Calcium (Ca2+)-evoked release of neurotransmitters from synaptic vesicles requires mechanisms both to prevent un-initiated fusion of vesicles (clamping) and to trigger fusion following Ca2+-influx. The principal components involved in these processes are the vesicular fusion machinery (SNARE proteins) and the regulatory proteins, Synaptotagmin-1 and Complexin. Here, we use a reconstituted single-vesicle fusion assay under physiologically-relevant conditions to delineate a novel mechanism by which Synaptotagmin-1 and Complexin act synergistically to establish Ca2+-regulated fusion. We find that under each vesicle, Synaptotagmin-1 oligomers bind and clamp a limited number of ‘central’ SNARE complexes via the primary interface and introduce a kinetic delay in vesicle fusion mediated by the excess of free SNAREpins. This in turn enables Complexin to arrest the remaining free ‘peripheral’ SNAREpins to produce a stably clamped vesicle. Activation of the central SNAREpins associated with Synaptotagmin-1 by Ca2+ is sufficient to trigger rapid (<100 msec) and synchronous fusion of the docked vesicles.

The buttressed‐ring hypothesis, supported by recent cryo‐electron tomography analysis of docked synaptic‐like vesicles in neuroendocrine cells, postulates that prefusion SNARE pins are stabilized and organized by Synaptotagmin (Syt) ring‐like oligomers. Here, we use a reconstituted single‐vesicle fusion analysis to test the prediction that destabilizing the Syt1 oligomers destabilizes the clamp and results in spontaneous fusion in the absence of Ca 2+ . Vesicles in which Syt oligomerization is compromised by a ring‐destabilizing mutation dock and diffuse freely on the bilayer until they fuse spontaneously, similar to vesicles containing only v‐ SNARE s. In contrast, vesicles containing wild‐type Syt are immobile as soon as they attach to the bilayer and remain frozen in place, up to at least 1 h until fusion is triggered by Ca 2+ .

SummaryMutations in proline-rich transmembrane protein 2 (PRRT2) are associated with a range of paroxysmal neurological disorders. PRRT2 predominantly localizes to the pre-synaptic terminals and is believed to regulate neurotransmitter release. However, the mechanism of action is unclear. Here, we use reconstituted single vesicle and bulk fusion assays, combined with live cell imaging of single exocytotic events in PC12 cells and biophysical analysis, to delineate the physiological role of PRRT2. We report that PRRT2 selectively blocks the trans SNARE complex assembly and thus negatively regulates synaptic vesicle priming. This inhibition is actualized via weak interactions of the N-terminal proline-rich domain with the synaptic SNARE proteins. Furthermore, we demonstrate that paroxysmal dyskinesia-associated mutations in PRRT2 disrupt this SNARE-modulatory function and with efficiencies corresponding to the severity of the disease phenotype. Our findings provide insights into the molecular mechanisms through which loss-of-function mutations in PRRT2 result in paroxysmal neurological disorders.

Experimental setups to produce and to monitor model membranes have been successfully used for decades and brought invaluable insights into many areas of biology. However, they all have limitations that prevent the full in vitro mimicking and monitoring of most biological processes. Here, a suspended physiological bilayer‐forming chip is designed from 3D‐printing techniques. This chip can be simultaneously integrated to a confocal microscope and a path‐clamp amplifier. It is composed of poly(dimethylsiloxane) and consists of a ≈100 µm hole, where the horizontal planar bilayer is formed, connecting two open crossed‐channels, which allows for altering of each lipid monolayer separately. The bilayer, formed by the zipping of two lipid leaflets, is free‐standing, horizontal, stable, fluid, solvent‐free, and flat with the 14 types of physiologically relevant lipids, and the bilayer formation process is highly reproducible. Because of the two channels, asymmetric bilayers can be formed by making the two lipid leaflets of different composition. Furthermore, proteins, such as transmembrane, peripheral, and pore‐forming proteins, can be added to the bilayer in controlled orientation and keep their native mobility and activity. These features allow in vitro recapitulation of membrane process close to physiological conditions.

In vivo membrane fusion primarily occurs between highly curved vesicles and planar membranes. A better understanding of fusion entails an accurate in vitro reproduction of the process. To date, supported bilayers have been commonly used to mimic the planar membranes. Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins that induce membrane fusion usually have limited fluidity when embedded in supported bilayers. This alters the kinetics and prevents correct reconstitution of the overall fusion process. Also, observing content release across the membrane is hindered by the lack of a second aqueous compartment. Recently, a step toward resolving these issues was achieved by using membranes spread on holey substrates. The mobility of proteins was preserved but vesicles were prone to bind to the substrate when reaching the edge of the hole, preventing the observation of many fusion events over the suspended membrane. Building on this recent advance, we designed a method for the formation of pore-spanning lipid bilayers containing t-SNARE proteins on Si/SiO holey chips, allowing the observation of many individual vesicle fusion events by both lipid mixing and content release. With this setup, proteins embedded in the suspended membrane bounced back when they reached the edge of the hole which ensured vesicles did not bind to the substrate. We observed SNARE-dependent membrane fusion with the freestanding bilayer of about 500 vesicles. The time between vesicle docking and fusion is ∼1 s. We also present a new multimodal open-source software, Fusion Analyzer Software, which is required for fast data analysis.

Previously we reported that Synaptotagmin-1 and Complexin synergistically clamp the SNARE assembly process to generate and maintain a pool of docked vesicles that fuse rapidly and synchronously upon Ca2+ influx (Ramakrishnan et al., 2020). Here, using the same in vitro single-vesicle fusion assay, we determine the molecular details of the Complexin-mediated fusion clamp and its role in Ca2+-activation. We find that a delay in fusion kinetics, likely imparted by Synaptotagmin-1, is needed for Complexin to block fusion. Systematic truncation/mutational analyses reveal that continuous alpha-helical accessory-central domains of Complexin are essential for its inhibitory function and specific interaction of the accessory helix with the SNAREpins enhances this functionality. The C-terminal domain promotes clamping by locally elevating Complexin concentration through interactions with the membrane. Independent of their clamping functions, the accessory-central helical domains of Complexin also contribute to rapid Ca2+-synchronized vesicle release by increasing the probability of fusion from the clamped state.

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Despite extensive recent research efforts on material-specific peptides, the fundamental problem to be explored yet is the molecular interactions between peptides and inorganic surfaces. Here we used computer simulations (density functional theory and classical molecular dynamics) to investigate the adsorption mechanism of silicon-binding peptides and the role of individual amino acids in the affinity of peptides for an n-type silicon (n(+)-Si) semiconductor. Three silicon binding 12-mer peptides previously elaborated using phage display technology have been studied. The peptides' conformations close to the surface have been determined and the best-binding amino acids have been identified. Adsorption energy calculations explain the experimentally observed different degrees of affinity of the peptides for n(+)-Si. Our residual scanning analysis demonstrates that the binding affinity relies on both the identity of the amino acid and its location in the peptide sequence.