Tomosyn negatively regulates both synaptic transmitter and neuropeptide release at the C. elegans neuromuscular junction

Abstract

The SNARE proteins, syntaxin, SNAP-25 and synaptobrevin form a tertiary complex essential for vesicle fusion. Proteins that influence SNARE complex assembly are therefore likely to be important regulators of fusion events. In this study we have focused on tomosyn, a highly conserved, neuronally enriched, syntaxin-binding protein that has been implicated in the regulation of vesicle exocytosis. To directly test the role of tomosyn in neurosecretion we analysed loss-of-function mutants in the single Caenorhabditis elegans tomosyn gene, tom-1. These mutants exhibit enhanced synaptic transmission based on electrophysiological analysis of neuromuscular junction activity. This phenotype is the result of increased synaptic vesicle priming. In addition, we present evidence that tom-1 mutants also exhibit enhanced peptide release from dense core vesicles. These results indicate that tomosyn negatively regulates secretion for both vesicle types, possibly through a common mechanism, interfering with SNARE complex formation, thereby inhibiting vesicle fusion.

Figure 1. TOM-1 negatively regulates UNC-13-dependent synaptic vesicle priming

A, evoked postsynaptic responses at the neuromuscular junction of C. elegans demonstrate the opposing phenotypes of unc-13 and tom-1 mutants. The complete loss of evoked responses in unc-13 mutants, due to a loss of vesicle priming, results in a reduction in morphologically docked synaptic vesicles in EM synaptic profiles (green asterisk, presynaptic density of motor neuron terminal; red arrows, membrane-contacting synaptic vesicles; SV, synaptic vesicle; DCV, dense-core vesicle). tom-1 mutants have prolonged evoked responses due to increased vesicle priming, seen by EM as increased numbers of docked vesicles. B, the evoked defect of unc-13 mutants is partially by-passed in tom-1–unc13 double mutants. C, the primed vesicle pool assessed by applying hyperosmotic saline that is absent in unc-13 mutants is partially restored in tom1–unc-13 double mutants. Data in A–C are adapted from Gacheva et al. (2006). D, model: UNC-13 binding to the N-terminal of syntaxin promotes the availability of the syntaxin SNARE domain for assembly with SNAP-25 and synaptobrevin during vesicle priming, the formation of the SNARE complex bringing the vesicle into close apposition with the plasma membrane (morphologically docked). The C-terminal of TOM-1 shown in the model, interacts with syntaxin, inhibiting SNARE complex assembly by competing with synaptobrevin. Additional predicted interactions between the N-terminal of TOM-1 (not shown) and SNAP-25 may also contribute to the inhibitory actions of TOM-1.