Emma Connell

Mechanism of arachidonic acid action on syntaxin-Munc18

Syntaxin and Munc18 are, in tandem, essential for exocytosis in all eukaryotes. Recently, it was shown that Munc18 inhibition of neuronal syntaxin 1 can be overcome by arachidonic acid, indicating that this common second messenger acts to disrupt the syntaxin–Munc18 interaction. Here, we show that arachidonic acid can stimulate syntaxin 1 alone, indicating that it is syntaxin 1 that undergoes a structural change in the syntaxin 1–Munc18 complex. Arachidonic acid is incapable of dissociating Munc18 from syntaxin 1 and, crucially, Munc18 remains associated with syntaxin 1 after arachidonic‐acid‐induced syntaxin 1 binding to synaptosomal‐associated protein 25 kDa (SNAP25). We also show that the same principle operates in the case of the ubiquitous syntaxin 3 isoform, highlighting the conserved nature of the mechanism of arachidonic acid action. Neuronal soluble N‐ethyl maleimide sensitive factor attachment protein receptors (SNAREs) can be isolated from brain membranes in a complex with endogenous Munc18, consistent with a proposed function of Munc18 in vesicle docking and fusion.

DOC2B Acts as a Calcium Switch and Enhances Vesicle Fusion

Calcium-dependent exocytosis is regulated by a vast number of proteins. DOC2B is a synaptic protein that translocates to the plasma membrane (PM) after small elevations in intracellular calcium concentration. The aim of this study was to investigate the role of DOC2B in calcium-triggered exocytosis. Using biochemical and biophysical measurements, we demonstrate that the C2A domain of DOC2B interacts directly with the PM in a calcium-dependent manner. Using a combination of electrophysiological, morphological, and total internal reflection fluorescent measurements, we found that DOC2B acts as a priming factor and increases the number of fusion-competent vesicles. Comparing secretion during repeated stimulation between wild-type DOC2B and a mutated DOC2B that is constantly at the PM showed that DOC2B enhances catecholamine secretion also during repeated stimulation and that DOC2B has to translocate to the PM to exert its facilitating effect, suggesting that its activity is dependent on calcium. The hypothesis that DOC2B exerts its effect at the PM was supported by the finding that DOC2B affects the fusion kinetics of single vesicles and interacts with the PM SNAREs (soluble NSF attachment receptors). We conclude that DOC2B is a calcium-dependent priming factor and its activity at the PM enables efficient expansion of the fusion pore, leading to increased catecholamine release.

Phospholipases and fatty acid signalling in exocytosis.

Vesicle fusion is a ubiquitous biological process involved in general membrane trafficking and a variety of specialized events, for example release of neurotransmitters and hormones, sperm acrosome exocytosis, plasma membrane repair and neurite outgrowth. Many vesicle fusion events have long been known to be activated by phospholipases and products of their activity, such as polyunsaturated arachidonic acid. Polyunsaturated fatty acids (PUFAs) have been proposed to have a number of multiple effectors, including ion channels and the cytoskeleton, but the precise mechanism of PUFA action is still unclear. It was recently reported that omega‐3 and omega‐6 PUFAs can act on syntaxin, a plasma membrane protein directly involved in vesicle fusion. In this review, we will discuss the role of this new mode of PUFA action in exocytosis.

Synaptotagmin Interaction with SNAP-25 Governs Vesicle Docking, Priming, and Fusion Triggering

SNARE complex assembly constitutes a key step in exocytosis that is rendered Ca2+-dependent by interactions with synaptotagmin-1. Two putative sites for synaptotagmin binding have recently been identified in SNAP-25 using biochemical methods: one located around the center and another at the C-terminal end of the SNARE bundle. However, it is still unclear whether and how synaptotagmin-1 × SNARE interactions at these sites are involved in regulating fast neurotransmitter release. Here, we have used electrophysiological techniques with high time-resolution to directly investigate the mechanistic ramifications of proposed SNAP-25 × synaptotagmin-1 interaction in mouse chromaffin cells. We demonstrate that the postulated central binding domain surrounding layer zero covers both SNARE motifs of SNAP-25 and is essential for vesicle docking, priming, and fast fusion-triggering. Mutation of this site caused no further functional alterations in synaptotagmin-1-deficient cells, indicating that the central acidic patch indeed constitutes a mechanistically relevant synaptotagmin-1 interaction site. Moreover, our data show that the C-terminal binding interface only plays a subsidiary role in triggering but is required for the full size of the readily releasable pool. Intriguingly, we also found that mutation of synaptotagmin-1 interaction sites led to more pronounced phenotypes in the context of the adult neuronal isoform SNAP-25B than in the embryonic isoform SNAP-25A. Further experiments demonstrated that stronger synaptotagmin-1 × SNAP-25B interactions allow for the larger primed vesicle pool supported by SNAP-25 isoform B. Thus, synaptotagmin-1 × SNARE interactions are not only required for multiple mechanistic steps en route to fusion but also underlie the developmental control of the releasable vesicle pool.

Synaptic Vesicle Docking: Sphingosine Regulates Syntaxin1 Interaction with Munc18

Consensus exists that lipids must play key functions in synaptic activity but precise mechanistic information is limited. Acid sphingomyelinase knockout mice (ASMko) are a suitable model to address the role of sphingolipids in synaptic regulation as they recapitulate a mental retardation syndrome, Niemann Pick disease type A (NPA), and their neurons have altered levels of sphingomyelin (SM) and its derivatives. Electrophysiological recordings showed that ASMko hippocampi have increased paired-pulse facilitation and post-tetanic potentiation. Consistently, electron microscopy revealed reduced number of docked vesicles. Biochemical analysis of ASMko synaptic membranes unveiled higher amounts of SM and sphingosine (Se) and enhanced interaction of the docking molecules Munc18 and syntaxin1. In vitro reconstitution assays demonstrated that Se changes syntaxin1 conformation enhancing its interaction with Munc18. Moreover, Se reduces vesicle docking in primary neurons and increases paired-pulse facilitation when added to wt hippocampal slices. These data provide with a novel mechanism for synaptic vesicle control by sphingolipids and could explain cognitive deficits of NPA patients.

Regulation of SNARE fusion machinery by fatty acids

Abstract.Vesicle fusion is a ubiquitous biological process involved in membrane trafficking and a variety of specialised events such as exocytosis and neurite outgrowth. The energy to drive biological membrane fusion is provided by fusion proteins called SNAREs. Indeed, SNARE proteins play critical roles in neuronal development as well as neurotransmitter and hormone release. SNARE proteins form a very tight alpha-helical bundle that can pull two membranes together, thereby initiating fusion. Whereas a great deal of attention has been paid to partner proteins that can affect SNARE function, recent genetic and biochemical evidence suggests that local lipid environment may be as important in SNARE regulation. Direct lipid modification of SNARE fusion proteins and their regulation by fatty acids following phospholipase action will be discussed here in detail. Our analysis highlights the fact that lipids are not a passive platform in vesicle fusion but intimately regulate SNARE function.

Cross-linking of Phospholipid Membranes is a Conserved Property of Calcium-sensitive Synaptotagmins

Synaptotagmins are vesicular proteins implicated in many membrane trafficking events. They are highly conserved in evolution and the mammalian family contains 16 isoforms. We now show that the tandem C2 domains of several calcium-sensitive synaptotagmin isoforms tested, including Drosophila synaptotagmin, rapidly cross-link phospholipid membranes. In contrast to the tandem structure, individual C2 domains failed to trigger membrane cross-linking in several novel assays. Large-scale liposomal aggregation driven by tandem C2 domains in response to calcium was confirmed by the following techniques: turbidity assay, dynamic light-scattering and both confocal and negative stain electron microscopy. Firm cross-linking of membranes was evident from laser trap experiments. High-resolution cryo-electron microscopy revealed that membrane cross-linking by tandem C2 domains results in a constant distance of ∼9 nm between the apposed membranes. Our findings show the conserved nature of this important property of synaptotagmin, demonstrate the significance of the tandem C2 domain structure and provide a plausible explanation for the accelerating effect of synaptotagmins on membrane fusion.

Real-time assay for monitoring membrane association of lipid-binding domains

The C2 domain is a common protein module which mediates calcium-dependent phospholipid binding. Several assays have previously been developed to measure membrane association. However, these assays either have technical drawbacks or are laborious to carry out. We now present a simple solution-based turbidity method for rapidly assaying membrane association of single lipid-binding domains in real time. We used the first C2 domain of synaptotagmin1 (C2A) as a model lipid-binding moiety. Our use of the common dimeric glutathione-S-transferase (GST) fusion tag allowed two C2A domains to be brought into close proximity. Consequently, calcium-triggered phospholipid binding by this artificially dimerized C2A resulted in liposomal aggregation, easily assayed by following absorbance of the solution at 350 nm. The assay is simple and sensitive and can be scaled up conveniently for use in a multiwell plate format, allowing high-throughput screening. In our screens, we identified nickel as a novel activator of synaptotagmin1 C2A domain membrane association. Finally, we show that the turbidity method can be applied to the study of other GST-tagged lipid-binding proteins such as epsin, protein kinase C-β, and synaptobrevin.

N-Terminal Acetylation of the Neuronal Protein SNAP-25 Is Revealed by the SMI81 Monoclonal Antibody

The monoclonal antibody SMI81 binds SNAP-25, a major player in neurotransmitter release, with high affinity and has previously been used to follow changes in the levels of this protein in neuropsychiatric disorders. We report here that the SMI81 epitope is present at the extreme N-terminus of SNAP-25 and, unusually, cannot be recognized when present as an internal sequence. Although it is known that SNAP-25 can be palmitoylated and phosphorylated in brain, we now reveal the existence of a third modification, acetylation of the N-terminus. This acetylation event greatly increases the efficiency of SMI81 antibody binding. We show that this highly specific antibody can be used for studying brain function in many vertebrate organisms.

Functional Characterization of Putative Synaptotagmin-Binding Interfaces in SNAP-25

SNARE complexes possess multiple negative surface charges that probably mediate association with regulatory factors during exocytosis. Several distinct groups of negatively charged residues in SNAP-25 have been identified as essential for binding synaptotagmin-1 (syt-1). One group exists at the C-terminal end (D172/D179/D186/D193), while another site extends around Layer 0 (D51/E52/E55). A third potential group may consist of D166/E170, as we previously found that E170 is essential for fast release. To investigate the nature of the binding interface, we introduced alanine-substitutions at the three putative binding sites and characterized the resulting mutant variants after expression in SNAP-25 -/- chromaffin cells. Secretion was induced by photolysis of caged-Ca2+ and assayed by capacitance measurements and amperometric recordings. We found substantial differences in the phenotypes of these mutant variants, which excludes that all sites cooperate in the same mode of syt-1 interaction. Though no variant fully phenocopied the changes seen in syt-1 -/- cells, SNAP-25A D51A/E52A/E55A caused a slowdown of secretion reminiscent of the syt-1 -/- phenotype. SNAP-25A D166A/E170A decreased total release substantially and abolished the fast burst component, while SNAP-25A D172A/D179A/D186A/D193A had little effect on secretion. Pull-down assays demonstrated that mutation of the two groups around Layer 0 indeed restricted syt-1 binding, while mutation of the C-terminal site did not decrease affinity towards syt-1. By electron microscopy we found that all mutations significantly affected docking of vesicles, which depends on both SNAP-25 and syt-1 (De Wit et al., 2009, Cell 138:935-946). This implies that SNAP-25/Syt-1 assisted docking might rely on another mode of interaction between SNAP-25 and syt-1 than fusion triggering. Our data thus suggest that the interaction between SNAREs and syt-1 might involve multiple interaction modes, and that negative charges around Layer 0 most likely function as a syt-1 binding interface during exocytosis triggering.