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Analysis of Myosin VI in Drosophila melanogaster Synaptic Function and DevelopmentKisiel, Marta 10 January 2014 (has links)
Myosin VI, encoded by jaguar (jar) in Drosophila melanogaster, is the only member of the myosin superfamily of actin-based motor proteins known to move towards the minus ends of actin filaments. In vitro studies demonstrate that Myosin VI has the ability to perform distinct functions as a cargo transporter and anchor in the cell, however which of these roles Myosin VI plays in the nervous system has yet to be determined. A locomotor defect, observed as sluggish movement in severe jar mutant larvae, was confirmed by behavioural assays. As this can indicate problems at the neuromuscular synapse, microscopy and electrophysiology were used to investigate neuromuscular junction (NMJ) structure and function in jar loss of function mutants of varying severity. Confocal imaging studies revealed a decrease in NMJ length, a reduction in bouton number per NMJ, alterations to the microtubule cytoskeleton and mislocalization of the synaptic vesicle protein Synaptotagmin in jar mutant boutons. FM dye labeling was consistent with the immunostaining data revealing vesicles endocytosed following electrical stimulation occupy the bouton centre in jar mutants. The data is indicative of a function for Myosin VI in maintaining proper peripheral vesicle localization. Electrophysiological experiments revealed a role for Myosin VI in basal synaptic transmission, with a reduction in low frequency nerve-evoked responses and spontaneous release in severe jar mutants. Changes in short-term synaptic plasticity were also observed in Myosin VI mutants by using both paired-pulse experiments to examine release probability and high-frequency stimulation paradigms to recruit vesicles from different functional pools. Taken together, the data suggest that Myosin VI functions as an anchor to peripherally localize vesicles within the bouton enabling their efficient release during nerve stimulation. Synaptic vesicles are mobile at the Drosophila NMJ; thus if Myosin VI is acting as a vesicle tether, it would normally be expected to restrain vesicle mobility at the synapse. FRAP analysis revealed a significant increase in synaptic vesicle mobility in jar mutant boutons. This study elucidates novel roles for Myosin VI function in the nervous system via regulation of the synaptic microtubule architecture and localization of synaptic vesicles within the nerve terminal.
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Analysis of Myosin VI in Drosophila melanogaster Synaptic Function and DevelopmentKisiel, Marta 10 January 2014 (has links)
Myosin VI, encoded by jaguar (jar) in Drosophila melanogaster, is the only member of the myosin superfamily of actin-based motor proteins known to move towards the minus ends of actin filaments. In vitro studies demonstrate that Myosin VI has the ability to perform distinct functions as a cargo transporter and anchor in the cell, however which of these roles Myosin VI plays in the nervous system has yet to be determined. A locomotor defect, observed as sluggish movement in severe jar mutant larvae, was confirmed by behavioural assays. As this can indicate problems at the neuromuscular synapse, microscopy and electrophysiology were used to investigate neuromuscular junction (NMJ) structure and function in jar loss of function mutants of varying severity. Confocal imaging studies revealed a decrease in NMJ length, a reduction in bouton number per NMJ, alterations to the microtubule cytoskeleton and mislocalization of the synaptic vesicle protein Synaptotagmin in jar mutant boutons. FM dye labeling was consistent with the immunostaining data revealing vesicles endocytosed following electrical stimulation occupy the bouton centre in jar mutants. The data is indicative of a function for Myosin VI in maintaining proper peripheral vesicle localization. Electrophysiological experiments revealed a role for Myosin VI in basal synaptic transmission, with a reduction in low frequency nerve-evoked responses and spontaneous release in severe jar mutants. Changes in short-term synaptic plasticity were also observed in Myosin VI mutants by using both paired-pulse experiments to examine release probability and high-frequency stimulation paradigms to recruit vesicles from different functional pools. Taken together, the data suggest that Myosin VI functions as an anchor to peripherally localize vesicles within the bouton enabling their efficient release during nerve stimulation. Synaptic vesicles are mobile at the Drosophila NMJ; thus if Myosin VI is acting as a vesicle tether, it would normally be expected to restrain vesicle mobility at the synapse. FRAP analysis revealed a significant increase in synaptic vesicle mobility in jar mutant boutons. This study elucidates novel roles for Myosin VI function in the nervous system via regulation of the synaptic microtubule architecture and localization of synaptic vesicles within the nerve terminal.
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Characterising the interaction between metastasis-associated protein S100A4 and non-muscle myosin IIA in vitro and in vivoIrvine, Andrew Francis January 2012 (has links)
S100A4 is a member of the S100 family of proteins and increases the motility of many cell types. This is also thought to explain its association with the epithelial-mesenchymal transition (EMT), a developmental program re-activated during tumourigenesis. Mechanistically, S100A4 interacts with a number of targets including Smad3 and liprin-β1; however, the best characterised is non-muscle myosin IIA (NMIIA) which regulates many aspects of the cytoskeleton. There is a large body of in vitro data indicating that S100A4 promotes the monomeric state of NMIIA; however, in vivo evidence for the interaction in cells is lacking. Accordingly, the first aim of this study was to determine if S100A4 interacts with, and promotes the monomeric state of NMIIA in A431 cells undergoing SIP1-induced EMT. Intriguingly, co-localisation analysis of S100A4 and NMIIA in A431-SIP1 cells using immunoelectron microscopy indicated that NMIIA is present in a folded, 10S state, and unfolded 6S state, and S100A4 interacts with both. This represents the first evidence of 10S and 6S states of NMIIA in non-muscle cells. In addition, FRAP analysis demonstrated that cells with attenuated expression of S100A4 turned over NMIIA with a slower rate, consistent with S100A4 promoting the monomeric state. The second part of the study explored the mechanism of the S100A4-NMMIA interaction. In vitro analysis of phosphomimetic S1916D and S1943D NMIIA showed no differences in binding affinity with S100A4 compared to WT NMIIA, contrary to the published literature. Based on the NMR structure of S100A4 and NMIIA, V77 and C81 were identified as key S100A4 residues that mediated the interaction with NMIIA. Mutation of these sites abolished the interaction with NMIIA, an effect reflected in null-phenotypes for both proteins when over-expressed in A431 cells compared to WT S100A4. In conclusion, this study suggests S100A4 is an important regulator of NMIIA dynamics in cells.
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The role of the sarcoplasmic reticulum in the regulation of [Ca'2'+]â†i in rat gastric myocytesWhite, Carl January 1999 (has links)
No description available.
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Structural aspects of an inhibited smooth muscle myosin conformerSalzameda, Bridget. January 2006 (has links)
Thesis (Ph. D.)--University of Nevada, Reno, 2006. / "August, 2006." Includes bibliographical references (leaves 102-110). Online version available on the World Wide Web.
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Regulation of adiponectin secretion by endothelin-I and myosin IIBedi, Deepa, January 2007 (has links) (PDF)
Thesis (Ph.D.)--Auburn University, 2007. / Abstract. Vita. Includes bibliographic references (ℓ. 104-133)
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Investigation of the 10S-filament equilibrium in smooth muscle myosinSchneck, Amy N. January 2007 (has links)
Thesis (M.S.)--University of Nevada, Reno, 2007. / "May, 2007." Includes bibliographical references (leaf 40). Online version available on the World Wide Web.
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Biased sampling methods for free energy computation with molecular dynamics simulationsHarris, Michael James. January 2008 (has links)
Thesis (M.S.)--University of Nevada, Reno, 2008. / "May, 2008." Includes bibliographical references (leaves 50-56). Online version available on the World Wide Web.
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Komponenten des Cytoskeletts der einzelligen Grünalge Chlamydomonas reinhardtii : molekulare Analyse zweier Myosine und eines neuen Mikrotubuli-assoziierten Proteins /Pfannenschmid, Frank. January 2001 (has links) (PDF)
Univ., Diss.--Regensburg, 2001.
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Effect of mechanical loading and ageing on myosin heavy chain turnover rate in fast-twitch skeletal muscle /Pehme, Ando. January 1900 (has links) (PDF)
Thesis (Ph. D.)--Tartu University, 2003. / Includes bibliography, p. 50-56.
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