Abstract
Neurotransmitter release requires SNARE complexes to bring membranes together, NSF-SNAPs to recycle the SNAREs, Munc18-1 and Munc13s to orchestrate SNARE complex assembly, and Synaptotagmin-1 to trigger fast Ca(2+)-dependent membrane fusion. However, it is unclear whether Munc13s function upstream and/or downstream of SNARE complex assembly, and how the actions of their multiple domains are integrated. Reconstitution, liposome-clustering and electrophysiological experiments now reveal a functional synergy between the C1, C2B and C2C domains of Munc13-1, indicating that these domains help bridging the vesicle and plasma membranes to facilitate stimulation of SNARE complex assembly by the Munc13-1 MUN domain. Our reconstitution data also suggest that Munc18-1, Munc13-1, NSF, αSNAP and the SNAREs are critical to form a 'primed' state that does not fuse but is ready for fast fusion upon Ca(2+) influx. Overall, our results support a model whereby the multiple domains of Munc13s cooperate to coordinate synaptic vesicle docking, priming and fusion.
Highlights
The release of neurotransmitters by Ca2+-evoked synaptic vesicle exocytosis is a key event for communication between neurons and involves several steps, including vesicle docking at presynaptic active zones, a priming reaction(s) that leaves the vesicles ready for release, and fast Ca2+-triggered fusion of the vesicle and plasma membranes (Sudhof, 2013)
Studies of the complex machinery that controls release have shown that eight proteins are important and have established defined roles for them (Rizo and Xu, 2015; Jahn and Fasshauer, 2012; Brunger et al, 2015; Sudhof and Rothman, 2009): i) the soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) syntaxin-1, SNAP-25 and synaptobrevin bring the membranes together by forming a four-helix bundle called SNARE complex (Sollner et al, 1993; Poirier et al, 1998; Sutton et al, 1998), which is critical for membrane fusion (Hanson et al, 1997); ii) N-ethylmaleimide sensitive factor (NSF) and soluble NSF attachment proteins (SNAPs; no relation to SNAP-25) disassemble the SNARE complex (Sollner et al, 1993) to recycle the SNAREs (Mayer et al, 1996; Banerjee et al, 1996); iii) Munc18-1 and Munc13s orchestrate SNARE complex
In experiments that followed our recent reconstitution study (Ma et al, 2013) and were directed at analyzing how different factors affect the efficiency of membrane fusion, we first analyzed lipid mixing between synaptobrevin-liposomes (V-liposomes) and syntaxin-1-SNAP-25 liposomes (T-liposomes) by monitoring de-quenching of the fluorescence of NBD-labeled lipids incorporated in the synaptobrevin-liposomes (Weber et al, 1998) (Figure 1)
Summary
The release of neurotransmitters by Ca2+-evoked synaptic vesicle exocytosis is a key event for communication between neurons and involves several steps, including vesicle docking at presynaptic active zones, a priming reaction(s) that leaves the vesicles ready for release, and fast Ca2+-triggered fusion of the vesicle and plasma membranes (Sudhof, 2013). Biophysics and structural biology Neuroscience eLife digest In the brain, neurons communicate with each other using small molecules called neurotransmitters. Electrical signals in one neuron trigger the release of the neurotransmitters, which bind to receptor proteins on another neuron nearby. Neurotransmitters are packaged into small compartments called synaptic vesicles and are released from the neuron when these vesicles fuse with the membrane that surrounds the cell. Many proteins are involved in regulating this process to ensure that neurotransmitters are released at the right place and time
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