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.
| Identifer | oai:union.ndltd.org:TORONTO/oai:tspace.library.utoronto.ca:1807/43617 |
| Date | 10 January 2014 |
| Creators | Kisiel, Marta |
| Contributors | Stewart, Bryan |
| Source Sets | University of Toronto |
| Language | en_ca |
| Detected Language | English |
| Type | Thesis, Video |
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