InteractiveFly: GeneBrief

Synapse protein 25: Biological Overview | References

Molecular mechanisms determining conserved properties of short-term synaptic depression revealed in NSF and SNAP-25 conditional mutants

Current models of synaptic vesicle trafficking implicate a core complex of proteins comprised of N-ethylmaleimide-sensitive factor (NSF), soluble NSF attachment proteins (SNAPs), and SNAREs (an acronym derived from 'SNAP (Soluble NSF Attachment Protein) REceptor', consisting of proteins that mediated vesicular fusion with the target membrane) in synaptic vesicle fusion and neurotransmitter release. Despite this progress, major challenges remain in establishing the in vivo functions of these proteins and their roles in determining the physiological properties of synapses. The present study employs glutamatergic adult neuromuscular synapses of Drosophila, which exhibit conserved properties of short-term synaptic plasticity with respect to mammalian glutamatergic synapses, to address these issues through genetic analysis. The findings establish an in vivo role for the target-Snare (t-Snare) protein SNAP-25, one part of the four helix bundle of proteins referred to as SNAPs, in synaptic vesicle priming, and support a zippering model of SNARE function in this process. Moreover, these studies define the contribution of SNAP-25-dependent vesicle priming to the detailed properties of short-term depression elicited by paired-pulse (PP) and train stimulation. In contrast, NSF is shown here not to be required for wild-type PP depression, but to be critical for maintaining neurotransmitter release during sustained stimulation. In keeping with this role, disruption of NSF function results in activity-dependent redistribution of the t-SNARE proteins, Syntaxin and SNAP-25, away from neurotransmitter release sites (active zones). These findings support a role for NSF in replenishing active zone t-SNAREs for subsequent vesicle priming, and provide new insight into the spatial organization of SNARE protein cycling during synaptic activity. Together, the results reported in this study establish in vivo contributions of SNAP-25 and NSF to synaptic vesicle trafficking and define molecular mechanisms determining conserved functional properties of short-term depression (Kawasaki, 2009).

The present study further defines the in vivo roles of NSF and SNAP-25 in synaptic vesicle trafficking and their contributions to conserved properties of short-term synaptic depression. Insight is also gained into the spatial organization of activity-dependent SNARE protein cycling with respect to active zones. The findings support a model incorporating in vivo molecular mechanisms of synaptic vesicle priming as important determinants of short-term depression and maintenance of neurotransmitter release during synaptic activity. These priming mechanisms include a direct role for SNAP-25 and an indirect contribution involving NSF-dependent replenishment of active zone free t-SNAREs for subsequent vesicle priming and fusion (see Working model of SNARE protein cycling during synaptic activity). In this model, recovery after a high frequence stimulation, in a measurement called paired pulse depression (PPD), depends on a limited pool of free active zone t-SNAREs (A), which is sufficient to support formation of trans-SNARE complexes (B) and refilling of the release-ready vesicle pool. This priming process is impaired in the SNAP-25TS mutant with respect to WT. The location of the SNAP-25TS mutation within the N-terminal region of the SNARE four helix bundle is consistent with a zippering model of SNARE complex function. On train stimulation, cis-SNARE complexes accumulate in the periactive zone (PAZ), resulting in depletion of the free t-SNARE pool at the active zone (AZ). Disassembly of cis-SNARE complexes by NSF and SNAP restores active zone t-SNARES for subsequent vesicle priming and fusion events. This aspect of synaptic vesicle priming is impaired in comatose, which encodes an enzyme that facilitates priming (Kawasaki, 2009).

The findings of this study reveal a role for SNAP-25 in determining the properties of recovery from short-term depression. An underlying function for SNAP-25 in synaptic vesicle priming was inferred from the following aspects of the SNAP-25^TS synaptic phenotype: (1) activity dependence as indicated by a WT initial EPSC amplitude and release-ready synaptic vesicle pool size, (2) preservation of the WT EPSC waveform, (3) loss of fast recovery in PPD, which is associated with fast refilling of the release-ready pool and exhibits a time course comparable with that of synaptic vesicle priming, (4) dependence on previous synaptic vesicle fusion, and (5) slowed refilling of the release-ready vesicle pool. Thus, although SNAP-25 is required for evoked synaptic vesicle fusion (Washbourne, 2002), the SNAP-25^TS mutation can selectively affect synaptic vesicle priming. This finding is consistent with systematic structure-function analysis of SNAP-25 in adrenal chromaffin cells, which provided strong support for the zippering model of SNARE function (Sorensen, 2004). In this model, vesicle priming involves initial assembly at the N-terminal end of the SNARE four helix bundle to form a loose trans-SNARE complex. Final assembly of its C-terminal end, which may be triggered by calcium influx, is critical for vesicle fusion. Notably, the SNAP-25^TS mutation is positioned within the N-terminal region of the four helix bundle, consistent with a TS impairment of trans-SNARE complex formation associated with synaptic vesicle priming. Previous studies of SNAP-25^TS indicate that synaptic vesicle docking at active zones is not disrupted at larval neuromuscular synapses, and that in vivo levels of SDS-resistant (likely cis) SNARE complexes are not altered in this mutant. Finally, it will be of great interest to further investigate the relationship of SNAP-25-dependent mechanisms underlying fast and slow components of recovery. These components are thought to involve calcium-dependent regulation of kinetically distinct synaptic vesicle pools exhibiting different release probabilities (Kawasaki, 2009).

The close resemblance of comatose (NSF) and WT synapses with respect to the initial EPSC amplitude and PPD suggests that mutant comatose (NSF) initially exhibits a WT release-ready vesicle pool and release probability. The strictly activity-dependent reduction in neurotransmitter release observed in comatose (NSF) mutants requires a brief period of synaptic activity as indicated by delayed onset of the synaptic phenotype during train stimulation. The underlying mechanism appears to involve activity-dependent redistribution of plasma membrane t-SNAREs away from the active zone. Together with systematic biochemical studies of comatose (NSF) demonstrating TS accumulation of (SDS-resistant) ternary SNARE complexes on the plasma membrane, these findings suggest that SNAREs accumulate in plasma membrane cis-SNARE complexes that are not retained within the active zone (see the working model). NSF disassembly of post-fusion plasma membrane cis-SNARE complexes has been directly demonstrated in studies of yeast exocytosis and the underlying heterotypic fusion of secretory vesicles with the plasma membrane. Previous and present efforts to address this issue at Drosophila synapses, including analysis of v-SNARE distribution in the presynaptic plasma membrane. Finally, an alternative view of NSF function favoring prefusion disassembly of SNARE complexes was reported in a study employing caged NSF peptides to acutely disrupt NSF function at the squid giant synapse. However, the activity-dependent effects of the peptide on EPSC amplitude are largely consistent with the working model presented in this study, in which the timing of the NSF requirement is not constrained by the total vesicle cycling time. Although the NSF peptide was also found to slow EPSC rise and decay times, no analogous effects were observed in the present study (Kawasaki, 2009).

The adult DLM neuromuscular synapse has had an important role in analysis of TS mutations in comatose (NSF), the presynaptic calcium channel α1 subunit gene, cacophony, and the Dynamin gene, shibire. This work has revealed that several functional characteristics of adult neuromuscular synapses are distinct from those described in the larva. In the present study, comparison of glutamatergic DLM neuromuscular synapses and cerebellar CF-PC synapses revealed a surprising degree of conservation in the detailed properties of synaptic function. These synapses exhibit morphological similarities as well, including extensive branching of axons and spatial isolation of active zones. Initial characterization of short-term depression at CF-PC synapses has been followed by a series of studies progressively defining the underlying factors in greater detail. The present study provides a basis for analysis of DLM neuromuscular synapses to characterize analogous factors and their molecular determinants. Finally, the results reported in this study complement previous and ongoing studies at DLM neuromuscular synapses of the Dynamin TS mutant, shibire, which exhibits a rapid enhancement of short-term depression as observed in SNAP-25^TS. Such similarities may reflect interactions of the in vivo molecular mechanisms governing synaptic vesicle endocytosis and exocytosis (Kawasaki, 2009).

Trapping of Syntaxin1a in presynaptic nanoclusters by a clinically relevant general anesthetic

Propofol is the most commonly used general anesthetic in humans. Understanding of its mechanism of action has focused on its capacity to potentiate inhibitory systems in the brain. However, it is unknown whether other neural mechanisms are involved in general anesthesia. This study demonstrates that the synaptic release machinery is also a target. Using single-particle tracking photoactivation localization microscopy, it was shown that clinically relevant concentrations of propofol and etomidate restrict syntaxin1A mobility on the plasma membrane, whereas non-anesthetic analogs produce the opposite effect and increase syntaxin1A mobility. Removing the interaction with the t-SNARE partner SNAP-25 abolishes propofol-induced Syntaxin1A confinement, indicating that Syntaxin1A and SNAP-25 together form an emergent drug target. Impaired Syntaxin1A mobility and exocytosis under propofol are both rescued by co-expressing a truncated Syntaxin1A construct that interacts with SNAP-25. These results suggest that propofol interferes with a step in SNARE complex formation, resulting in non-functional Syntaxin1A nanoclusters (Bademosi, 2018).

This study demonstrates that clinical concentrations of a commonly used GABA-acting general anesthetic, propofol, also restrict syntaxin1A mobility on the plasma membrane. The contrast seen with the effect of propofol analogs is particularly striking, with the non-anesthetic analogs significantly increasing syntaxin1A mobility instead. These results indicate that propofol acts like its non-anesthetic analogs when the interaction between syntaxin1A and SNAP-25 is lost, suggesting that propofol targets this interaction to immobilize syntaxin1A. It seems plausible that syntaxin1A confinement to nanoclusters could lead to impaired neurotransmission, which was also observed under propofol. However, more work is needed to establish causality here. How exactly propofol impairs syntaxin1A mobility remains unclear, although the requirement for SNAP-25 interaction suggests the nanoclusters are composed of syntaxin1A/SNAP-25 heterodimers that have been blocked from proceeding to a subsequent step in SNARE complex formation due to the presence of the general anesthetic. It is also unclear how a truncated syntaxin1A protein might preserve this process against the effects of propofol on syntaxin1A mobility and exocytosis. The finding that the truncated syntaxin1A molecule simultaneously interacts with both SNAP-25 and wild-type syntaxin1A suggests occupancy of a site that might otherwise be targeted by propofol. In this regard, future experiments with other truncation constructs employing propofol resistance as a readout will be helpful toward determining whether the effects on syntaxin1A mobility and exocytosis are indeed correlated (Bademosi, 2018).

In addition to identifying an alternative target process for this widely used sedative, the current findings may provide a more complete understanding of general anesthesia. Every neuron communicates with other neurons by way of syntaxin1A-mediated neurotransmission, which is highly conserved from worms to humans. Although these experiments were focused on the intravenous drugs propofol and etomidate, it will be interesting to see in future studies whether other general anesthetics have the same effect on syntaxin1A mobility. There is already considerable evidence that a broader range of general anesthetics affect synaptic release mechanisms, and a previous study using nuclear magnetic resonance found that clinical concentrations of these drugs interact with syntaxin1A and SNAP-25, but not VAMP2, which is consistent with the conclusion that propofol acts before completed SNARE formation. One hypothesis consistent with these findings would be that a general anesthetic target emerges only when syntaxin1A and SNAP-25 interact on the plasma membrane and that the association of propofol with this emergent target interferes with subsequent steps in SNARE formation. This would lead to a 'traffic jam' of syntaxin1A/SNAP-25 heterodimers (or another pre-SNARE moiety), which would manifest as syntaxin1A nanoclusters in this analysis. Another explanation for the decrease in syntaxin1A mobility could be that propofol promotes its recruitment into nonfunctional SNARE complexes that do not promote vesicle fusion. Whereas the data suggest interactions in the membrane, this need not be the only explanation for altered syntaxin1A mobility. An alternative possibility is that anesthetics might alter syntaxin1A mobility by more specifically interfering with other key protein interactions leading to SNARE formation, such as between syntaxin1A/SNAP-25 and Munc-13, which is a crucial mediator in forming the final tetrameric complex with VAMP2. Future experiments testing the effects of mutating candidate residues in the syntaxin1A SNARE motif should reveal the exact nature of this propofol-binding target, as has been revealed for other propofol targets, such as GABAA receptors (Bademosi, 2018).

Like sleep, general anesthesia resembles a reversible switch, and the search for mechanisms of anesthesia has justifiably focused on proteins that exert major effects on neuronal excitability, such as inhibitory GABAA receptors, which are indeed targets of many general anesthetics. However, the current results and the work of others show that clinically relevant concentrations of general anesthetics also compromise neurotransmitter release, and the current set of results with intravenous drugs suggests this may be consequence of effects on syntaxin1A mobility in the plasma membrane. However, general anesthetics do not abolish neurotransmission; they only decrease quantal content. So how could this be relevant to the behavioral endpoint that is general anesthesia? With most animal brains comprising anywhere between millions and trillions of synapses, it seems plausible that normal brain functions would be compromised if syntaxin1A mobility became globally restricted across a variety of synapses following exposure to general anesthetics. While a decrease in quantal content may not significantly impair some muscular (or spinal cord) functions, it is likely that a similar effect on central synapses would dramatically change temporal dynamics in the brain, leading to a loss of functional connectivity. In support of this view, recent electroencephalogram (EEG) and fMRI studies have shown that functional connectivity throughout the brain is significantly altered in patients undergoing general anesthesia. Thus, other manipulations that compromise presynaptic communication, including effects on presynaptic excitability , might fall into the same category of anesthetic mechanisms as the syntaxin1A-mediated effects described in this study, that may be considered a class of effects that is distinct from GABAergic sleep-related mechanisms. One possibility, which has been raised previously, is that GABAergic processes (e.g., sedation and loss of consciousness) are induced at lower drug doses (e.g., < 1 µM propofol), while the presynaptic processes discussed in this study are affected at the slightly higher concentrations required for surgery. It remains unknown however whether other general anesthetics target presynaptic mechanisms. A recent study using hippocampal cultures found that isoflurane inhibits synaptic vesicle exocytosis through reduced Ca2+ influx rather than Ca2+-exocytosis coupling. In contrast, the current results suggest that propofol and etomidate-mediated presynaptic effects might be directly coupled to the exocytosis machinery. Whether this is a difference between intravenous and volatile anesthetics is unclear. Nevertheless, a set of distinct presynaptic mechanisms linked to exocytosis might explain why recovery from general anesthesia appears to involve a different process than anesthesia induction; re-establishing functional connectivity after neurotransmission has returned to normal levels across the brain would likely involve a different process than falling asleep or waking up. It will be interesting in future research to use transgenic syntaxin1A animals to link the local effects found at the presynapse with consequent changes in global readouts, such as whole-brain connectivity and coherence (Bademosi, 2018).

Arg206 of SNAP-25 is essential for neuroexocytosis at the Drosophila melanogaster neuromuscular junction

An analysis of SNAP-25 isoform sequences indicates that there is a highly conserved arginine residue (198 in vertebrates, 206 in the genus Drosophila) within the C-terminal region, which is cleaved by botulinum neurotoxin A, with consequent blockade of neuroexocytosis. The possibility that it may play an important role in the function of the neuroexocytosis machinery was tested at neuromuscular junctions of Drosophila larvae expressing SNAP-25 in which Arg206 had been replaced by alanine. Electrophysiological recordings of spontaneous and evoked neurotransmitter release under different conditions as well as testing for the assembly of the SNARE complex indicate that this residue, which is at the P₁' position of the botulinum neurotoxin A cleavage site, plays an essential role in neuroexocytosis. Computer graphic modelling suggests that this arginine residue mediates protein-protein contacts within a rosette of SNARE complexes that assembles to mediate the fusion of synaptic vesicles with the presynaptic plasma membrane (Megighian, 2010).

SNARE proteins are the main molecular constituents of the synaptic vesicle (SV) fusion machinery. A four helix bundle spontaneously forms by coil-coiling of the cytoplasmic domains of the three SNARE proteins: VAMP (also known as synaptobrevin), syntaxin and SNAP-25. The assembly of the SNARE complex is essential for the synaptic vesicle fusion process (Jahn, 2006; Sudhof, 2009). It has been estimated that the formation of three or more SNARE complexes provides sufficient free energy to drive membrane fusion, but the energetic yield of the process is not known. Additional proteins (rab-GTPases, MUNC and RIM proteins, complexin, syntaxin and others) are involved in regulation binding of the SV to the cytosolic face of the presynaptic membrane and in Ca²⁺-triggered fusion; the local Ca²⁺ concentration rises following the opening of voltage-gated Ca²⁺ channels located close to the active zones where fusion takes place. The time delay between the cytosolic Ca²⁺ trigger and the fusion of docked and ready-to-release SVs varies for different synapses, but it is always very short (>100 microseconds). This very short time period indicates that the ready-to-fuse vesicles may be actually in a state of hemifusion with the presynaptic membrane. This would require that the SVs are actually stably juxtaposed on the cytosolic face of the presynaptic membrane and this appears to involve several SNARE complexes at the same site (Megighian, 2010).

Convincing evidence for the key role of the three neuronal SNARE proteins in neurotransmitter release derives from its complete blockade exerted by tetanus and botulinum neurotoxins. Botulinum neurotoxin type A (BoNT/A) cleaves SNAP-25 nine residues from the C-terminus (Schiavo, 1993; Binz, 1994) suggesting that this part of the molecule plays a central role in the assembly and/or function of the neuroexocytosis machinery. This study begins a detailed investigation of this SNAP-25 region by site-directed mutagenesis in Drosophila coupled to electrophysiological recording of the Drosophila larva neuromuscular junction (NMJ). The study started with an analysis of the available sequences of neuronal SNAP-25 and related isoforms reported in databases. As expected, mapping of the conservation profiles into the structure of the SNARE complex (Chen, 2002) shows a high conservation for residues involved in intermolecular contacts within each SNARE four-helix bundle. External residues were also considered that are essential for possible protein-protein contacts that are necessary to form a rosette of SNARE complexes. The formation and involvement of such a rosette in neuroexocytosis is suggested by several findings and considerations. There is a single amino acidic residue that is fully conserved among species and isoforms of SNAP-25, not involved in the zippering of the SNARE complex, and this is Arg198 (mouse and human SNAP-25 numbering), which points outside of the four-helix bundle SNARE complex in the C-terminal of SNAP-25. This residue is at the P₁' site of the peptide bond cleaved by BoNT/A (Megighian, 2010).

On this basis, it was hypothesized that site-directed substitution of Arg198 with a neutral residue could interfere, in vivo, with the function of the neuroexocytosis apparatus in such a way as to be detectable by electrophysiological recordings at the neuromuscular junction. To test this possibility, Drosophila was used as an animal model. Arg198 of human SNAP-25 corresponds to Arg206 of Drosophila SNAP-25. Transgenic lines of Drosophila were generated carrying SNAP-25 with an alanine at position 206. The transgene was expressed in a wild-type background and resulted in a significant reduction of both evoked and spontaneous neurotransmitter release at the neuromuscular junction in third instar larvae, relative to that in controls, in the absence of overt NMJ morphological defects. This finding is discussed and interpreted in terms of a rosette model for the apparatus that mediates fusion of synaptic vesicles with the presynaptic membrane (Megighian, 2010).

The present finding that the replacement of the conserved Arg198 residue of SNAP-25 with alanine affects the probability of neurotransmitter release is very relevant because it identifies a key residue in this process of paramount importance for the function of the nervous system. However, as the process is a complex one, the end result is open to different interpretations. Evidence is provided against the possibilities that: (1) this change might have affected the assembly of the SNARE complex; (2) the calcium concentration dependence of the phenomenon could have been altered; (3) the morphology and/or morphometry of the NMJ was changed. This residue is not involved in the protein-protein interactions between the three SNARE proteins, and points outward, suggesting that it may be involved in the interaction with other proteins of the neuroexocytosis machinery. There are several scattered pieces of evidence that a number of SNARE complexes are involved in the exocytosis of vesicles and granules. Multimers containing a variable number of SNARE complexes have been observed under various circumstances in vitro and were isolated from detergent-treated squid synaptosomes. The Ca²⁺-cooperativity of neurotransmitter release was found to be linked to the number of SNARE proteins. Remarkably, star-shaped oligomers, comprising three to four SNARE complexes, were isolated from detergent-treated homogenized bovine brain with a monoclonal antibody directed toward the acetylated N-terminus of SNAP-25 (Rickman, 2005). In vitro experiments performed with different biophysical approaches indicate the formation of oligomers of three to ten SNARE complexes (Karatekin, 2010; Yersin, 2003; Lu, 2008; Megighian, 2010 and references therein).

Using a peptide that inhibits granule fusion in a chromaffin cell line, it has been estimated that a minimum of three SNARE complexes is sufficient to support exocytosis. In the same PC12 cells, mutagenesis of the transmembrane domain of the SNARE presynaptic membrane protein syntaxin led to an estimate of five to eight syntaxin molecules being involved in catecholamine release. However, careful experiments performed with mouse spinal cord motoneurons and with the frog NMJ intoxicated with botulinum neurotoxins suggested a higher figure. The involvement of multi SNARE complexes in neuroexocytosis also accounts for the strikingly long duration of BoNT/A poisoning (>3 months in human skeletal muscles) and for the fact that transfection of BoNT/A-truncated SNAP-25 inhibits exocytosis (for a discussion see Montecucco, 2005). On the basis of this rich set of data indicating the role of an oligomeric SNARE supercomplex in exocytosis, a ten-SNARE-complex rosette was modeled and molecular dynamics simulations were run. Such modelling is justified by the fact that the three SNARE proteins are sufficient to promote membrane fusion in vitro and can, therefore provide relevant information on protein-protein contacts among SNARE complexes in membranes (Sudhof, 2009; Karatekin, 2010). Given the necessity of forming a ring of 'petals' to define a central area where membrane fusion may take place, such an approach shows that few protein-protein contacts between SNARE complexes are involved, and Arg198 is at the centre of the contact areas between the petals of the rosette. Changing the number of petals of the rosette between eight and 13 results in little alteration in the amount of protein-protein contacts between each SNARE complex, while Arg198 remains in a central position within the area of contact. The simple replacement of this charged residue with a helix-promoting, but uncharged, alanine residue does not appear to change the secondary structure, and indeed no effect was found of the alanine replacement in the rate and extent of assembly of the SNARE complex in vitro. However, this single mutated SNAP-25 is sufficient to decrease the number of miniature end plate potential events as well as evoked end plate potentials. It is even more remarkable that it does so in a wild-type SNAP-25 background at the Drosophila larva NMJ. Although further analysis with expression of the mutant SNAP-25 in a null background is required to substantiate the present results, the consistency of the data reported in this study suggests that the presence of a single mutant SNARE complex in the very critical region of protein-protein contact between the petals of the rosette is sufficient to block the activity of the rosette. This does explain the dominant-negative nature of the mutation introduced here with respect to neuroexocytosis, and fits well with the specific action of BoNT/A and BoNT/C which cleave SNAP-25 just before and after Arg198, respectively, causing a dominant-negative effect with the characteristic long duration of action of these two neurotoxins. The present model also explains the remarkable findings that the replacements of Lys201 and Leu203 with a negatively charged glutamate residue do not affect SNARE assembly but inhibits exocytosis in chromaffin cells as these changes are disturbing the protein-protein contacts between adjacent SNARE complexes (Megighian, 2010).

Synaptotagmin-1 docks secretory vesicles to syntaxin-1/SNAP-25 acceptor complexes

Docking, the initial association of secretory vesicles with the plasma membrane, precedes formation of the SNARE complex, which drives membrane fusion. For many years, the molecular identity of the docked state, and especially the vesicular docking protein, has been unknown, as has the link to SNARE complex assembly. This study, using adrenal chromaffin cells, identifies the vesicular docking partner as synaptotagmin-1, the calcium sensor for exocytosis, and SNAP-25 as an essential plasma membrane docking factor, which, together with the previously known docking factors Munc18-1 and syntaxin, form the minimal docking machinery. Moreover, the requirement for Munc18-1 in docking, but not fusion, can be overcome by stabilizing syntaxin/SNAP-25 acceptor complexes. These findings, together with cross-rescue, double-knockout, and electrophysiological data, led to a proposal that vesicles dock when synaptotagmin-1 binds to syntaxin/SNAP-25 acceptor complexes, whereas Munc18-1 is required for the downstream association of synaptobrevin to form fusogenic SNARE complexes (de Wit, 2009).

These data identify two genes, Snap-25 and synaptotagmin-1, that, together with two previously characterized genes, munc18-1 and syntaxin-1, are required for docking of secretory vesicles. This study addressed the involvement of the syntaxin-1/SNAP-25 acceptor complex and found that two conditions that favor the formation of syntaxin-1/SNAP-25 acceptor complexes rescue the docking defects in munc18-1 null mutants: SNAP-25 overexpression and expression of truncated synaptobrevin. Furthermore, null mutations for SNAP-25 and the vesicular protein synaptotagmin-1 abolish docking, and SNAP-25 no longer rescues docking in synaptotagmin-1/munc18-1 double-null mutants. By using synaptotagmin-1 and SNAP-25 mutations that affect their interaction, both proteins were confirmed to act in concert for correct anchoring of secretory vesicles to fusion sites. Moreover, the rescue of docking, but not fusion, after expression of SNAP-25 or the synaptobrevin-2 C-terminal fragment on the munc18-1 null background indicates that Munc18-1 is not an essential constituent of the docking complex itself, but plays an essential downstream role. Together, the null mutation and (cross-) rescue experiments indicate that the corresponding four proteins work together to dock vesicles and at the same time suggest that Munc18-1 plays a unique, orchestrating role. While docking is established between syntaxin-1/SNAP-25 acceptor complexes at the target membrane and synaptotagmin-1 on the vesicle membrane, Munc18-1 promotes the formation or stability of the correct acceptor SNARE complexes (de Wit, 2009).

Munc18-1 can interact with both 'closed' and 'open' syntaxin-1, but it is unclear which binding mode is essential to perform its function in docking. Munc18-1 binding to 'open' syntaxin-1 involves an interaction with the N-terminal H(abc) domain of syntaxin-1 and the four-helical bundle of the assembled SNARE complex. It has been shown that N-terminal interaction is not sufficient for docking, since a docking phenotype similar to syntaxin-1 and munc18-1 null was observed in chromaffin cells from knockin mice that express a mutant syntaxin-1 that only allows N-terminal interaction. In addition, when the well-characterized D34N/M38V double mutant of Munc18-1 that is known to perturb the interaction with 'closed' syntaxin was expressed, it was observed that docking was not restored in munc18-1 null chromaffin cells. Other studies have shown that Munc18-1 binding to 'open' syntaxin is essential to execute fusion. In the present study, docking and fusion phenotypes were experimentally separated in munc18-1 null chromaffin cells. The observations that SNAP-25 and SybCT overexpression, which both increase the number of syntaxin-1/SNAP-25 dimers, restore docking implies that Munc18-1 promotes the existence/stability of intermediate syntaxin-1/SNAP-25 dimers at the target membrane and therefore probably binds to these intermediate complexes. This increased number of acceptor complexes is not sufficient to restore fusion in the absence of Munc18-1, which firmly establishes a postdocking role for Munc18-1 in SNARE-dependent fusion. Currently, it is unclear whether Munc18-1's function downstream of docking requires either binding to intermediate syntaxin-1/SNAP-25 dimers alone or also binding to assembled SNARE complexes (containing synaptobrevin-2) to promote fusion as shown previously in vitro. In addition, these experiments with synaptotagmin-1 and SNAP-25 mutations, which have been shown to impair secretion, show that in the presence of Munc18-1 a correlation exists between mutations that impair secretion and those that impair docking. This is not the case in the absence of Munc18-1, emphasizing its postdocking role in SNARE-dependent fusion (de Wit, 2009).

This study identifies synaptotagmin-1 as a vesicular docking factor that binds to the assembled docking acceptor discussed above and has the capacity to anchor vesicles to the target membrane. This docking role of synaptotagmin-1 is consistent with previous findings in invertebrate synapses, which, however, have not been specifically interpreted in terms of docking because of additional phenotypes in these synapses: large effects on undocked vesicle populations near the active zone, which has been related to the increased mini rate observed in these mutant synapses, and/or impaired recruitment. Interestingly, a mutation used in the latter study is in an area of the molecule that was later identified to interact with SNAP-25 (de Wit, 2009).

The docking role of synaptotagmin-1 proposed in this study does not conflict with its well-established role in fusion. However, while its role in fusion is strictly Ca²⁺ dependent, its role in docking is probably Ca²⁺ independent, since resting chromaffin cells have a strong docking phenotype in the absence of synaptotagmin-1 and its Ca²⁺ affinity is insufficient to be activated by resting Ca²⁺ levels in the cytosol. This is in line with a Ca²⁺-independent, upstream role previously suggested in rescue experiments in fly neuromuscular junction (Loewen, 2006). It is tempting to speculate that on top of this principally Ca²⁺-independent docking role, synaptotagmins may also contribute to the well-known but incompletely understood Ca²⁺-dependent acceleration of vesicle recruitment/docking/priming (de Wit, 2009).

Secretory systems typically express multiple synaptotagmins. In chromaffin cells, synaptotagmin-7 can partially compensate for the loss of synaptotagmin-1, but the secretion phenotype of the synaptotagmin-1 null cells is still drastic. In analogy, the docking phenotype in synaptotagmin-1 null cells is also drastic, but still slightly less severe than the munc18-1 null phenotype. This may be explained by a partial compensation by other synaptotagmins. The presence of multiple synaptotagmins, with different Ca²⁺ sensitivities and the new evidence that they are not only involved in fusion (and endocytosis), but also in docking, may require reinterpretation of previous studies on these proteins. Most studies assess upstream processes by measuring the final one (fusion) and thereby sample a composite measure of the combined effects of experimental manipulations on all upstream steps. For these combined effects to be dissected, new methodologies may be required to directly assess these upstream steps and to go beyond what current secretion assays have revealed about the complexity of the secretory pathway (de Wit, 2009).

Invertebrate synapses, docking phenotypes for Munc18-1, syntaxin-1, SNAP-25, and synaptotagmin-1 have not been described or are at least less evident. It is possible that these proteins are dispensable for synaptic vesicle docking and that distinct mechanisms dock vesicles in synapses. However, it seems more likely that docking principles are conserved among secretory systems. This idea is strongly supported by the fact that docking phenotypes have been observed in invertebrate synapses upon mutations in three of the four genes. However, these phenotypes are generally subtle and sometimes require advanced methodology and new docking definitions to become evident. In the case of synaptotagmin, invertebrate phenotypes are robust, but additional phenotypes were observed that prevented a specific interpretation in terms of docking. It is likely that docking phenotypes are less evident in vertebrate synapses either because of redundancy arising from the expression of multiple isoforms for some of the docking genes identified here or because structurally unrelated proteins that are not expressed in chromaffin cells restrict undocking of synaptic vesicles even when essential docking factors are not expressed. Finally, it is plausible that undocking and docking phenotypes are simply not as evident in the densely packed nerve terminal (de Wit, 2009).

With the currently identified four genes for docking and the link to SNARE complex assembly, a consistent (minimal) working model for the exocytotic pathway from the initial docking step until the final fusion reaction can now be synthesized for the first time, proposing the following four steps: (1) Munc18-1 binds the closed conformation of syntaxin-1. Munc18-1 interacts with two epitopes in syntaxin-1, the Habc domain, and the N-terminal domain. (2) SNAP-25 binds the syntaxin-1/Munc18-1 heterodimer. (3) Secretory vesicles reach the target membrane area and associate via synaptotagmin-1 to this trimeric syntaxin-1/Munc18-1/SNAP-25 complex, which effectuates docking. This binding requires the C2B domain of synaptotagmin-1, and recent studies suggest that Munc18-1's function here is to further help stabilize the syntaxin-1/SNAP-25 (1:1) acceptor complex for subsequent binding of synaptobrevin-2. In addition, since only vesicles docked in the presence of Munc18-1 are able to fuse, Munc18-1 might help restrict fusion to specific sites on the plasma membrane. By attaching the vesicle to the plasma membrane, the calcium sensor for exocytosis (synaptotagmin-1) has the additional function of localizing vesicles close to calcium channels. (4) Synaptobrevin-2 then binds to the synaptotagmin-1/syntaxin-1/Munc18-1/SNAP-25 complex and the four helical SNARE bundle forms, which subsequently allows complexins to associate with the four helical SNARE bundle, and ultimately the vesicle fuses upon Ca²⁺ entry. It has been proposed that synaptobrevin-2 replaces Munc18-1, but, given the proposed fusion-promoting actions of Munc18-1 while associated to SNARE complexes, Munc18-1 may also continue to associate with the ternary SNARE complex until fusion is triggered (de Wit, 2009).

A Drosophila SNAP-25 null mutant reveals context-dependent redundancy with SNAP-24 in neurotransmission

The synaptic protein SNAP-25 is an important component of the neurotransmitter release machinery, although its precise function is still unknown. Genetic analysis of other synaptic proteins has yielded valuable information on their role(s) in synaptic transmission. In this study, a mutagenesis screen was performed to identify new SNAP-25 alleles that fail to complement SNAP-25^ts, a previously isolated recessive temperature-sensitive allele of SNAP-25. In a screen of 100,000 flies, 26 F₁ progeny failed to complement SNAP-25^ts and 21 of these were found to be null alleles of SNAP-25. These null alleles die at the pharate adult stage and electroretinogram recordings of these animals reveal that synaptic transmission is blocked. At the third instar larval stage, SNAP-25 nulls exhibit nearly normal neurotransmitter release at the neuromuscular junction. This is surprising since SNAP-25^ts larvae exhibit a much stronger synaptic phenotype. A related protein, SNAP-24, can substitute for SNAP-25 at the larval stage in SNAP-25 nulls. However, if a wild-type or mutant form of SNAP-25 is present, then SNAP-24 does not appear to take part in neurotransmitter release at the larval NMJ. These results suggest that the apparent redundancy between SNAP-25 and SNAP-24 is due to inappropriate genetic substitution. In this situation, two related protein isoforms perform similar functions in distinct biochemical pathways. Normally, an individual isoform does not participate in the other pathway, and so hypomorphic mutations disrupting each protein show a distinct phenotype. However, a null allele that completely abolishes the expression of one protein could allow an isoform to step in and compensate for it. In this way, a null allele of a gene may paradoxically show a phenotype that is much weaker than that of a hypomorphic allele of that gene (Vilinsky, 2002).

What could account for the unexpectedly mild effects of abolishing SNAP-25 at the larval stage? Since SNAP-25 is thought to be a crucial component of the exocytosis machinery and since these mutants die just prior to eclosion, it was reasoned that another related protein, SNAP-24, may be substituting for SNAP-25 function in the larvae. The distributions of SNAP-24 and SNAP-25 in mutant and control animals was examined using an antibody raised against a peptide sequence from exon 4 of SNAP-25 that is identical in the SNAP-25 and SNAP-24 proteins. The Exon 4 antibody recognizes both proteins equally when used on a Western blot containing lanes of equal amounts of SNAP-25 and SNAP-24. Western blot analysis of different tissues in larvae shows that, while SNAP-25 is specifically neuronal, SNAP-24 is found in all tissues examined except the larval gut. In fact, SNAP-24 is found at relatively high levels within the CNS of both control and mutant animals. In the larvae, the level of SNAP-24 in the nervous system is at least as high as that of SNAP-25. This ratio shifts during metamorphosis, so that pharate adult heads express more SNAP-25 than SNAP-24. No clear evidence was found that levels of SNAP-24 in mutant animals are upregulated to compensate for the lack of SNAP-25 (Vilinsky, 2002).

The larval CNS and neuromuscular junctions were probed for immunoreactivity to the Exon 4 antibody. Staining in SNAP-25 null larvae revealed that SNAP-24 is indeed found within synaptic boutons at the neuromuscular junction and in synaptic regions of the CNS. The pattern of staining in SNAP-25 nulls is similar to that of controls, but control larvae show greater staining intensity due to the presence of both SNAP-24 and SNAP-25. Due to the fact that SNAP-24 is also present in larval muscle, detection of SNAP-24 in synaptic boutons required confocal microscopy to separate out the signal in muscle from that in boutons (Vilinsky, 2002).

If SNAP-24 can substitute for the function of SNAP-25, how well does SNAP-24 interact with other SNARE proteins? SNAP-24 can form the characteristic 73-kD SNARE complex with syntaxin and neuronal-synaptobrevin. The ability of SNAP-24, SNAP-25, and the temperature-sensitive form of SNAP-25, SNAP-25^ts, to form SNARE complexes was compared using an in vitro assay. While SNAP-24 forms less 73-kD complex than does SNAP-25, it forms more complex than does SNAP-25^ts. Interestingly, SNAP-24 is much better than either SNAP-25 or SNAP-25^ts at forming a higher-order SNARE complex, indicating that the biochemical functions of SNAP-24 and SNAP-25 may have differences more subtle than those apparent in electrophysiological assays. In addition, SNARE complexes containing SNAP-24 migrate faster on SDS-PAGE gels than do those containing SNAP-25. The apparent size difference of SNARE complexes containing SNAP-24 cannot be accounted for by the small difference in molecular weight between SNAP-25 and SNAP-24. Thus, the size differences likely represent structural differences between complexes formed by these two proteins (Vilinsky, 2002).

While SNAP-25 is critical for adult Drosophila, the data suggest that SNAP-24 is sufficient for the animals during the larval stage. This division of function between developmental stages is not unprecedented for proteins involved in synaptic transmission and exocytosis. In mammals, SNAP-25 is alternatively spliced into two forms, SNAP-25a and SNAP-25b, which differ in the composition of the palmitoylated cysteine-rich domain thought to be responsible for association of the protein with membranes. SNAP-25a is the dominant form throughout embryonic development, while levels of SNAP-25b rise and exceed SNAP-25a postnatally. There is evidence that the developmentally significant SNAP-25a form is expressed in adult neurons that retain morphological plasticity or undergo regrowth. In Drosophila, SNAP-24 also differs from SNAP-25 in the cysteine-rich domain, where it contains three instead of four cysteine residues that could potentially affect its membrane association dynamics. SNAP-24 is found at high levels relative to SNAP-25 during the larval stage; during metamorphosis into adulthood SNAP-25 expression rises significantly relative to SNAP-24. Therefore, the roles of SNAP-24 and SNAP-25 in Drosophila may be in some ways analogous to the roles of SNAP-25a and SNAP-25b in mammals (Vilinsky, 2002).

Another parallel may be drawn between SNAP-24 and mammalian SNAP-23, a protein that is expressed widely throughout the body and is not restricted to the nervous system. While SNAP-23 is not highly expressed in the nervous system, it has been demonstrated that SNAP-23 can functionally substitute for SNAP-25 in at least some exocytosis processes, and it is found in regions of the hippocampus and cortex where SNAP-25 is absent. In flies, SNAP-24 is concentrated in mushroom body neuropil in adult brains, a region where SNAP-25 levels are very low. This segregation of expression between specific neuropils may point to more subtle differences in functional requirements for vesicle fusion. Thus, the role of SNAP-24 in the adult CNS may have parallels with the role of SNAP-23 in the mammalian brain (Vilinsky, 2002 and references therein).

In Drosophila other synaptic proteins have developmentally specific isoforms that roughly parallel those of SNAP-25 and SNAP-24. One example is N-ethylmaleimide-sensitive factor (NSF), a chaperone that uses ATP to dissociate SNARE complexes and is thought to be important for recycling SNARE proteins after a round of vesicle exocytosis. NSF has two isoforms in Drosophila, dNSF1 and dNSF2. dNSF1 and dNSF2 are expressed in the larval and adult CNS, with dNSF-2 also being expressed more widely in nonneuronal tissues. Like SNAP-25 nulls, dNSF1 loss-of-function mutants die at the pharate adult stage and are rescued by neuronal expression of a wild-type transgene. However, unlike the SNAP-25^ts allele, temperature-sensitive paralytic alleles of dNSF1 have no synaptic phenotype at the third instar larval NMJ, presumably due to the presence of dNSF2 in the larval nervous system. Future studies may resolve how different isoforms of NSF may interact with different members of the SNAP-25 gene family (Vilinsky, 2002).

Syntaxins and the sequential assembly of the SNARE assembly in exocytosis

Changes in SNARE conformations during MgATP-dependent priming of cracked PC12 cells were probed by their altered accessibility to various inhibitors. Dominant negative soluble syntaxin and, to a much lesser extent, VAMP coil domains inhibit exocytosis more efficiently after priming. Neurotoxins and an anti-SNAP25 antibody inhibit exocytosis less effectively after priming. It is proposed that SNAREs partially and reversibly assemble during priming, and that the syntaxin H3 domain is prevented from fully joining the complex until the arrival of the Ca2+ trigger. Furthermore, mutation of hydrophobic residues of the SNAP25 C-terminal coil that contribute to SNARE core interactions affects the maximal rate of exocytosis, while mutation of charged residues on the surface of the complex affects the apparent affinity of the coil domain for the partially assembled complex (Chen, 2001).

In neurons and neuroendocrine cells, exocytosis is highly regulated, and therefore SNARE-complex formation must also be highly regulated, since this complex formation catalyzes a late step or perhaps the final step of the membrane fusion reaction. How is the formation of the SNARE complex regulated in Ca2+-triggered exocytosis? Based on functional data obtained in the cracked PC12 cell system, the following is proposed: (1) SNAREs partially and reversibly assemble during priming, so that full assembly can occur very rapidly once the cell is triggered; (2) the syntaxin coil is likely to be less tightly associated than the VAMP and SNAP25 coils and it is proposed that the syntaxin coil is held back by a calcium sensor until the arrival of the Ca2+ trigger; (3) surface and core hydrophobic residues of the SNARE complex play different roles in the assembly process, with only the hydrophobic ones being important for the maximal membrane fusion rates, while the surface residues are important in the initial formation of partially assembled complexes (Chen, 2001).

The first two proposals are supported by the following observations: (1) VAMP-cleaving neurotoxins and an anti-SNAP25 antibody show decreased inhibition efficiency after priming, suggesting reduced accessibility to these two SNAREs. Note that these inhibitors are proteins of relatively large size (>50 kDa) compared to the H3 and V2 coils (8-9 kDa). The fact that the toxins are able to inhibit in primed cells also suggests that trans-SNARE complexes are reversibly (and therefore likely only partially) zippered after priming, since fully zippered complexes resist toxin cleavage. (2) Primed cells are more sensitive to inhibition by the syntaxin H3 coil, and, to a lesser extent, the VAMP2 coil than unprimed cells, suggesting that the SNAREs are more free to bind each other or are loosely associated after priming. However, the inhibition by H3 was increased much more dramatically than that by V2 after priming, suggesting that endogenous VAMP and SNAP25 preferentially associate during priming in vivo. Because the inhibition results obtained with toxins and antibody show effects opposite to those of V2 and H3, it is unlikely that probe accessibility, such as loss of a diffusion barrier, or an artifact of data normalization is the explanation for the enhanced H3 or V2 inhibition. (3) The experiments using soluble V2 + S25 or H3 + S25 as the inhibitors suggest that, whereas endogenous syntaxin is not readily available for exogenous SNARE binding regardless of priming, endogenous VAMP becomes less available for exogenous SNARE binding after priming. Perhaps in unprimed cells, n-sec1 binding to syntaxin prevents it from forming a core complex with V2 + S25, while VAMP is available to bind H3 + S25. In primed cells, however, VAMP and syntaxin both become reversibly complexed with other SNAREs, making both V2 + S25 and H3 + S25 ineffective inhibitors. It is also possible that the binding of a calcium sensor to syntaxin after priming prevents strong binding of V2 + S25 to syntaxin (Chen, 2001).

Taken together, these results are most consistent with the existence of a dynamic, partially assembled SNARE complex lacking the full syntaxin H3 domain. This is consistent with earlier electrophysiological data proposing the existence of loose or partially zippered SNARE complexes. It is proposed that, in vivo, syntaxin might be regulated to fully join the SNARE complex only after VAMP and SNAP25 partially and reversibly assemble. Previous biochemical studies demonstrate a moderate affinity (1-1.4 µM) between SNAP25 and VAMP in vitro, and circular dichroism experiments show that their binding causes an increase in the observed alpha-helicity, suggesting that the VAMP and SNAP25 interaction may occur in vivo, particularly if stabilized by other proteins. Moreover, since syntaxin is the only neuronal SNARE that has a long N-terminal region beyond the coiled-coil-forming domain, it may be the most suitable for regulation by other proteins, such as a Ca2+ sensor. Unfortunately, due to the high propensity of the SNAREs to form unproductive cis complexes in vitro, it is not possible to directly study the nature of the partial or trans-SNARE complexes involved in the priming and triggering steps of exocytosis by common biochemical methods such as immunoprecipitation or SDS-PAGE analysis (Chen, 2001).

Two distinct effects on neurotransmission in a temperature-sensitive SNAP-25 mutant

Vesicle fusion in eukaryotic cells is mediated by SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors). In neurons, the t-SNARE SNAP-25 is essential for synaptic vesicle fusion but its exact role in this process is unknown. A SNAP-25 temperature-sensitive paralytic mutant has been isolated in Drosophila, SNAP-25^ts. The mutation causes a Gly50 to Glu change in SNAP-25's first amphipathic helix. A similar mutation in the yeast homologue SEC9 also results in temperature sensitivity, implying a conserved role for this domain in secretion. In vitro-generated 70 kDa SNARE complexes containing SNAP-25^ts are thermally stable but the mutant SNARE multimers (of approximately 120 kDa) rapidly dissociate at 37±C. The SNAP-25^ts mutant has two effects on neurotransmitter release depending upon temperature. At 22±C, evoked release of neurotransmitter in SNAP-25^ts larvae is greatly increased, and at 37±C, the release of neurotransmitter is reduced as compared with controls. These data suggest that at 22±C the mutation causes the SNARE complex to be more fusion competent but, at 37±C the same mutation leads to SNARE multimer instability and fusion incompetence (Rao, 2001; full text of article).

SNAP-24, a Drosophila SNAP-25 homologue on granule membranes, is a putative mediator of secretion and granule-granule fusion in salivary glands

Fusion of vesicles with target membranes is dependent on the interaction of target (t) and vesicle (v) SNARE proteins located on opposing membranes. For fusion at the plasma membrane, the t-SNARE SNAP-25 is essential. In Drosophila, the only known SNAP-25 isoform is specific to neuronal axons and synapses and additional t-SNAREs must exist that mediate both non-synaptic fusion in neurons and constitutive and regulated fusion in other cells. The identification and characterization is reported of SNAP-24, a closely related Drosophila SNAP-25 homolog, that is expressed throughout development. The spatial distribution of SNAP-24 in the nervous system is punctate and, unlike SNAP-25, is not concentrated in synaptic regions. In vitro studies, however, show that SNAP-24 can form core complexes with syntaxin and both synaptic and non-synaptic v-SNAREs. High levels of SNAP-24 are found in larval salivary glands, where SNAP-24 localizes mainly to granule membranes rather than the plasma membrane. During glue secretion, the massive exocytotic event of these glands, SNAP-24 containing granules fuse with one another and the apical membrane, suggesting that glue secretion utilizes compound exocytosis and that SNAP-24 mediates secretion (Niemeyer, 2000).

Complex gene organization of synaptic protein SNAP-25 in Drosophila melanogaster

The evolutionarily conserved protein SNAP-25 [synaptosome-associated protein 25 kDa (kilodaltons)] is a component of the protein complex involved in the docking and/or fusion of synaptic vesicles in nerve terminals. The SNAP-25 gene (Snap) in Drosophila has a complex organization with eight exons spanning more than 120 kb (kilobases). The exon boundaries coincide with those of the chicken SNAP-25 gene. Only a single exon 5 has been found in Drosophila, whereas human, rat, chicken, zebrafish and goldfish have two alternatively spliced versions of this exon. In situ hybridization and immunocytochemistry to whole mount embryos show that SNAP-25 mRNA and protein are detected in stage 14 and later developmental stages, and are mainly localized to the ventral nerve cord. Thus, Snap has an evolutionarily conserved and complex gene organization, and its onset of expression in Drosophila melanogaster correlates with a time in neuronal development when synapses begin to be formed and when other synapse-specific genes are switched on (Risinger, 1997).

Evolutionary conservation of synaptosome-associated protein 25 kDa (SNAP-25) shown by Drosophila and Torpedo cDNA clones

The neuron-specific proteins SNAP-25 (synaptosome-associated protein 25 kDa), synaptobrevin and syntaxin, are localized to presynaptic terminals in mammals and have been found to associate with proteins involved in vesicle docking and membrane fusion. This study describes SNAP-25 cDNA clones from Drosophila and the ray Torpedo marmorata. In situ hybridization shows that SNAP-25 mRNA is exclusively found in brain and ganglia in Drosophila with a pattern suggesting expression in most neurons. The Drosophila and Torpedo proteins show 61 and 81% amino acid identity to mouse SNAP-25, a degree of conservation similar to that previously reported for synaptobrevin. None of the SNAP-25 sequences has a membrane-spanning region, but all contain a cluster of cysteine residues that can be palmitoylated for membrane attachment. SNAP-25 displays sequence similarity to syntaxin A and B. These data show that SNAP-25 and synaptobrevin, which are both implicated in vesicle docking and/or membrane fusion, have both been highly conserved during evolution. This supports the existence of a basic molecular machinery for synaptic vesicle docking in vertebrate and invertebrate synapses (Risinger, 1993).

A novel Netrin-1-sensitive mechanism promotes local SNARE-mediated exocytosis during axon branching

Developmental axon branching dramatically increases synaptic capacity and neuronal surface area. Netrin-1 promotes branching and synaptogenesis, but the mechanism by which Netrin-1 stimulates plasma membrane expansion is unknown. This study demonstrates that SNARE-mediated exocytosis is a prerequisite for axon branching and identifies the E3 ubiquitin ligase TRIM9 as a critical catalytic link between Netrin-1 and exocytic SNARE machinery in murine cortical neurons. TRIM9 ligase activity promotes SNARE-mediated vesicle fusion and axon branching in a Netrin-dependent manner. A direct interaction was identified between TRIM9 and the Netrin-1 receptor DCC as well as a Netrin-1-sensitive interaction between TRIM9 and the SNARE component SNAP25. The interaction with SNAP25 negatively regulates SNARE-mediated exocytosis and axon branching in the absence of Netrin-1. Deletion of TRIM9 elevated exocytosis in vitro and increased axon branching in vitro and in vivo. These data provide a novel model for the spatial regulation of axon branching by Netrin-1, in which localized plasma membrane expansion occurs via TRIM9-dependent regulation of SNARE-mediated vesicle fusion (Winkle, 2014).

Search PubMed for articles about Drosophila SNAP-25

Bademosi, A. T., Steeves, J., Karunanithi, S., Zalucki, O. H., Gormal, R. S., Liu, S., Lauwers, E., Verstreken, P., Anggono, V., Meunier, F. A. and van Swinderen, B. (2018), Trapping of Syntaxin1a in presynaptic nanoclusters by a clinically relevant general anesthetic. Cell Rep 22(2): 427-440. PubMed ID: 29320738

Binz, T., et al. (1994). Proteolysis of SNAP-25 by types E and A botulinal neurotoxins. J. Biol. Chem. 269: 1617-1620. PubMed ID: 8294407

Chen, X., et al. (2002). Three-dimensional structure of the complexin/SNARE complex. Neuron 33: 397-409. PubMed ID: 11832227

Chen, Y. A., Scales, S. J. Scheller, R. H. (2001). Sequential SNARE assembly underlies priming and triggering of exocytosis. Neuron 30: 161-170. PubMed ID: 11343652

de Wit, H., et al. (2009). Synaptotagmin-1 docks secretory vesicles to syntaxin-1/SNAP-25 acceptor complexes. Cell 138(5): 935-46. PubMed ID: 19716167

Jahn, R. and Scheller, R. H. (2006). SNAREs-engines for membrane fusion. Nat. Rev. Mol. Cell Biol. 7: 631-643. PubMed ID: 16912714

Karatekin, E., et al. (2010). A fast, single-vesicle fusion assay mimics physiological SNARE requirements. Proc. Natl. Acad. Sci. 107: 3517-3521. PubMed ID: 20133592

Kawasaki, F. and Ordway, R. W. (2009). Molecular mechanisms determining conserved properties of short-term synaptic depression revealed in NSF and SNAP-25 conditional mutants. Proc. Natl. Acad. Sci. 106(34): 14658-63. PubMed ID: 19706552

Lu, X., Zhang, Y. and Shin, Y. K. (2008). Supramolecular SNARE assembly precedes hemifusion in SNARE-mediated membrane fusion. Nat. Struct. Mol. Biol. 15: 700-706. PubMed ID: 18552827

Megighian, A., et al. (2010). Arg206 of SNAP-25 is essential for neuroexocytosis at the Drosophila melanogaster neuromuscular junction. J. Cell Sci. 123(Pt 19): 3276-83. PubMed ID: 20826463

Montecucco, C., Schiavo, G. and Pantano, S. (2005). SNARE complexes and neuroexocytosis: how many, how close? Trends Biochem. Sci. 30: 367-372. PubMed ID: 15935678

Niemeyer, B. A. and Schwarz, T. L. (2000). SNAP-24, a Drosophila SNAP-25 homologue on granule membranes, is a putative mediator of secretion and granule-granule fusion in salivary glands. J. Cell Sci. 113: 4055-64. PubMed ID: 11058092

Rao, S. S., et al. (2000). Two distinct effects on neurotransmission in a temperature-sensitive SNAP-25 mutant. EMBO J. 20(23): 6761-71. PubMed ID: 11726512

Rickman, C., Hu, K., Carroll, J. and Davletov, B. (2005). Self-assembly of SNARE fusion proteins into star-shaped oligomers. Biochem. J. 388: 75-79. PubMed ID: 15877547

Risinger, C., et al. (1993). Evolutionary conservation of synaptosome-associated protein 25 kDa (SNAP-25) shown by Drosophila and Torpedo cDNA clones. J. Biol. Chem. 268(32): 24408-14. PubMed ID: 8226991

Risinger, C., et al. (1997). Complex gene organization of synaptic protein SNAP-25 in Drosophila melanogaster. Gene 194(2): 169-77. PubMed ID: 9272858

Schiavo, G., et al. (1993). Botulinum neurotoxins serotypes A and E cleave SNAP-25 at distinct COOH-terminal peptide bonds. FEBS Lett. 335: 99-103. PubMed ID: 8243676

Sorensen, J. B., et al. (2006). Sequential N- to C-terminal SNARE complex assembly drives priming and fusion of secretory vesicles. EMBO J 25: 955-966. PubMed ID: 16498411

Sudhof, T. C. and Rothman, J. E. (2009). Membrane fusion: grappling with SNARE and SM proteins. Science 323: 474-477. PubMed ID: 19164740

Vilinsky, I., et al. (2002). A Drosophila SNAP-25 null mutant reveals context-dependent redundancy with SNAP-24 in neurotransmission. Genetics 162: 259-271. PubMed ID: 12242238

Washbourne, P., et al. (2002). Genetic ablation of the t-SNARE SNAP-25 distinguishes mechanisms of neuroexocytosis. Nat. Neurosci. 5: 19-26. PubMed ID: 11753414

Winkle, C. C., McClain, L. M., Valtschanoff, J. G., Park, C. S., Maglione, C. and Gupton, S. L. (2014). A novel Netrin-1-sensitive mechanism promotes local SNARE-mediated exocytosis during axon branching. J Cell Biol 205: 217-232. PubMed ID: 24778312

Yersin, A., et al. (2003). Interactions between synaptic vesicle fusion proteins explored by atomic force microscopy. Proc. Natl. Acad. Sci. 100: 8736-8741. PubMed ID: 12853568

date revised: 23 July 2014

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