Synaptic vesicle exocytosis is a highly specialized vesicle trafficking process in which calcium triggers fusion of synaptic vesicles with the plasma membrane, resulting in neurotransmitter release. SNARE complex assembly between synaptobrevin, SNAP-25 and syntaxin is a critical requirement preceding this vesicle fusion event. Several SNARE-interacting proteins have been shown to profoundly influence the strength of synaptic transmission, through their regulatory effects on the SNARE complex. Recently, a new SNARE binding partner, tomosyn was isolated from rat brain cytosol. Tomosyn has a SNARE binding domain that can compete with synaptobrevin for assembly into a tomosyn SNARE complex with syntaxin and SNAP-25. Based on these biochemical observations as well as tomosyn overexpression data, tomosyn is proposed to regulate vesicle release through an undefined mechanism. There are presently no loss-of-functions mutants available in any organism other than C. elegans. Therefore, we intend to examine the mechanism of tomosyn action at synapses in this powerful genetic model organism.
Aim 1) Characterize the synaptic phenotype of tom-1 deletion mutants. We have obtained two tom-1 deletion mutants that have phenotypes consistent with increased synaptic transmission. We will conduct a detailed characterization of these tom-1 mutants including behavioral, cytoarchitectural, pharmacological, electrophysiological and ultrastructural analyses.
Aim 2) Determine which TOM-1 isoforms regulate synaptic transmission. C. elegans tom-1 encodes three isoforms. The isoform expression patterns will be ascertained and mosaic analysis and tissue specific rescue experiments will be performed.
Aim 3) Genetic analysis of TOM-1 function. We hypothesize that tomosyn regulates the priming step of exocytosis. To test this model we will generate and characterize double mutants between tom-1 and several mutants known to affect the vesicle primed pool (unc-13, unc-10, open-syntaxin and unc-18).
Aim 4) Identify TOM-1 domains required for the regulation of synaptic transmission. TOM-1 protein domains essential for the regulation of exocytosis will be identified using a genetic screen for mutants that fail to complement the tom-1 mutation. These experiments are likely to further our understanding of neurotransmission, a foundation that may contribute to our understanding of neurological diseases and vesicle trafficking disorders.
Information flow within nervous systems occurs via specialized cell-cell contacts called 'synapses'. The mechanisms controlling information flow through synapses are incompletely understood. Here we propose supplemental work to study a protein called 'tomosyn', which our previous work, as well as work from other labs, has shown is an important regulator of synapse function. Specifically, our supplemental work will extend our previously funded work from worms into another genetic model organism, the fruitfly (Drosophila melanogaster), where we can probe the mechanism by which tomosyn works in more detail, and explore whether tomosyn plays a role in learning. A molecular understanding of tomosyn could contribute to treatments for a variety of neurological and learning disorders.
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