Docking and fusion of synaptic vesicles in cell-free model system of exocytosis

The present study involves the testing and characterization of synaptic vesicle (SV) docking and fusion as the steps of exocytosis using two different approaches in vitro.The interaction of SVs was determined by the changing of particles size in suspensions by the method of dynamic light scattering (DLS). Fluorescence assay is represented for studying the mechanism of SV membrane fusion. The sizes of membrane particles were shown to increase in the medium containing cytoplasmic proteins of synaptosomes. Therefore, the cytosolic proteins are suggested to promote the SVs into close proximity where they may become stably bound or docked. The specific effect of synaptosomal cytosolic proteins on the interaction of SVs in the cell-free system was demonstrated. The incubation of SVs with liver cytosol proteins or in the bovine serum albumin solution did not lead to the enlargement of the particles size. The fusion reaction of the SVs membranes occurred within the micromolar range of Ca²⁺ concentrations. Our studies have shown that in vitro process of exocytosis can be divided into Ca²⁺-independent step, termed docking and followed by fusion step that is triggered by Ca²⁺. The role of cytosolic proteins of synaptosomes in docking and fusion of SVs in cell-free system was further confirmed.     

Effects of Antiepileptic Agents on Homotypic Fusion of Synaptic Vesicles

We studied the effects of three antiepileptic drugs (AEDs) in a cell-free model system containing isolated synaptic vesicles (SVs) and cytosolic proteins, which allowed us to reproduce one of the stages of complex exocytosis. Ethosuximide, sodium valproate, and gabapentin intensified calcium- and Mg²⁺-ATP-induced fusion of SVs; the effect was indicative of the ability of these agents to influence the processes of simple and/or complex exocytosis in synaptic connections in the CNS structures. Antiepileptic drugs did not change the intensity of calcium-dependent fusion of liposomes and SVs treated by proteases. Therefore, the effect of AEDs can be realized via their interaction with proteins of SVs. After decrease in the level of cholesterol in the membranes of SVs using treatment by methyl-β- cyclodextrin, the ability of AEDs to activate fusion of SVs remained unchanged. Therefore, the studied AEDs act via proteins localized beyond the borders of cholesterol-enriched microdomains of the membrane. Drugs that induce convulsions (corazole and picrotoxin) did not change the characteristics of fusion of SVs under the in vitro action of AEDs. This is indicative of the absence of molecular targets for the above chemoconvulsants in the SV membranes, as compared with those in the plasma membranes of nerve terminals. According to our experiments, just proteins of SVs are functional targets for ethosuximide, sodium valproate, and gabapentin providing their anticonvulsant actions. The proposed model, which allows one to reproduce the membrane fusion, can be successfully used for the testing of drugs influencing a presynaptic link of synaptic contacts in the CNS.     

Synaptopathy under conditions of altered gravity: Changes in synaptic vesicle fusion and glutamate release

Glutamate release and synaptic vesicle heterotypic/homotypic fusion were characterized in brain synaptosomes of rats exposed to hypergravity (10 G, 1 h). Stimulated vesicular exocytosis determined as KCl-evoked fluorescence spike of pH-sensitive dye acridine orange (AO) was decreased twice in synaptosomes under hypergravity conditions as compared to control. Sets of measurements demonstrated reduced ability of synaptic vesicles to accumulate AO (∼10% higher steady-state baseline level of AO fluorescence). Experiments with preloaded l-[¹⁴C]glutamate exhibited similar amount of total glutamate accumulated by synaptosomes, equal concentration of ambient glutamate, but the enlarged level of cytoplasmic glutamate measuring as leakage from digitonin-permeabilized synaptosomes in hypergravity. Thus, it may be suggested that +G-induced changes in stimulated vesicular exocytosis were a result of the redistribution of intracellular pool of glutamate, i.e. a decrease in glutamate content of synaptic vesicles and an enrichment of the cytoplasmic glutamate level. To investigate the effect of hypergravity on the last step of exocytosis, i.e. membrane fusion, a cell-free system consisted of synaptic vesicles, plasma membrane vesicles, cytosolic proteins isolated from rat brain synaptosomes was used. It was found that hypergravity reduced the fusion competence of synaptic vesicles and plasma membrane vesicles, whereas synaptosomal cytosolic proteins became more active to promote membrane fusion. The total rate of homo- and heterotypic fusion reaction initiated by Ca²⁺ or Mg²⁺/ATP remained unchanged under hypergravity conditions. Thus, hypergravity could induce synaptopathy that was associated with incomplete filling of synaptic vesicles with the neuromediator and changes in exocytotic release.     

Synaptopathy under conditions of altered gravity: Changes in synaptic vesicle fusion and glutamate release

Glutamate release and synaptic vesicle heterotypic/homotypic fusion were characterized in brain synaptosomes of rats exposed to hypergravity (10 G, 1 h). Stimulated vesicular exocytosis determined as KCl-evoked fluorescence spike of pH-sensitive dye acridine orange (AO) was decreased twice in synaptosomes under hypergravity conditions as compared to control. Sets of measurements demonstrated reduced ability of synaptic vesicles to accumulate AO (10% higher steady-state baseline level of AO fluorescence). Experiments with preloaded l-[¹⁴C]glutamate exhibited similar amount of total glutamate accumulated by synaptosomes, equal concentration of ambient glutamate, but the enlarged level of cytoplasmic glutamate measuring as leakage from digitonin-permeabilized synaptosomes in hypergravity. Thus, it may be suggested that +G-induced changes in stimulated vesicular exocytosis were a result of the redistribution of intracellular pool of glutamate, i.e. a decrease in glutamate content of synaptic vesicles and an enrichment of the cytoplasmic glutamate level. To investigate the effect of hypergravity on the last step of exocytosis, i.e. membrane fusion, a cell-free system consisted of synaptic vesicles, plasma membrane vesicles, cytosolic proteins isolated from rat brain synaptosomes was used. It was found that hypergravity reduced the fusion competence of synaptic vesicles and plasma membrane vesicles, whereas synaptosomal cytosolic proteins became more active to promote membrane fusion. The total rate of homo- and heterotypic fusion reaction initiated by Ca²⁺ or Mg²⁺/ATP remained unchanged under hypergravity conditions. Thus, hypergravity could induce synaptopathy that was associated with incomplete filling of synaptic vesicles with the neuromediator and changes in exocytotic release.     

Modulation of neurosecretion and approaches for its multistep analysis

Background

Neurosecretion is the multistep process occurring in separate spatial and temporal cellular boundaries which complicates its comprehensive analysis. Most of the research are focused on one distinct stage of synaptic vesicle recycling. Here, we describe approaches for complex analysis of synaptic vesicle (SV) endocytosis and separate steps of exocytosis at the level of presynaptic bouton and highly purified SVs.

Endocytic proteins: An expanding repertoire of presynaptic functions

From a presynaptic perspective, neuronal communication mainly relies on two interdependent events: The fast Ca²⁺-triggered fusion of neurotransmitter-containing synaptic vesicles (SVs) and their subsequent high-fidelity reformation. To allow rapid neurotransmission, SVs have evolved into fascinating molecular nanomachines equipped with a well-defined set of proteins. However, upon exocytosis, SVs fully collapse into the presynaptic plasma membrane leading to the dispersal of their molecular components. While the canonical function of endocytic proteins at the presynapse was believed to be the retrieval of SV proteins via clathrin-mediated endocytosis, it is now evident that clathrin-independent endocytic mechanisms predominate. We will highlight in how far these mechanisms still rely on the classical endocytic machinery and discuss the emerging functions of endocytic proteins in release site clearance and SV reformation from endosomal-like vacuoles.     

Fusion of isolated synaptic vesicles as a model of the terminal stage of regulated exocytosis

In the study of membrane fusion, which is the terminal stage of exocytosis, we used a simplified model consisting of homotypic membranes of isolated synaptic vesicles (SV) obtained from the synaptosomal fraction of rat brain tissue. It was shown that fusion of SV develops in the presence of cytoplasmic proteins and 10^–7 to 10^–5 M Ca²⁺ ions. This conclusion was made based on changes in the intensity of fluorescence of a probe, R18. Calcium ions were found to be the most effective activators of the membrane fusion when the effects of bivalent cations, Ca²⁺, Sr²⁺, and Ba²⁺, were compared. ATP induced membrane fusion both in the presence and in the absence of Ca²⁺, and the effects of ATP and Ca²⁺ were additive. These findings allow us to believe that there are factors in the system containing SV and soluble proteins of synaptosomes, which initiate fusion of the membranes under the influence of not only Ca²⁺ but also ATP. The intensity of Ca²⁺-dependent fusion of SV dropped after trypsin treatment, i.e., proteolysis resulted in modulation of the sensitivity of vesicular proteins and/or a change in their capability of evoking membrane fusion. Monoclonal antibodies against synaptotagmin and synaptobrevin inhibited fusion of SV, but only partly. Our results support the concept that Ca²⁺-regulated membrane fusion is possible without the involvement of the entire SNARE complex.Neirofiziologiya/Neurophysiology, Vol. 36, No. 4, pp. 272–280, July–August, 2004.This revised version was published online in April 2005 with a corrected cover date.     

Single vesicle assaying of SNARE-synaptotagmin-driven fusion reveals fast and slow modes of both docking and fusion and intrasample heterogeneity

Lipid mixing between vesicles functionalized with SNAREs and the cytosolic C2AB domain of synaptotagmin-1 recapitulates the basic Ca²⁺ dependence of neuronal exocytosis. However, in the conventional ensemble lipid mixing assays it is not possible to discriminate whether Ca²⁺ accelerates the docking or the fusion of vesicles. Here we report a fluorescence microscopy-based assay to monitor SNARE-mediated docking and fusion of individual vesicle pairs. In situ measurement of the concentration of diffusing particles allowed us to quantify docking rates by a maximum-likelihood approach. This analysis showed that C2AB and Ca²⁺ accelerate vesicle-vesicle docking with more than two orders of magnitude. Comparison of the measured docking rates with ensemble lipid mixing kinetics, however, suggests that in most cases bilayer fusion remains the rate-limiting step. Our single vesicle results show that only ∼60% of the vesicles dock and only ∼6% of docked vesicles fuse. Lipid mixing on single vesicles was fast (t_mix < 1 s) while an ensemble assay revealed two slow mixing processes with t_mix ∼ 1 min and t_mix ∼ 20 min. The presence of several distinct docking and fusion pathways cannot be rationalized at this stage but may be related to intrasample heterogeneities, presumably in the form of lipid and/or protein composition.     

Cyclic AMP potentiates Ca2+-dependent exocytosis in pancreatic duct epithelial cells

Exocytosis is evoked by intracellular signals, including Ca²⁺ and protein kinases. We determined how such signals interact to promote exocytosis in exocrine pancreatic duct epithelial cells (PDECs). Exocytosis, detected using carbon-fiber microamperometry, was stimulated by [Ca²⁺]_i increases induced either through Ca²⁺ influx using ionomycin or by activation of P2Y2 or protease-activated receptor 2 receptors. In each case, the exocytosis was strongly potentiated when cyclic AMP (cAMP) was elevated either by activating adenylyl cyclase with forskolin or by activating the endogenous vasoactive intestinal peptide receptor. This potentiation was completely inhibited by H-89 and partially blocked by Rp-8-Br-cAMPS, inhibitors of protein kinase A. Optical monitoring of fluorescently labeled secretory granules showed slow migration toward the plasma membrane during Ca²⁺ elevations. Neither this Ca²⁺-dependent granule movement nor the number of granules found near the plasma membrane were detectably changed by raising cAMP, suggesting that cAMP potentiates Ca²⁺-dependent exocytosis at a later stage. A kinetic model was made of the exocytosis stimulated by UTP, trypsin, and Ca²⁺ ionophores with and without cAMP increase. In the model, without a cAMP rise, receptor activation stimulates exocytosis both by Ca²⁺ elevation and by the action of another messenger(s). With cAMP elevation the docking/priming step for secretory granules was accelerated, augmenting the releasable granule pool size, and the Ca²⁺ sensitivity of the final fusion step was increased, augmenting the rate of exocytosis. Presumably both cAMP actions require cAMP-dependent phosphorylation of target proteins. cAMP-dependent potentiation of Ca²⁺-induced exocytosis has physiological implications for mucin secretion and, possibly, for membrane protein insertion in the pancreatic duct. In addition, mechanisms underlying this potentiation of slow exocytosis may also exist in other cell systems.     

The fusion of synaptic vesicle membranes studied by lipid mixing: the R18 fluorescence assay validity

The various experimental approaches and octadecyl rhodamine B chloride (R18) assay's capability to meet the criteria for examining the Ca²⁺dependent synaptic vesicles (SVs) fusion with target membranes have been investigated. The existence of at least two simultaneous processes one of which attributed to real Ca²⁺-dependent membrane fusion, while another is considered to be non-specific probe transfer has been shown. The differences in response to temperature changes were found for R18 fluorescence dequenching upon stimulation of membrane fusion or nonspecific probe transfer. The temperature dependences of the probe dequenching rate were the same for heterotypic and homotypic membrane systems and increased with the temperature growth. The combination of R18 fluorescence studies with the data obtained by dynamic light scattering (DLS) offers a unique opportunity for the determination of SVs aggregation and the membrane fusion. The cholesterol content of the synaptosomal plasma membrane was modulated by methyl-β-cyclodextrin (MCD). The MCD molecule has proven to bind directly the membrane cholesterol and interact with lipophilic probe R18 that affects its fluorescence. The obvious distinctions in probe dequenching due to the membrane mixing or the MCD effect were observed. The cholesterol depletion from the synaptosomal plasma membranes was found to inhibit the process of Ca²⁺-induced membrane fusion with SVs. Thus, the manipulations with conditions of R18 probe dequenching at the model conditions, specific for the Ca²⁺-triggered fusion steps of regulated exocytosis, allowed us to determine the relative contribution of probe transfer and genuine membrane fusion to the overall fluorescence signal.     

Presynaptic active zones in invertebrates and vertebrates

The regulated release of neurotransmitter occurs via the fusion of synaptic vesicles (SVs) at specialized regions of the presynaptic membrane called active zones (AZs). These regions are defined by a cytoskeletal matrix assembled at AZs (CAZ), which functions to direct SVs toward docking and fusion sites and supports their maturation into the readily releasable pool. In addition, CAZ proteins localize voltage‐gated Ca²⁺ channels at SV release sites, bringing the fusion machinery in close proximity to the calcium source. Proteins of the CAZ therefore ensure that vesicle fusion is temporally and spatially organized, allowing for the precise and reliable release of neurotransmitter. Importantly, AZs are highly dynamic structures, supporting presynaptic remodeling, changes in neurotransmitter release efficacy, and thus presynaptic forms of plasticity. In this review, we discuss recent advances in the study of active zones, highlighting how the CAZ molecularly defines sites of neurotransmitter release, endocytic zones, and the integrity of synapses.     

A novel dual Ca2+ sensor system regulates Ca2+-dependent neurotransmitter release

Ca²⁺-dependent neurotransmitter release requires synaptotagmins as Ca²⁺ sensors to trigger synaptic vesicle (SV) exocytosis via binding of their tandem C2 domains—C2A and C2B—to Ca²⁺. We have previously demonstrated that SNT-1, a mouse synaptotagmin-1 (Syt1) homologue, functions as the fast Ca²⁺ sensor in Caenorhabditis elegans. Here, we report a new Ca²⁺ sensor, SNT-3, which triggers delayed Ca²⁺-dependent neurotransmitter release. snt-1;snt-3 double mutants abolish evoked synaptic transmission, demonstrating that C. elegans NMJs use a dual Ca²⁺ sensor system. SNT-3 possesses canonical aspartate residues in both C2 domains, but lacks an N-terminal transmembrane (TM) domain. Biochemical evidence demonstrates that SNT-3 binds both Ca²⁺ and the plasma membrane. Functional analysis shows that SNT-3 is activated when SNT-1 function is impaired, triggering SV release that is loosely coupled to Ca²⁺ entry. Compared with SNT-1, which is tethered to SVs, SNT-3 is not associated with SV. Eliminating the SV tethering of SNT-1 by removing the TM domain or the whole N terminus rescues fast release kinetics, demonstrating that cytoplasmic SNT-1 is still functional and triggers fast neurotransmitter release, but also exhibits decreased evoked amplitude and release probability. These results suggest that the fast and slow properties of SV release are determined by the intrinsically different C2 domains in SNT-1 and SNT-3, rather than their N-termini–mediated membrane tethering. Our findings therefore reveal a novel dual Ca²⁺ sensor system in C. elegans and provide significant insights into Ca²⁺-regulated exocytosis.     

Two synaptic vesicle pools,vesicle recruitment and replenishment of pools at the Drosophila neuromuscular junction

Drosophila neuromuscular junctions (DNMJs) are malleable and its synaptic strength changes with activities. Mobilization and recruitment of synaptic vesicles (SVs), and replenishment of SV pools in the presynaptic terminal are involved in control of synaptic efficacy. We have studied dynamics of SVs using a fluorescent styryl dye, FM1-43, which is loaded into SVs during endocytosis and released during exocytosis, and identified two SV pools. The exo/endo cycling pool (ECP) is loaded with FM1-43 during low frequency nerve stimulation and releases FM1-43 during exocytosis induced by high K⁺. The ECP locates close to release sites in the periphery of presynaptic boutons. The reserve pool (RP) is loaded and unloaded only during high frequency stimulation and resides primarily in the center of boutons. The size of ECP closely correlates with the efficacy of synaptic transmission during low frequency neuronal firing. An increase of cAMP facilitates SV movement from RP to ECP. Post-tetanic potentiation (PTP) correlates well with recruitment of SVs from RP. Neither PTP nor post-tetanic recruitment of SVs from RP occurs in memory mutants that have defects in the cAMP/PKA cascade. Cyotochalasin D slows mobilization of SVs from RP, suggesting involvement of actin filaments in SV movement. During repetitive nerve stimulation the ECP is replenished, while RP replenishment occurs after tetanic stimulation in the absence of external Ca²⁺. Mobilization of internal Ca²⁺ stores underlies RP replenishment. SV dynamics is involved in synaptic plasticity and DNMJs are suitable for further studies.     

Identifying the targets of the amplifying pathway for insulin secretion in pancreatic beta-cells by kinetic modeling of granule exocytosis

A kinetic model for insulin secretion in pancreatic β-cells is adapted from a model for fast exocytosis in chromaffin cells. The fusion of primed granules with the plasma membrane is assumed to occur only in the “microdomain” near voltage-sensitive L-type Ca²⁺-channels, where [Ca²⁺] can reach micromolar levels. In contrast, resupply and priming of granules are assumed to depend on the cytosolic [Ca²⁺]. Adding a two-compartment model to handle the temporal distribution of Ca²⁺ between the microdomain and the cytosol, we obtain a unified model that can generate both the fast granule fusion and the slow insulin secretion found experimentally in response to a step of membrane potential. The model can simulate the potentiation induced in islets by preincubation with glucose and the reduction in second-phase insulin secretion induced by blocking R-type Ca²⁺-channels (Ca_V2.3). The model indicates that increased second-phase insulin secretion induced by the amplifying signal is controlled by the “resupply” step of the exocytosis cascade. In contrast, enhancement of priming is a good candidate for amplification of first-phase secretion by glucose, cyclic adenosine 3′:5′-cyclic monophosphate, and protein kinase C. Finally, insulin secretion is enhanced when the amplifying signal oscillates in phase with the triggering Ca²⁺-signal.     

Enhancement of the Ca2+-triggering steps of native membrane fusion via thiol-reactivity

Ca²⁺-triggered membrane fusion is the defining step of exocytosis. Isolated urchin cortical vesicles (CV) provide a stage-specific preparation to study the mechanisms by which Ca²⁺ triggers the merger of two apposed native membranes. Thiol-reactive reagents that alkylate free sulfhydryl groups on proteins have been consistently shown to inhibit triggered fusion. Here, we characterize a novel effect of the alkylating reagent iodoacetamide (IA). IA was found to enhance the kinetics and Ca²⁺ sensitivity of both CV-plasma membrane and CV–CV fusion. If Sr²⁺, a weak Ca²⁺ mimetic, was used to trigger fusion, the potentiation was even greater than that observed for Ca²⁺, suggesting that IA acts at the Ca²⁺-sensing step of triggered fusion. Comparison of IA to other reagents indicates that there are at least two distinct thiol sites involved in the underlying fusion mechanism: one that regulates the efficiency of fusion and one that interferes with fusion competency.     

SNARE complex alters the interactions of the Ca2+ sensor synaptotagmin 1 with lipid bilayers

Release of neuronal transmitters from nerve terminals is triggered by the molecular Ca²⁺ sensor synaptotagmin 1 (Syt1). Syt1 is a transmembrane protein attached to the synaptic vesicle (SV), and its cytosolic region comprises two domains, C2A and C2B, which are thought to penetrate into lipid bilayers upon Ca²⁺ binding. Before fusion, SVs become attached to the presynaptic membrane (PM) by the four-helical SNARE complex, which is thought to bind the C2B domain in vivo. To understand how the interactions of Syt1 with lipid bilayers and the SNARE complex trigger fusion, we performed molecular dynamics (MD) simulations at a microsecond scale. We investigated how the isolated C2 modules and the C2AB tandem of Syt1 interact with membranes mimicking either SV or PM. The simulations showed that the C2AB tandem can either bridge SV and PM or insert into PM with its Ca²⁺-bound tips and that the latter configuration is more favorable. Surprisingly, C2 domains did not cooperate in penetrating into PM but instead mutually hindered their insertion into the bilayer. To test whether the interaction of Syt1 with lipid bilayers could be affected by the C2B-SNARE attachment, we performed systematic conformational analysis of the C2AB-SNARE complex. Notably, we found that the C2B-SNARE interface precludes the coupling of C2 domains and promotes their insertion into PM. We performed the MD simulations of the prefusion protein complex positioned between the lipid bilayers mimicking PM and SV, and our results demonstrated in silico that the presence of the Ca²⁺ bound C2AB tandem promotes lipid merging. Altogether, our MD simulations elucidated the role of the Syt1-SNARE interactions in the fusion process and produced the dynamic all-atom model of the prefusion protein-lipid complex.     

Phosphatidylserine Regulation of Ca2+-triggered Exocytosis and Fusion Pores in PC12 Cells

The synaptic vesicle protein synaptotagmin I (Syt I) binds phosphatidylserine (PS) in a Ca²⁺-dependent manner. This interaction is thought to play a role in exocytosis, but its precise functions remain unclear. To determine potential roles for Syt I-PS binding, we varied the PS content in PC12 cells and liposomes and studied the effects on the kinetics of exocytosis and Syt I binding in parallel. Raising PS produced a steeply nonlinear, saturating increase in Ca²⁺-triggered fusion, and a graded slowing of the rate of fusion pore dilation. Ca²⁺-Syt I bound liposomes more tightly as PS content was raised, with a steep increase in binding at low PS, and a further gradual increase at higher PS. These two phases in the PS dependence of Ca²⁺-dependent Syt I binding to lipid may correspond to the two distinct and opposing kinetic effects of PS on exocytosis. PS influences exocytosis in two ways, enhancing an early step leading to fusion pore opening, and slowing a later step when fusion pores dilate. The possible relevance of these results to Ca²⁺-triggered Syt I binding is discussed along with other possible roles of PS.     

Regulation of synaptic activity by snapin‐mediated endolysosomal transport and sorting

Recycling synaptic vesicles (SVs) transit through early endosomal sorting stations, which raises a fundamental question: are SVs sorted toward endolysosomal pathways? Here, we used snapin mutants as tools to assess how endolysosomal sorting and trafficking impact presynaptic activity in wild-type and snapin^−/− neurons. Snapin acts as a dynein adaptor that mediates the retrograde transport of late endosomes (LEs) and interacts with dysbindin, a subunit of the endosomal sorting complex BLOC-1. Expressing dynein-binding defective snapin mutants induced SV accumulation at presynaptic terminals, mimicking the snapin^−/− phenotype. Conversely, over-expressing snapin reduced SV pool size by enhancing SV trafficking to the endolysosomal pathway. Using a SV-targeted Ca²⁺ sensor, we demonstrate that snapin–dysbindin interaction regulates SV positional priming through BLOC-1/AP-3-dependent sorting. Our study reveals a bipartite regulation of presynaptic activity by endolysosomal trafficking and sorting: LE transport regulates SV pool size, and BLOC-1/AP-3-dependent sorting fine-tunes the Ca²⁺ sensitivity of SV release. Therefore, our study provides new mechanistic insights into the maintenance and regulation of SV pool size and synchronized SV fusion through snapin-mediated LE trafficking and endosomal sorting.     

Synaptobrevin N-terminally bound to syntaxin–SNAP-25 defines the primed vesicle state in regulated exocytosis

Rapid neurotransmitter release depends on the ability to arrest the SNAP receptor (SNARE)–dependent exocytosis pathway at an intermediate “cocked” state, from which fusion can be triggered by Ca²⁺. It is not clear whether this state includes assembly of synaptobrevin (the vesicle membrane SNARE) to the syntaxin–SNAP-25 (target membrane SNAREs) acceptor complex or whether the reaction is arrested upstream of that step. In this study, by a combination of in vitro biophysical measurements and time-resolved exocytosis measurements in adrenal chromaffin cells, we find that mutations of the N-terminal interaction layers of the SNARE bundle inhibit assembly in vitro and vesicle priming in vivo without detectable changes in triggering speed or fusion pore properties. In contrast, mutations in the last C-terminal layer decrease triggering speed and fusion pore duration. Between the two domains, we identify a region exquisitely sensitive to mutation, possibly constituting a switch. Our data are consistent with a model in which the N terminus of the SNARE complex assembles during vesicle priming, followed by Ca²⁺-triggered C-terminal assembly and membrane fusion.     

Assay of dense-core vesicle exocytosis using permeabilized PC12 cells

Exocytosis plays an essential role in fundamental cellular events by secreting neurotransmitters, hormones, and cytokines. Although the minimal molecular components termed SNARE that govern membrane fusion have been identified, the precise mechanisms behind the finely-tuned regulation of exocytosis executed by many molecules in addition to the actions of SNARE remain to be fully identified. Here, we evaluated a model system for assaying catecholamine secretion from permeabilized rat pheochromocytoma PC12 cells, in which the structural integrity required was preserved adequately. Among several chemical reagents used for the cell permeabilization and freezing-thawing procedures, the treatment of cells with digitonin at concentrations of 7.5–15 μM was most suitable for the secretion assay, as it was considered to cause mild disruption of the plasma membrane, enabling free access to small molecules such as Ca²⁺ and ATP to the minimal membrane fusion machinery. No additional cytosolic proteins were required to reconstitute the secretion. In this assay model, ATP was necessary to maintain the priming state before Ca²⁺-triggered exocytosis but was not required for the Ca²⁺-triggered membrane fusion process itself. The present study provides a useful cell model for exploring novel molecules that may be implicated in exocytosis such as those playing regulatory roles in addition to the “minimal membrane fusion machinery for exocytosis”, which does not require any additional special apparatus.