Summary Background Synaptic transmission can occur in a binary or graded fashion depending on whether transmitter release is triggered by action potentials or by gradual changes in membrane potential. Molecular differences of these two types of fusion events and their differential regulation in a physiological context have yet to be addressed. Complexin is a conserved SNARE-binding protein that has been proposed to regulate both spontaneous and stimulus-evoked synaptic vesicle (SV) fusion. Results Here, we examine complexin function at a graded synapse in C. elegans. Null complexin (cpx-1) mutants are viable although nervous system function is significantly impaired. Loss of CPX-1 results in a 3-fold increase in the rate of tonic synaptic transmission at the neuromuscular junction while stimulus-evoked SV fusion is decreased 10-fold. A truncated CPX-1 missing its C-terminal domain can rescue stimulus-evoked synaptic vesicle exocytosis but fails to suppress tonic activity, demonstrating that these two modes of exocytosis can be distinguished at the molecular level. A CPX-1 variant with impaired SNARE-binding also rescues evoked but not tonic neurotransmitter release. Finally, tonic but not evoked release can be rescued in a syntaxin point mutant by removing CPX-1. Rescue of either form of exocytosis partially restores locomotory behavior indicating that both types of synaptic transmission are relevant. Conclusion These observations suggest a dual role for CPX-1: suppressing SV exocytosis driven by low levels of endogenous neural activity while promoting synchronous fusion of SVs driven by a depolarizing stimulus. Thus, patterns of synaptic activity regulate complexin's inhibitory and permissive roles at a graded synapse.
Summary Two models have been proposed for Endophilin function in synaptic vesicle (SV) endocytosis. The scaffolding model proposes that Endophilin’s SH3 domain recruits essential endocytic proteins whereas the membrane-bending model proposes that the BAR domain induces positively curved membranes. We show that mutations disrupting the scaffolding function do not impair endocytosis, while those disrupting membrane-bending cause significant defects. By anchoring Endophilin to the plasma membrane, we show that Endophilin acts prior to scission to promote endocytosis. Despite acting at the plasma membrane, the majority of Endophilin is targeted to the SV pool. Photoactivation studies suggest that the soluble pool of Endophilin at synapses is provided by unbinding from the adjacent SV pool and that the unbinding rate is regulated by exocytosis. Thus, Endophilin participates in an association-dissociation cycle with SVs that parallels the cycle of exo- and endocytosis. This Endophilin cycle may provide a mechanism for functionally coupling endocytosis and exocytosis.
Although C. elegans has been utilized extensively to study synapse formation and function, relatively little is known about synaptic plasticity in C. elegans. We show that a brief treatment with the cholinesterase inhibitor aldicarb induces a form of presynaptic potentiation whereby ACh release at neuromuscular junctions (NMJs) is doubled. Aldicarb-induced potentiation was eliminated by mutations that block processing of pro-neuropeptides, by mutations inactivating a single pro-neuropeptide (NLP-12), and by those inactivating an NLP-12 receptor (CKR-2). NLP-12 expression is limited to a single stretch-activated neuron, DVA. Analysis of a YFP-tagged NLP-12 suggests that aldicarb stimulates DVA secretion of NLP-12. Mutations disrupting the DVA mechanoreceptor (TRP-4) decreased aldicarb-induced NLP-12 secretion and blocked aldicarb-induced synaptic potentiation. Mutants lacking NLP-12 or CKR-2 have decreased locomotion rates. Collectively, these results suggest that NLP-12 mediates a mechanosensory feedback loop that couples muscle contraction to changes in presynaptic release, thereby providing a mechanism for proprioceptive control of locomotion.
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