high degree of sequence identity with key components of the eukaryotic actin cytoskeleton. One is actin itself; the other is profilin, an actin binding protein. This work presents the detailed characterization of both proteins and comparisons with the eukaryotic archetype. In contrast to bona fide actin, its cyanobacterial counterpart does not inhibit DNaseI. It forms polymers that can be visualized with labeled phalloidin, resembling eukaryotic actin in that respect. However, confocal microscopy reveals key differences between polymers of eukaryotic and cyanobacterial actin. Whereas the former appear as cylindrical filaments about 100 µm in length, the latter are shorter and wider arresting polymerization at 5-10 µm. Structural elucidation by Small-angle X-ray scattering shows that cyanobacterial actin polymers are ribbon-shaped. This work also shows fundamental differences between cyanobacterial and eukaryotic profilin. Most importantly, cyanobacterial profilin binds actin filaments and mediates their assembly into heteropolymeric sheets. GFP labeling experiments show that the co-expression of cyanobacterial profilin and actin results in the formation of large hollow enclosures in E.coli. These structures resemble the shell-like distribution of actin in Microcystis aeruginosa and may be based on the actin/profilin heteropolymers observed in vitro. This work shows that both cyanobacterial proteins are established in a natural bacterial community where they have gained properties unknown from their eukaryotic ancestors. Consequently, the adaptation to the confined space of a bacterial cell devoid of binding proteins usually regulating actin polymerization in eukaryotes may have driven the co-evolution of cyanobacterial actin and profilin, giving rise to an intracellular entity of potential structural relevance.
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Rosa, Jack. "Perturbation and Modulation of Microtubule Cytoskeletal Elements in Response to the Potentially Oncogenic Molecules, Survivin and P53, and Cytokinesis: A Dissertation." eScholarship@UMMS, 2006. https://escholarship.umassmed.edu/gsbs_diss/280.
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A complex network of protein filaments collectively known as the cytoskeleton carries out several crucial cellular processes. These functions include, but are not limited to, motility, cell shape, mitosis and organelle trafficking. The cytoskeleton is also highly responsive, allowing the cell to alter its shape in response to its immediate needs and environment. One of the major components of the cytoskeleton is the microtubule network. To refer to the array of micro tubules in the cell as a skeleton is a misnomer. Microtubules, by virtue of their structure and nature, are highly dynamic, continuously growing and shrinking. They also bind a variety of accessory molecules that aid in regulating and directing their dynamic activity. In this way they provide a structural basis for integral cell functions that require rapid assembly and disassembly. In some cases, perturbations of the microtubule network results in structural anomalies that lead to undesirable outcomes for the cell, namely chromosomal missegregation events and instability. The accumulation of these events may induce aneuploidy, which has been a fundamental component of tumorigenesis. This dissertation examines the role of the microtubule cytoskeleton within three distinct contexts. The first chapter investigates the association of the anti-apoptotic protein survivin with the microtubule network and its potential impact upon the cell from interphase to cytokinesis. The second chapter of this dissertation explores a little-studied, microtubule-dense organelle, referred to as the midbody, and the highly orchestrated events that take place within it during cytokinesis. The third and final chapter describes a unique experimental condition that may further our understanding of the interaction between the tumor suppressor p53 and the centrosome in cell cycle regulation and tumorigenesis.
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Sun, Ning. "Molecular interactions of the mammalian intermediate filament protein synemin with cytoskeletal proteins present in adhesion sites." [Ames, Iowa : Iowa State University], 2008.
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Fransson, Åsa. "Cell signaling by Rho and Miro GTPases : studies of Rho GTPases in cytoskeletal reorganizations and of Miro GTPases in mitochondrial dynamics /." Uppsala : Acta Universitatis Upsaliensis : Universitetsbiblioteket [distributör], 2008. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-8514.
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Fenton, Andrew Karl. "Roles of cytoskeletal proteins in the predatory life cycle of Bdellovibrio bacteriovorus." Thesis, University of Nottingham, 2010. http://eprints.nottingham.ac.uk/11537/.
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Bdellovibrio bacteriovorus are small, predatory bacteria that grow within the periplasmic space of a host bacterium. Bdellovibrio has a biphasic life-cycle switching from a uni-nucleoid, growth-senescent ‘attack-phase’ to a novel, multi-nucleoid filamentous ‘growth-phase’, which elongates and divides, growing saprophytically within the periplasmic space of their prey. Little is known to date about Bdellovibrio developmental processes and cell division within this periplasmic niche. Recent publications have demonstrated that bacterial cytoplasms house highly organised matrices of protein structures, called the bacterial cytoskeleton. The Bdellovibrio processes of prey-cell entry, filamentous cell growth and division coordination brings cellular morphological changes and challenges that could be coordinated by cytoskeletal elements. Green Fluorescent protein (GFP)-tagging and gene knock out approaches were used to gain insights into the function of these elements including: an Intermediate filament like protein Ccrp, which has a role in the maintenance of cell morphology; two actin homologues, which appear to function at different points in the predatory cycle, MreB1 and MreB2; and a new type of cytoskeletal element designated ‘bactofilin’, which may have a role in cell division control. Recent advances in GFP technologies have led to the development of optimised GFP variants, such as mTFP1 and mCherry. These have been used to reveal previously unseen detail of Bdellovibrio development within prey. Bdellovibrio do not follow the familiar pattern of bacterial cell division by binary fission, instead divide synchronously at multiple sites along their length, once prey resources are depleted. This yields both odd and even numbers of progeny Bdellovibrio.
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Treharne, Kate. "Studies on the interactions of cytoskeletal proteins with membranes in the brain." Thesis, University of Kent, 1989. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.330189.
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Chiu, Sheng-Wen. "Spatiotemporal dynamics of cytoskeletal and chemosensory proteins in the bacterium Rhodobacter sphaeroides." Thesis, University of Oxford, 2014. http://ora.ox.ac.uk/objects/uuid:d7d05b1a-07c5-4e26-9650-37bcfae2fade.
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The discovery of the prokaryotic cytoskeleton has revolutionized our thinking about spatial organisation in prokaryotes. However, the roles different bacterial cytoskeletal proteins play in the localisations of diverse biomolecules are controversial. Bacterial chemotaxis depends on signalling through large protein clusters and each cell must inherit a cluster on cytokinesis. In Escherichia coli the membrane chemosensory clusters are polar and new static clusters form at pre-cytokinetic sites, ensuring positioning at new poles after cytokinesis and suggesting a role for the bacterial FtsZ and MreB cytoskeletons. Rhodobacter sphaeroides has both polar, membrane-associated and cytoplasmic, chromosome-associated chemosensory clusters. This study sought to investigate the roles of FtsZ and MreB in the partitioning of the two chemosensory clusters in R. sphaeroides. The relative positioning between the two chemosensory systems, FtsZ and MreB in R. sphaeroides cells during the cell cycle was monitored using fluorescence microscopy. FtsZ forms polar spots after cytokinesis, which redistribute to the midcell forming nodes from which gradients of FtsZ extend circumferentially to form the Z-ring. The proposed node-precursor model might represent a common mechanism for the formation of cytokinetic rings. The MreB cytoskeleton continuously reorganizes between patchy and filamentous structures, and colocalises with FtsZ at midcell. Membrane chemosensory proteins form individual dynamic unit-clusters with mature clusters containing about 1000 CheW3 proteins. These unit-clusters diffuse randomly within the membrane but have a higher propensity for curved regions like cell poles. Membrane clusters do not colocalise with FtsZ and MreB and appear excluded from the Z-ring vicinity. The bipolar localisation of membrane clusters is established after cell division via random diffusion and polar trapping of clusters. The cytoplasmic chemosensory clusters colocalise with FtsZ at midcell in new-born cells. Before cytokinesis one cluster moves to a daughter cell, followed by the second moving to the other cell. FtsZ and MreB do not participate in the positioning of cytoplasmic clusters. Therefore the two homologous chemosensory clusters use different mechanisms to ensure partitioning, and neither system utilizes FtsZ or MreB for positioning.
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Crowley, Jessica Lynn. "Role of Supervillin, a Membrane Raft Protein, in Cytoskeletal Organization and Invadopodia Function." eScholarship@UMMS, 2009. https://escholarship.umassmed.edu/gsbs_diss/406.
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Crucial to a cell’s ability to migrate is the organization of its plasma membrane and associated proteins in a polarized manner to interact with and respond to its surrounding environment. Cells interact with the extracellular matrix (ECM) through specialized contact sites, including podosomes and invadopodia. Tumor cells use F-actin-rich invadopodia to degrade ECM and invade tissues; related structures, termed podosomes, are sites of dynamic ECM interaction and degradation. We show here that supervillin (SV), a peripheral membrane protein that binds F-actin and myosin II,reorganizes the actin cytoskeleton and potentiates invadopodial function. Overexpressed SV increases the number of F-actin punctae, which are highly dynamic and co-localize with markers of podosomes and invadopodia. Endogenous SV localizes to the cores of Src-generated podosomes in COS-7 cells and with invadopodia in MDA-MB-231 cells. EGFP-SV overexpression increases the average amount of matrix degradation; RNAi-mediated downregulation of SV decreases degradation. Cortactin, an essential component of both podosomes and invadopodia, binds SV sequences in vitro and contributes to the formation of EGFP-SV induced punctae. Additionally, SV affects cortactin localization,which could provide a mechanism for SV action at invadopodia.
The formation of cholesterol-rich membrane rafts is one method of plasma membrane organization. A property of membrane rafts is resistance to extraction with cold Triton X-100 and subsequent flotation to low buoyant densities. The actin cytoskeleton has been implicated in many signaling events localized to membrane rafts, but interactions between actin and raft components are not well characterized. Our laboratory isolated a heavy detergent resistant membrane fraction from neutrophils, called DRM-H, that contains at least 23 plasma membrane proteins. DRM-H is rich in cytoskeletal proteins, including fodrin, actin, myosin II, as well as supervillin. DRM-H also contains proteins implicated in both raft organization and membrane-mediated signaling. DRM-H complexes exhibit a higher buoyant density than do most DRMs (referred to as DRM-L), which are deficient in cytoskeletal proteins. By using similar purification methods, I find that COS-7 cells also contain cytoskeleton-associated DRMs. In addition, when transfected into COS-7 cells, estrogen receptor (ER)α associates with DRM-H, while ERβ is seen in both DRM-L and DRM-H populations, suggesting a role for DRM-H in nongenomic estrogen signaling. Thus, the cytoskeleton-associated DRM-H not limited to hematopoietic cells and could constitute a scaffold for membrane raftcytoskeleton signaling events in many cells.
Taken together, our results show that SV is a component of cytoskeleton-associated membrane rafts as well as podosomes and invadopodia, and that SV plays a role in invadopodial function. SV, with its connections to both membrane rafts and the cytoskeleton, is well situated to mediate cortactin localization, activation state, and/or dynamics of matrix metalloproteases at the ventral cell surface for proper matrix degradation through invadopodia. The molecular dissection of invadopodia formation and function may contribute to a greater understanding of in vivo invasion, and thus, tumor cell metastasis.
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Xing, Yi. "Structural and biochemical studies on the Wnt/[beta]-catenin signaling pathway and the PI3K/CISK signaling pathway /." Thesis, Connect to this title online; UW restricted, 2004. http://hdl.handle.net/1773/5680.
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Kunttas-Tatli, Ezgi. "Role of APC Proteins in Regulating Wnt Signaling and Cytoskeletal Organization in Drosophila." Research Showcase @ CMU, 2014. http://repository.cmu.edu/dissertations/423.
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Development of an embryo is a fascinating biological process that requires effective communication between neighboring cells and coordination of movement across the entire organism. At a cellular level, this is achieved by upstream signaling pathways ultimately regulating gene expression to provide cells with cues to perform certain tasks such as cell division, migration, cell rearrangements or changes in cell shape. All of these diverse tasks ultimately rely on rearrangements of the cytoskeleton. However, it is unclear what the molecular connections are between signaling and cytoskeletal dynamics. Adenomatous Polyposis Coli (APC) is a multifunctional protein that plays vital roles both in regulating the canonical Wnt signaling pathway and the cytoskeleton. Mutations of APC are associated with more than 80% of both familial and sporadic colorectal cancer cases. APC is one of few cytoskeletal proteins with direct links to cancer. However, as a multi-domain, multifunctional protein, a comprehensive understanding of APC biology has been difficult to achieve. While we have known for almost 20 years that APC proteins are essential negative regulators of Wnt signaling, the precise role they play both in regulating Wnt signaling and cytoskeletal have been unclear. In order for APC proteins to perform these diverse tasks and regulate both signaling and the cytoskeleton, it needs to be highly regulated itself. In my Ph.D. project, I approached APC proteins from many different angles, in many different developmental contexts, to gain insights into the precise role they play and how they are regulated in both the Wnt signaling and the cytoskeletal context. To better understand how APC proteins are regulated, I used Drosophila as a simpler, more tractable model. The large size of the vertebrate APCs (~300 kDa) makes it difficult to perform structure/function studies in the context of a full-length protein. Similar to vertebrates, flies have two highly conserved APC proteins (APC1 and APC2). Thus, I chose to study the fly APC homologs and mostly focused on the smaller member of the family, APC2, in my studies. To elucidate how APC proteins are regulated in the context of Wnt signaling, first we dissected the role of phosphorylation in the context of Wnt signaling. APC proteins are highly phosphorylated, and this plays a role in APCs activity in Wnt signaling. As a part of the destruction complex, APC targets the key effector of the pathway, ß-catenin for degradation. Phosphorylation of the central 20 amino acid repeats (20Rs) has received the most attention over the last decades, and has been shown to change the affinity of β-catenin binding in vitro. However, many of these in vitro models lacked an in vivo model. To test the functional significance of 20R phosphorylation in Wnt signaling, we used Drosophila APC2 and took advantage of the awesome power of genetics in this model organism. Our studies showed for the first time in an intact animal that 20R phosphorylation played an essential role. This study also suggested functional diversity among different 20Rs as well as gave us hints about the presence of macromolecular destruction complex, which we coined the term “destructosome’ (see Chapter 2). Besides the phosphorylation of the 20Rs, phosphorylation of other APC domains, such as the Axin binding SAMP repeats, had not been investigated before. Therefore, I also studied the phosphorylation of SAMP repeats and tested if it played a functionally significant role in APCs Wnt function. Similar to the 20Rs, I’ve shown that SAMP phosphorylation plays a previously uncharacterized role in APCs Wnt signaling function and proposed a novel idea of functional diversity among different SAMP repeats (see Chapter 3). As mentioned above, while studying the importance of 20R phosphorylation, I got interested in the idea of higher order destruction complex structures, or destructosome. This led me to think about the role of APC proteins in the assembly of this complex. Although it has been long appreciated that human APC can self-associate, the precise role of self-association in Wnt signaling hasn’t been explored in part due to the complexity of self-association in the vertebrate APC (vAPC) proteins. By using Drosophila APC2, I’ve identified a novel self-association domain (ASAD) and uncovered a new role for APC proteins in promoting the assembly and stability of the destructosome (see Chapter 4). I was interested in APC phosphorylation not only in the context of Wnt signaling but also in APCs cytoskeletal roles. One of the emerging themes in APCs role in regulating the actin cytoskeleton is its interaction with the formin Diaphanous (Dia). Previous work from our laboratory suggested that Drosophila APC2 and Dia cooperated during the formation of actin based structures during embryogenesis and this interaction was regulated. In order to understand this relationship further, I tested the role of phosphorylation as potential regulatory mechanism. My studies showed, in deed phosphorylation played a role in APCs activity in this context too (see Chapter 5). This study also revealed a potential cross talk between two pools of actin (linear and branched). In summary, studying APC, an exciting and highly complex protein, allowed me to think about many different biological questions from signaling to cytoskeleton in various developmental contexts. The findings from my Ph.D. research uncovered new aspects of APC biology, and showed how various regulatory mechanisms weather it’s phosphorylation or self-association, affect its functions, both during Wnt signaling and also in regulating the actin cytoskeleton. My studies will also help better understand the disease relevance of human APC proteins and provide novel insights.
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Priddle, Helen. "The use of gene disruption to study the cytoskeletal proteins talin and vinculin." Thesis, University of Leicester, 1996. http://hdl.handle.net/2381/35134.
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Talin and vinculin are cytoskeletal proteins co-localising with the integrin family of adhesion receptors at sites of adhesion to the extracellular matrix. To investigate the role of these proteins in cell adhesion, gene disruption by homologous recombination has been used in an attempt to produce cells deficient in talin or vinculin. A C129 mouse embryonic stem (ES) cell ? library was screened with a 5' mouse talin cDNA and talin genomic clones isolated. A promoterless neo gene was inserted into the genomic DNA within the first two coding exons, forming a talin gene disruption vector. This vector failed to confer G418 resistance to ES cells, possibly due to the use of a neo gene containing a mutation which reduces the phosphotransferase activity of the encoded protein. A further vector was constructed using the same section of talin genomic DNA with a wild type neo gene driven by a PGK promoter. A tk cassette was included for selection against random integration. This vector was used successfully to isolate ES cells with one allele of the talin gene disrupted. However, talin levels were not reduced in these cells. This cell line will be useful to produce talin deficient cells or mice for the study of talin function. A vinculin gene targeting vector containing a tk negative selection marker and a neo gene driven by a PGK promoter was used unsuccessfully to disrupt the vinculin gene in ES cells. An alternative vector was constructed with a promoterless neo to reduce random integration, but this approach was also unsuccessful. The original vector used the mutant version of neo. The use of a disruption vector employing a wild type neo cassette has since led to the disruption of the vinculin gene. Wistar Furth rats have a point mutation (pro 1176 thr) in the talin gene. The mutation was introduced into a talin cDNA encoding residues 565-1328. The mutant and wild type cDNAs were expressed as GST-fusion proteins. The vinculin- and actin- binding activities of the expressed fusion proteins were assayed in vitro. The mutation did not prevent the binding of the talin polypeptide to vinculin or actin and the mutant polypeptide was still able to localise to focal adhesions when microinjected into fibroblasts.
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Nicholson, Leigh. "A molecular study on cytoskeletal proteins in the uterus during pregnancy and cancer." Thesis, The University of Sydney, 2018. http://hdl.handle.net/2123/18820.
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The uterus has the unique ability to undergo morphological and functional changes in different biological contexts. Through the investigation of four cytoskeletal proteins, α-parvin, β-parvin, lasp-1 and palladin, during two separate events specific to the uterus, early pregnancy and endometrial carcinoma, this thesis aimed to contribute to both fields of study, whilst additionally determining comparisons and similarities in the molecular processes and proteins involved. Palladin and lasp-1 were both found to increase during the time of implantation, compared to the time of fertilisation. Lasp-1 exhibited an apical and lateral junctional localisation on both studied days of early pregnancy, however predominantly during fertilisation, indicating a potential role for the protein in the disruption of the actin terminal web and loss of adherens junctions seen during implantation. α- and β-parvin were found to play a reciprocal role during implantation, with α-parvin decreasing and β-parvin increasing during this time. Additionally, the study found that β-parvin significantly increased in the underlying stroma of the uterus during decidualisation, and that this increase was dependent on decidualisation occurring. In addition to the studies of α-parvin, β-parvin, lasp-1 and palladin in the uterus during early pregnancy, these four proteins were investigated in the glandular epithelial cells (GECs) of human endometrial cancer tissue to determine possible roles in the morphological changes seen during this event. Our results suggest a potential for the localisation of lasp-1 as an indicator for different aggressive characteristics. Phosphorylated α-parvin was additionally investigated and displayed a strong basal and apical location in low grade GECs, which was lost in type 2 serous GECs when the protein became localised to the nucleus. In combination, this thesis contributes to two different fields of study, whilst additionally elucidating similarities in the molecular processes of early pregnancy and endometrial tumorigenesis. Together this work highlights the importance of studying the role cytoskeletal proteins, α-parvin, β-parvin, lasp-1 and palladin, have in cellular changes involving morphological and proliferative transformations.
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Ramachandran, Preethi. "Cytoskeletal Regulation and Morphogen Signaling During Synaptic Outgrowth at the Drosophila Larval Neuromuscular Junction : A Dissertation." eScholarship@UMMS, 2009. https://escholarship.umassmed.edu/gsbs_diss/442.
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Synaptic plasticity, in its broadest sense, can be defined as the ability of synapses to be modified structurally and functionally in response to various internal and external factors. Growing evidence has established that at the very core of these modifications are alterations in the cytoskeletal architecture. This discovery has led to the unearthing of a number of signaling pathways that might be involved in cytoskeletal regulation and also in the regulation of other aspects of synapse development and plasticity. In this regard, polarity proteins and secreted morphogens such as the Wnt proteins, typically involved in embryonic development, are emerging as critical determinants of synaptic growth and plasticity. However, their mechanism of action at synapses needs further investigation. Additionally, not much is known about how these morphogens are secreted or transported across synapses. Using the Drosophila larval NMJ as a model system, I have addressed aspects related to the issues mentioned above in the subsequent body of work. In the first half of my thesis, I have uncovered a role for the aPKC/Baz/Par-6 polarity protein complex in the regulation of the postsynaptic actin cytoskeleton in conjunction with the lipid and protein phosphatase PTEN. In the second half of my thesis, I have contributed to the elucidation of mechanisms underlying the secretion of Wg, the Drosophila Wnt homolog. Our findings suggest that Wnts might be secreted via a previously unidentified mechanism involving the release of exosome like vesicles from the presynapse and this process requires Evi/Wntless (Evi), a protein dedicated to Wnt secretion. Alterations in signaling pathways and aberrant cytoskeletal regulation lead to a variety of neurological disorders. The body of work in this thesis will provide a deeper understanding of the mechanisms involved in synaptic plasticity and provide a basis for uncovering similar pathways in the context of vertebrate synapses.
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Ramusi, Matome Jack. "Computational studies of structural properties of both calciumoxide and calcium suphide." Thesis, University of Limpopo (Turfloop Campus), 2006. http://hdl.handle.net/10386/747.
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Thesis (M.Sc. (Physics)) --University of Limpopo, 2006In this work, we are studying the properties of CaS and CaO structures in both atomistic simulation and Density Functional Theory. Defects formation (vacancies, impurity and interstitial) will be mechanism studied by using atomistic simulation method. In this approach, Mott-Littleton method will be used since it is a good ap- proach of defects studies, and further explanation will be given on how the introduction of defects contribute on the stability of the bulk material. Diffusion of different atoms from one lattice site via interstitial path to vacancy lattice site, and how it segregates through the material, is also part of this study. The surface properties will be studied using both methods mentioned. Surface energies calculations of different surface layers (e.g. CaS (100), CaS (110), CaS (111), CaO (100), CaO (110) and CaO (111)) is the approach we used to determine the most stable surface. In atomistic simulation, we further studied how percentage coverage of atoms contributes on the stability of the surfaces. We further used Density Functional Theory to calculate surface energies of the above-mentioned surfaces. As in atomistic simulation method, we used surface ener- gies to determine the most stable surface. In DFT we used only the most stable surface of both CaS and CaO to study the adsorption of molecules, namely H2O, H2S, HS and S2 on CaO (100) and CaS (100). The most/least-adsorbed molecule on both surfaces is explained in this study.
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Sutton, Deborah Helen. "Analysis of the function of the cytoskeletal proteins vinculin and talin using gene disruption." Thesis, University of Leicester, 2001. http://hdl.handle.net/2381/29665.
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Talin, a 270 kDa cytoskeletal protein is thought to be a key player in the formation of focal adhesions. Talin (-/-) embryonic stem (ES) cells expressing no intact talin spread less efficiently on gelatin, laminin and fibronectin compared to wild-type ES cells, exhibited extensive membrane blebbing, showed a decrease in cell polarity, and were unable to form focal adhesions when planted on fibronectin. They also showed reduced adhesion to laminin (but not fibronectin), probably due to reduced expression of 1-integrins. The phenotypic characteristics of the talin (-/-) ES cells could be revealed to wild-type by expression of the full length mouse talin cDNA. Indeed, adhesion to laminin and fibronectin was greater than seen with the wild-type cells. These results provide strong support to the conclusion that talin is essential for integrin-mediated cell spreading and the assembly of focal adhesions. However, expression of a talin polypeptide lacking the C-terminal actin-binding site failed to rescue the mutant phenotype demonstrating the importance of this site in talin function. The talin (-/-) ES cells formed normal embryoid bodies, and when these were plated on gelatin, a population of spread cells slowly emerged from the central cell mass. Interestingly, these cells formed small vinculin and paxillin-containing focal adhesions, raising the possibility that talin is not essential for focal adhesion formation in all cell types. The talin-binding protein vinculin is also localised to focal adhesions. Although vinculin (-/-) null mouse embryo fibroblasts (MEFs) spread less efficiently on fibronectin, they were more motile compared to vinculin (+/-) MEFs.
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Gardiner, Fiona Claire. "Elucidating the biological role of actin cytoskeletal proteins in the budding yeast Saccharomyces cerevisiae." Thesis, University of Glasgow, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.418345.
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Vater, Ruth. "The fate of myofibrillar and cytoskeletal proteins during degeneration and regeneration of skeletal muscle." Thesis, University of Newcastle Upon Tyne, 1993. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.334746.
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Director, Laura Taylor. "A Novel Approach For The Identification of Cytoskeletal and Adhesion A-Kinase Anchoring Proteins." ScholarWorks @ UVM, 2014. http://scholarworks.uvm.edu/graddis/264.
Full textAbstract:
A-kinase anchoring proteins (AKAPs) are signaling scaffolds which provide spatial and temporal organization of signaling pathways in discrete subcellular compartments. Through tethering the cyclic-AMP dependent protein kinase A (PKA), AKAPs target PKA activity to distinct regions in the cell, bringing PKA in close proximity to its target proteins. This provides a high level of specificity and regulation of PKA and its role in mediating a number of biological processes, one of which is cell migration. Cell migration is a highly dynamic and fundamental process, when misregulated can lead to a number of pathologies. The process of cell migration requires integration and coordination of actin cytoskeletal dynamics, adhesion turnover, and contractility. The important role of PKA in regulating the cellular processes involved in cell migration has been extensively studied. Our lab has shown that PKA activity and spatial distribution through AKAPs are localized to the leading edge of migrating cells and are required for effective cell migration, yet the specific AKAPs responsible remain unknown.
Traditional methods for identifying AKAPs suffer from a number of limitations. Therefore the objective of the enclosed work is to establish and characterize a novel approach for the identification of cytoskeletal and adhesion-associated AKAPs. We show for the first time, an in vitro approach to identify cytoskeletal AKAPs which may be responsible for localizing PKA to the leading edge of migrating cells.
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Stubberfield, Lisa Marie. "Interactions of Plasmodium falciparum proteins at the membrane skeleton of infected erythrocytes." Monash University, Dept. of Microbiology, 2003. http://arrow.monash.edu.au/hdl/1959.1/9433.
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Osterloh, Jeannette M. "dSarm/Sarm1 Governs a Conserved Axon Death Program: A Dissertation." eScholarship@UMMS, 2013. https://escholarship.umassmed.edu/gsbs_diss/668.
Full textAbstract:
Axonal and synaptic degeneration is a hallmark of peripheral neuropathy, brain injury, and neurodegenerative disease. Axonal degeneration has been proposed to be mediated by an active autodestruction program, akin to apoptotic cell death; however, loss-of-function mutations capable of potently blocking axon self-destruction have not been described. Using a forward genetic screen in Drosophila, we identified that loss of the Toll receptor adaptor dSarm (sterile a/Armadillo/Toll-Interleukin receptor homology domain protein) cell-autonomously suppresses Wallerian degeneration for weeks after axotomy. Severed mouse Sarm1 null axons exhibit remarkable long-term survival both in vivo and in vitro, indicating that Sarm1 prodegenerative signaling is conserved in mammals. Our results provide direct evidence that axons actively promote their own destruction after injury and identify dSarm/Sarm1 as a member of an ancient axon death signaling pathway. This death signaling pathway can be activated without injury by loss of the N-terminal self-inhibitory domain, resulting in spontaneous neurodegeneration. To investigate the role of axon self-destruction in disease, we assessed the effects of Sarm1 loss on neurodegeneration in the SOD1-G93A model of amyotrophic lateral sclerosis (ALS), a lethal condition resulting in progressive motor neuron death and paralysis. Loss of Sarm1 potently protects motor axons and synapses from degeneration, but only extends animal survival by 10%. Thus, there appears to be at least two driving forces in place during ALS disease progression: (1) Sarm1 mediated axon death, and (2) cell body destruction via some unknown mechanism.
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Osterloh, Jeannette M. "dSarm/Sarm1 Governs a Conserved Axon Death Program: A Dissertation." eScholarship@UMMS, 2006. http://escholarship.umassmed.edu/gsbs_diss/668.
Full textAbstract:
Axonal and synaptic degeneration is a hallmark of peripheral neuropathy, brain injury, and neurodegenerative disease. Axonal degeneration has been proposed to be mediated by an active autodestruction program, akin to apoptotic cell death; however, loss-of-function mutations capable of potently blocking axon self-destruction have not been described. Using a forward genetic screen in Drosophila, we identified that loss of the Toll receptor adaptor dSarm (sterile a/Armadillo/Toll-Interleukin receptor homology domain protein) cell-autonomously suppresses Wallerian degeneration for weeks after axotomy. Severed mouse Sarm1 null axons exhibit remarkable long-term survival both in vivo and in vitro, indicating that Sarm1 prodegenerative signaling is conserved in mammals. Our results provide direct evidence that axons actively promote their own destruction after injury and identify dSarm/Sarm1 as a member of an ancient axon death signaling pathway. This death signaling pathway can be activated without injury by loss of the N-terminal self-inhibitory domain, resulting in spontaneous neurodegeneration. To investigate the role of axon self-destruction in disease, we assessed the effects of Sarm1 loss on neurodegeneration in the SOD1-G93A model of amyotrophic lateral sclerosis (ALS), a lethal condition resulting in progressive motor neuron death and paralysis. Loss of Sarm1 potently protects motor axons and synapses from degeneration, but only extends animal survival by 10%. Thus, there appears to be at least two driving forces in place during ALS disease progression: (1) Sarm1 mediated axon death, and (2) cell body destruction via some unknown mechanism.
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Herron, Lissa Rocha. "A study of the behaviour and interactions of the novel FERM protein Willin." Thesis, St Andrews, 2008. http://hdl.handle.net/10023/418.
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