Abstract
BackgroundAt the Drosophila neuromuscular junction (NMJ), synaptic vesicles are mobile; however, the mechanisms that regulate vesicle traffic at the nerve terminal are not fully understood. Myosin VI has been shown to be important for proper synaptic physiology and morphology at the NMJ, likely by functioning as a vesicle tether. Here we investigate vesicle dynamics in Myosin VI mutants of Drosophila.ResultsIn Drosophila, Myosin VI is encoded by the gene, jaguar (jar). To visualize active vesicle cycling we used FM dye loading and compared loss of function alleles of jar with controls. These studies revealed a differential distribution of vesicles at the jar mutant nerve terminal, with the newly endocytosed vesicles observed throughout the mutant boutons in contrast to the peripheral localization visualized at control NMJs. This finding is consistent with a role for Myosin VI in restraining vesicle mobility at the synapse to ensure proper localization. To further investigate regulation of vesicle dynamics by Myosin VI, FRAP analysis was used to analyze movement of GFP-labeled synaptic vesicles within individual boutons. FRAP revealed that synaptic vesicles are moving more freely in the jar mutant boutons, indicated by changes in initial bleach depth and rapid recovery of fluorescence following photobleaching.ConclusionThis data provides insights into the role for Myosin VI in mediating synaptic vesicle dynamics at the nerve terminal. We observed mislocalization of actively cycling vesicles and an apparent increase in vesicle mobility when Myosin VI levels are reduced. These observations support the notion that a major function of Myosin VI in the nerve terminal is tethering synaptic vesicles to proper sub-cellular location within the bouton.
Highlights
At the Drosophila neuromuscular junction (NMJ), synaptic vesicles are mobile; the mechanisms that regulate vesicle traffic at the nerve terminal are not fully understood
New synaptic vesicles are known to be transported to the nerve terminal from the cell body along microtubule tracks, less is known about the regulation of vesicle traffic within the nerve terminal itself
By way of fluorescence recovery after photobleaching (FRAP) analysis, that a reduction in Myosin VI expression corresponds to an increase in synaptic vesicle mobility
Summary
At the Drosophila neuromuscular junction (NMJ), synaptic vesicles are mobile; the mechanisms that regulate vesicle traffic at the nerve terminal are not fully understood. It was previously thought that upon delivery to the nerve terminal, synaptic vesicles remained relatively static until they were mobilized for neurotransmitter release [1,2]. This brief period of free mobility was attributed to disassembly of the actin cytoskeleton, which was thought to otherwise cage the vesicles when the synapse was at rest [3]. Synaptic vesicles at goldfish ribbon synapses were shown to be highly mobile and this mobility is not related to changes in calcium concentration or the actin cytoskeleton [5]
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