UNC-18 and Tomosyn Antagonistically Control Synaptic Vesicle Priming Downstream of UNC-13 in Caenorhabditis elegans

Abstract

Munc18-1/UNC-18 is believed to prime SNARE-mediated membrane fusion, yet the underlying mechanisms remain enigmatic. Here, we examine how potential gain-of-function mutations of Munc18-1/UNC-18 affect locomotory behavior and synaptic transmission, and how Munc18-1-mediated priming is related to Munc13-1/UNC-13 and Tomosyn/TOM-1, positive and negative SNARE regulators, respectively. We show that a Munc18-1(P335A)/UNC-18(P334A) mutation leads to significantly increased locomotory activity and acetylcholine release in , as well as enhanced synaptic neurotransmission in cultured mammalian neurons. Importantly, similar to null mutants, () mutants partially bypass the requirement of UNC-13. Moreover, () and null mutations confer a strong synergy in suppressing the phenotypes of mutants. Through biochemical experiments, we demonstrate that Munc18-1(P335A) exhibits enhanced activity in SNARE complex formation as well as in binding to the preformed SNARE complex, and partially bypasses the Munc13-1 requirement in liposome fusion assays. Our results indicate that Munc18-1/UNC-18 primes vesicle fusion downstream of Munc13-1/UNC-13 by templating SNARE complex assembly and acts antagonistically with Tomosyn/TOM-1. At presynaptic sites, SNARE-mediated membrane fusion is tightly regulated by several key proteins including Munc18/UNC-18, Munc13/UNC-13, and Tomosyn/TOM-1. However, how these proteins interact with each other to achieve the precise regulation of neurotransmitter release remains largely unclear. Using as an model, we found that a gain-of-function mutant of UNC-18 increases locomotory activity and synaptic acetylcholine release, that it partially bypasses the requirement of UNC-13 for release, and that this bypass is synergistically augmented by the lack of TOM-1. We also elucidated the biochemical basis for the gain-of-function caused by this mutation. Thus, our study provides novel mechanistic insights into how Munc18/UNC-18 primes synaptic vesicle release and how this protein interacts functionally with Munc13/UNC-13 and Tomosyn/TOM-1.

Open syntaxin overcomes synaptic transmission defects in diverseC. elegansexocytosis mutants

“…We first examined whether the effects of open syntaxin and tom-1 on acetylcholine release and thrashing. As previously reported 15, 45 , tom-1 null exhibited slightly reduced motility and thrashing compared to N2 wild-type despite the increased acetylcholine release (Fig. 6a, b).…”

“…As we previously reported, the aldicarb sensitivity of unfurled unc-18(sks2) KI ; tom-1(ok285) ; unc-13(s69) reached N2 wild-type levels (Fig. 6f) 45 . These results suggest that open syntaxin and tom-1 null exhibit additive, not synergistic effects on the rescue of unc-13(s69) .…”

“…In this context, the tom-1 mutant that lacks Tomosyn, a protein that competes with synaptobrevin for SNARE complex formation 44 , partially rescued unc-13 phenotypes, and multicopy expression of the open syntaxin mutant further increased the rescue to a modest extent 15 . In contrast, we recently discovered an interesting synergism between a KI of an unc-18 mutant containing a point mutation (P334A) that is believed to unfurl a loop involved in synaptobrevin binding and the tom-1(ok285) null mutant in rescuing the unc-13(s69) mutant in synaptic vesicle exocytosis 45 . That is, although unc-18(P334A) KI or tom-1(ok285) had a limited rescuing effect in aldicarb sensitivity of the unc-13(s69) mutant, in combination they restored aldicarb sensitivity to the wild-type level.…”

“…The e81 allele contains a C1582T mutation in exon 9, resulting in a Q530Stop mutation and is thus likely null of the unc-18 gene 51 . Finally, the sks1 strain contains a large insertion containing the genes for selection markers GFP and neomycin that disrupts proper transcription of the unc-18 gene 45 .…”

Mechanism of neurotransmitter release coming into focus

“…(A)], in addition to being critical for release itself. It is interesting to note that a gain‐of‐function mutation that is believed to unfurl the loop in domain 3a of unc18 can partially rescue the severe phenotypes observed in unc13 nulls in C. elegans , which is reminiscent of the rescue of these nulls by the open syntaxin‐1 mutant . These findings show that, without the energy barriers caused by the closed conformation of syntaxin‐1 and the furled conformation of the Munc18‐1/unc18 loop, Munc13s/unc13 would not be essential for neurotransmitter release; in wild type animals, the presence of these energy barriers leads to the essential nature of Munc13s/unc13 and enables the multiple forms of regulation that depend on Munc13s/unc13.…”