Analysis of Caenorhabditis elegans tomosyn.

摘要

Tomosyn is a syntaxin-binding protein proposed to negatively regulate synaptic vesicle priming. Here we show mutants of C. elegans tomosyn (TOM-1) exhibit prolonged evoked release associated with an increase in morphologically docked synaptic vesicles at the ultrastructural level. Our analyses show that docked vesicles largely represent UNC-13-dependent primed vesicles. This exuberant priming can be rescued by expressing full-length TOM-1 in motor neurons. These data indicate that in the intact nervous system, TOM-1 negatively regulates SV priming.;We also examined the role of tomosyn in peptide secretion in C. elegans. Ultrastructural analysis of tom-1 mutants revealed a 50% reduction in presynaptic dense-core vesicles (DCVs) corresponding to enhanced neuropeptide release. Conversely, overexpression of TOM-1 led to an accumulation of DCVs. Together, these data provide the first in vivo evidence that TOM-1 negatively regulates DCV exocytosis.;The molecular mechanism by which tomosyn inhibits SV and DCV exocytosis has yet to be fully elucidated. To directly test the role of the SNARE motif and establish the functional significance of the WD40 repeats that are highly-conserved we took the following approaches. (1) we examined mutants with in-frame deletions in these domains of C. elegans tom-1 and (2) we expressed truncated TOM-1 proteins in the tom-1 mutant background. Our results show that the ability of tomosyn to regulate release in C. elegans is dependent on the physical integrity of the TOM-1 protein; mutants lacking either domain were non-functional and neither the SNARE domain alone nor the entire protein without the SNARE domain rescued function.;To identify potential binding partners of the tomosyn WD40 repeat domain, we used the yeast-two hybrid approach. 3 independent clones of the same protein, VPS39/VAM6, were obtained. We began an initial characterization of the C. elegans vps-39 gene and showed that vps-39 deletion decreases cholinergic release at the NMJ. In the double mutant with tom-1 aldicarb hypersensitivity is partially suppressed, therefore our initial conclusion is that VPS-39 may work antagonistically with tomosyn by promoting release.;Together this research creates a more complete picture of tomosyn function in exocytosis and raises new questions for future exploration.