Ca(2+)-synaptotagmin directly regulates t-SNARE function during reconstituted membrane fusion

In nerve terminals, exocytosis is mediated by SNARE proteins and regulated by Ca(2+) and synaptotagmin-1 (syt). Ca(2+) promotes the interaction of syt with anionic phospholipids and the target membrane SNAREs (t-SNAREs) SNAP-25 and syntaxin. Here, we have used a defined reconstituted fusion assay to determine directly whether syt-t-SNARE interactions couple Ca(2+) to membrane fusion by comparing the effects of Ca(2+)-syt on neuronal (SNAP-25, syntaxin and synaptobrevin) and yeast (Sso1p, Sec9c and Snc2p) SNAREs. Ca(2+)-syt aggregated neuronal and yeast SNARE liposomes to similar extents via interactions with anionic phospholipids. However, Ca(2+)-syt was able to bind and stimulate fusion mediated by only neuronal SNAREs and had no effect on yeast SNAREs. Thus, Ca(2+)-syt regulates fusion through direct interactions with t-SNAREs and not solely through aggregation of vesicles. Ca(2+)-syt drove assembly of SNAP-25 onto membrane-embedded syntaxin, providing direct evidence that Ca(2+)-syt alters t-SNARE structure.     

Analysis of the synaptotagmin family during reconstituted membrane fusion. Uncovering a class of inhibitory isoforms

Ca(2+)-triggered exocytosis in neurons and neuroendocrine cells is regulated by the Ca(2+)-binding protein synaptotagmin (syt) I. Sixteen additional isoforms of syt have been identified, but little is known concerning their biochemical or functional properties. Here, we assessed the abilities of fourteen syt isoforms to directly regulate SNARE (soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor)-catalyzed membrane fusion. One group of isoforms stimulated neuronal SNARE-mediated fusion in response to Ca(2+), while another set inhibited SNARE catalyzed fusion in both the absence and presence of Ca(2+). Biochemical analysis revealed a strong correlation between the ability of syt isoforms to bind 1,2-dioleoyl phosphatidylserine (PS) and t-SNAREs in a Ca(2+)-promoted manner with their abilities to enhance fusion, further establishing PS and SNAREs as critical effectors for syt action. The ability of syt I to efficiently stimulate fusion was specific for certain SNARE pairs, suggesting that syts might contribute to the specificity of intracellular membrane fusion reactions. Finally, a subset of inhibitory syts down-regulated the ability of syt I to activate fusion, demonstrating that syt isoforms can modulate the function of each other.     

Synaptotagmin isoforms couple distinct ranges of Ca2+, Ba2+, and Sr2+ concentration to SNARE-mediated membrane fusion

Ca2+-triggered exocytosis of synaptic vesicles is controlled by the Ca2+-binding protein synaptotagmin (syt) I. Fifteen additional isoforms of syt have been identified. Here, we compared the abilities of three syt isoforms (I, VII, and IX) to regulate soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-mediated membrane fusion in vitro in response to divalent cations. We found that different isoforms of syt couple distinct ranges of Ca2+, Ba2+, and Sr2+ to membrane fusion; syt VII was approximately 400-fold more sensitive to Ca2+ than was syt I. Omission of phosphatidylserine (PS) from both populations of liposomes completely abrogated the ability of all three isoforms of syt to stimulate fusion. Mutations that selectively inhibit syt.target-SNARE (t-SNARE) interactions reduced syt stimulation of fusion. Using Sr2+ and Ba2+, we found that binding of syt to PS and t-SNAREs can be dissociated from activation of fusion, uncovering posteffector-binding functions for syt. Our data demonstrate that different syt isoforms are specialized to sense different ranges of divalent cations and that PS is an essential effector of Ca2+.syt action.     

The C2 domains of synaptotagmin--partners in exocytosis

Rapid signaling between neurons relies on the Ca(2+)-triggered exocytosis of neurotransmitters. Release is mediated by 'kiss-and-run' or complete fusion of secretory organelles with the plasma membrane. Current models indicate that exocytosis is regulated by synaptotagmin I (syt) and mediated by SNARE (soluble NSF-attachment protein receptor) proteins. Syt senses Ca(2+) via two conserved motifs, C2A and C2B. C2B engages a wider array of effector molecules than C2A and appears to play a more crucial role in synaptic transmission. However, it has recently become clear that the tandem C2 domains of syt influence each another in unexpected ways. Here, we focus on recent structure-function studies that are beginning to provide insights into the mechanism through which the C2 domains of syt trigger exocytosis.     

Complexin arrests a pool of docked vesicles for fast Ca(2+)-dependent release

Regulated exocytosis requires that the assembly of the basic membrane fusion machinery is temporarily arrested. Synchronized membrane fusion is then caused by a specific trigger-a local rise of the Ca(2+) concentration. Using reconstituted giant unilamellar vesicles (GUVs), we have analysed the role of complexin and membrane-anchored synaptotagmin 1 in arresting and synchronizing fusion by lipid-mixing and cryo-electron microscopy. We find that they mediate the formation and consumption of docked small unilamellar vesicles (SUVs) via the following sequence of events: Synaptotagmin 1 mediates v-SNARE-SUV docking to t-SNARE-GUVs in a Ca(2+)-independent manner. Complexin blocks vesicle consumption, causing accumulation of docked vesicles. Together with synaptotagmin 1, complexin synchronizes and stimulates rapid fusion of accumulated docked vesicles in response to physiological Ca(2+) concentrations. Thus, the reconstituted assay resolves both the stimulatory and inhibitory function of complexin and mimics key aspects of synaptic vesicle fusion.     

Conformation and membrane position of the region linking the two C2 domains in synaptotagmin 1 by site-directed spin labeling

Synaptotagmin 1 (syt1) is an integral membrane protein localized on the synaptic vesicle that acts as the Ca(2+) sensor for neuronal exocytosis. Synaptotagmin 1 contains two C2 domains, C2A and C2B, which bind Ca(2+) ions, membranes, and SNAREs. Here, site-directed spin labeling (SDSL) was used to determine the position and dynamics of the region that links the two C2 domains in a water soluble construct encompassing the two C2 domains (syt1C2AB). An analysis of the EPR line shapes from this region indicates that the linker is flexible and unstructured when syt1 is in solution or bound to lipid bilayers. The nanosecond dynamics of the linker does not change, in the presence or absence of Ca(2+), suggesting that there is no Ca(2+)-dependent intramolecular association between the two domains. When syt1C2AB is membrane-bound, the position of the linker relative to the membrane interface was determined by measuring parameters for the collision of the spin-labeled syt1C2AB mutants with both soluble and membrane-bound Ni(II) chelates. These data indicate that the linker does not penetrate the membrane surface but lies approximately 7-10 A from the bilayer surface. In addition, the linker remains flexible when syt1C2AB binds to the SNARE complex, indicating that direct interactions between this linker and the SNAREs do not mediate association. These data suggest that the two C2 domains of syt1 interact independently on the membrane interface, or when bound to SNAREs.     

Phosphatidylinositol 4,5-bisphosphate alters synaptotagmin 1 membrane docking and drives opposing bilayers closer together

Synaptotagmin 1 (syt1) is a synaptic vesicle-anchored membrane protein that acts as the calcium sensor for the synchronous component of neuronal exocytosis. Using site-directed spin labeling, the position and membrane interactions of a fragment of syt1 containing its two C2 domains (syt1C2AB) were assessed in bilayers containing phosphatidylcholine (PC), phosphatidylserine (PS), and phosphatidylinositol 4,5-bisphosphate (PIP(2)). Addition of 1 mol % PIP(2) to a lipid mixture of PC and PS results in a deeper membrane penetration of the C2A domain and alters the orientation of the C2B domain so that the polybasic face of C2B comes into the proximity of the bilayer interface. The C2B domain is found to contact the membrane interface in two regions, the Ca(2+)-binding loops and a region opposite the Ca(2+)-binding loops. This suggests that syt1C2AB is configured to bridge two bilayers and is consistent with a model generated previously for syt1C2AB bound to membranes of PC and PS. Point-to-plane depth restraints, obtained by progressive power saturation, and interdomain distance restraints, obtained by double electron-electron resonance, were obtained in the presence of PIP(2) and used in a simulated annealing routine to dock syt1C2AB to two membrane interfaces. The results yield an average structure different from what is found in the absence of PIP(2) and indicate that bilayer-bilayer spacing is decreased in the presence of PIP(2). The results indicate that PIP(2), which is necessary for bilayer fusion, alters C2 domain orientation, enhances syt1-membrane electrostatic interactions, and acts to drive vesicle and cytoplasmic membrane surfaces closer together.     

Synaptotagmin C2B domain regulates Ca2+-triggered fusion in vitro: critical residues revealed by scanning alanine mutagenesis

Synaptotagmin (syt) 1 is localized to synaptic vesicles, binds Ca2+, and regulates neuronal exocytosis. Syt 1 harbors two Ca2+-binding motifs referred to as C2A and C2B. In this study we examine the function of the isolated C2 domains of Syt 1 using a reconstituted, SNARE (soluble N-ethylmaleimide-sensitive factor attachment receptor)-mediated, fusion assay. We report that inclusion of phosphatidylethanolamine into reconstituted SNARE vesicles enabled isolated C2B, but not C2A, to regulate Ca2+-triggered fusion. The isolated C2B domain had a 6-fold lower EC50 for Ca2+-activated fusion than the intact cytosolic domain of Syt 1 (C2AB). Phosphatidylethanolamine increased both the rate and efficiency of C2AB- and C2B-regulated fusion without affecting their abilities to bind membrane-embedded syntaxin-SNAP-25 (t-SNARE) complexes. At equimolar concentrations, the isolated C2A domain was an effective inhibitor of C2B-, but not C2AB-regulated fusion; hence, C2A has markedly different effects in the fusion assay depending on whether it is tethered to C2B. Finally, scanning alanine mutagenesis of C2AB revealed four distinct groups of mutations within the C2B domain that play roles in the regulation of SNARE-mediated fusion. Surprisingly, substitution of Arg-398 with alanine, which lies on the opposite end of C2B from the Ca2+/membrane-binding loops, decreases C2AB t-SNARE binding and Ca2+-triggered fusion in vitro without affecting Ca2+-triggered interactions with phosphatidylserine or vesicle aggregation. In addition, some mutations uncouple the clamping and stimulatory functions of syt 1, suggesting that these two activities are mediated by distinct structural determinants in C2B.     

Synaptotagmin 1 modulates lipid acyl chain order in lipid bilayers by demixing phosphatidylserine

Synaptotagmin 1 (syt1) functions as the Ca(2+) sensor in neuronal exocytosis, and it has been proposed to act by modulating lipid bilayer curvature. Here we examine the effect of the two C2 domains (C2A and C2B) of syt1 on membrane lipid order and lateral organization. In mixtures of phosphatidylcholine and phosphatidylserine (PS), attenuated total internal reflection Fourier transform infrared spectroscopy indicates that a fragment containing both domains (C2AB) or C2B alone disorders the lipid acyl chains, whereas the C2A domain has little effect upon chain order. Two observations suggest that these changes reflect a demixing of PS. First, the changes in acyl chain order are reversed at higher protein concentration; second, selective lipid deuteration demonstrates that the changes in lipid order are associated only with the PS component of the bilayer. Independent evidence for lipid demixing is obtained from fluorescence self-quenching of labeled lipid and from natural abundance (13)C NMR, where heteronuclear single quantum correlation spectra reveal Ca(2+)-dependent chemical shift changes for PS, but not for phosphatidylcholine, in the presence of the syt1 C2 domains. The ability of syt1 to demix PS is observed in a range of lipid mixtures that includes cholesterol, phosphatidylethanolamine, and varied PS content. These data suggest that syt1 might facilitate SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptors)-mediated membrane fusion by phase separating PS, a process that is expected to locally buckle bilayers and disorder lipids due to the curvature tendencies of PS.     

SNAREpin Assembly by Munc18-1 Requires Previous Vesicle Docking by Synaptotagmin 1

Regulated exocytosis requires the general membrane fusion machinery-soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) and Sec1/Munc18 (SM) proteins. Using reconstituted giant unilamellar vesicles containing preassembled t-SNARE proteins (syntaxin 1·SNAP-25), we determined how Munc18-1 controls the docking, priming, and fusion of small unilamellar vesicles containing the v-SNARE VAMP2 and the Ca(2+) sensor synaptotagmin 1. In vitro assays allowed us to position Munc18-1 in the center of a sequential reaction cascade; vesicle docking by synaptotagmin 1 is a prerequisite for Munc18-1 to accelerate trans-SNARE complex (SNAREpin) assembly and membrane fusion. Complexin II stalls SNAREpin zippering at a late stage and, hence, contributes to synchronize membrane fusion in a Ca(2+)- and synaptotagmin 1-dependent manner. Thus, at the neuronal synapse, the priming factor Munc18-1 may accelerate the conversion of docked synaptic vesicles into a readily releasable pool by activating SNAREs for efficient membrane fusion.     

Synaptotagmin I and Ca(2+) promote half fusion more than full fusion in SNARE-mediated bilayer fusion

Synaptic membrane fusion, which is necessary for neurotransmitter release, may be mediated by SNAREs and regulated by synaptotagmin (Syt) and Ca(2+). Fusion of liposomes mediated by reconstituted SNAREs produces full fusion and hemifusion, a membrane structure in which outer leaflets are mixed but the inner leaflets remain intact. Here, using the liposome fusion assay, it is shown that Syt promoted both hemifusion and full fusion in a Ca(2+)-dependent manner. Syt.Ca(2+) increased hemifusion more than full fusion, modulating the ratio of hemifusion to full fusion. Unlike the case of neuronal SNAREs, stimulation of fusion by Syt.Ca(2+) was not seen for other SNAREs involved in trafficking in yeast, indicating that the Syt.Ca(2+) stimulation was SNARE-specific. We constructed hybrid SNAREs in which transmembrane domains were swapped between neuronal and yeast SNAREs. With these hybrid SNAREs, we demonstrated that the interaction between the SNARE motifs of neuronal proteins and Syt.Ca(2+) was required for the stimulation of fusion.     

Synaptotagmin perturbs the structure of phospholipid bilayers

Synaptotagmin I (syt), an integral protein of the synaptic vesicle membrane, is believed to act as a Ca2+ sensor for neuronal exocytosis. Syt's cytoplasmic domain consists largely of two C2 domains, C2A and C2B. In response to Ca2+ binding, the C2 domains interact with membranes, becoming partially embedded in the lipid bilayer. We have imaged syt C2AB in association with lipid bilayers under fluid, using AFM. As expected, binding of C2AB to bilayers required both an anionic phospholipid [phosphatidylserine (PS)] and Ca2+. C2AB associated with bilayers in the form of aggregates of varying stoichiometries, and aggregate size increased with an increase in PS content. Repeated scanning of bilayers revealed that as C2AB dissociated it left behind residual indentations in the bilayer. The mean depth of these identations was 1.81 nm, indicating that they did not span the bilayer. Individual C2 domains (C2A and C2B) also formed aggregates and produced bilayer indentations. Binding of C2AB to bilayers and the formation of indentations were significantly compromised by mutations that interfere with binding of Ca2+ to syt or reduce the positive charge on the surface of C2B. We propose that bilayer perturbation by syt might be significant with respect to its ability to promote membrane fusion.     

Fusion pore dynamics are regulated by synaptotagmin*t-SNARE interactions

Exocytosis involves the formation of a fusion pore that connects the lumen of secretory vesicles with the extracellular space. Exocytosis from neurons and neuroendocrine cells is tightly regulated by intracellular [Ca2+] and occurs rapidly, but the molecular events that mediate the opening and subsequent dilation of fusion pores remain to be determined. A putative Ca2+ sensor for release, synaptotagmin I (syt), binds directly to syntaxin and SNAP-25, which are components of a conserved membrane fusion complex. Here, we show that Ca2+-triggered syt*SNAP-25 interactions occur rapidly. The tandem C2 domains of syt cooperate to mediate binding to syntaxin/SNAP-25; lengthening the linker that connects C2A and C2B selectively disrupts this interaction. Expression of the linker mutants in PC12 cells results in graded reductions in the stability of fusion pores. Thus, the final step of Ca2+-triggered exocytosis is regulated, at least in part, by direct contacts between syt and SNAP-25/syntaxin.     

C2B polylysine motif of synaptotagmin facilitates a Ca2+-independent stage of synaptic vesicle priming in vivo

Synaptotagmin I, a synaptic vesicle protein required for efficient synaptic transmission, contains a highly conserved polylysine motif necessary for function. Using Drosophila, we examined in which step of the synaptic vesicle cycle this motif functions. Polylysine motif mutants exhibited an apparent decreased Ca2+ affinity of release, and, at low Ca2+, an increased failure rate, increased facilitation, and increased augmentation, indicative of a decreased release probability. Disruption of Ca2+ binding, however, cannot account for all of the deficits in the mutants; rather, the decreased release probability is probably due to a disruption in the coupling of synaptotagmin to the release machinery. Mutants exhibited a major slowing of recovery from synaptic depression, which suggests that membrane trafficking before fusion is disrupted. The disrupted process is not endocytosis because the rate of FM 1-43 uptake was unchanged in the mutants, and the polylysine motif mutant synaptotagmin was able to rescue the synaptic vesicle depletion normally found in syt(null) mutants. Thus, the polylysine motif functions after endocytosis and before fusion. Finally, mutation of the polylysine motif inhibits the Ca2+-independent ability of synaptotagmin to accelerate SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor)-mediated fusion. Together, our results demonstrate that the polylysine motif is required for efficient Ca2+-independent docking and/or priming of synaptic vesicles in vivo.     

Identification of synaptotagmin effectors via acute inhibition of secretion from cracked PC12 cells

The synaptotagmins (syts) are a family of membrane proteins proposed to regulate membrane traffic in neuronal and nonneuronal cells. In neurons, the Ca2+-sensing ability of syt I is critical for fusion of docked synaptic vesicles with the plasma membrane in response to stimulation. Several putative Ca2+-syt effectors have been identified, but in most cases the functional significance of these interactions remains unknown. Here, we have used recombinant C2 domains derived from the cytoplasmic domains of syts I-XI to interfere with endogenous syt-effector interactions during Ca2+-triggered exocytosis from cracked PC12 cells. Inhibition was closely correlated with syntaxin-SNAP-25 and phosphatidylinositol 4,5-bisphosphate (PIP2)-binding activity. Moreover, we measured the expression levels of endogenous syts in PC12 cells; the major isoforms are I and IX, with trace levels of VII. As expected, if syts I and IX function as Ca2+ sensors, fragments from these isoforms blocked secretion. These data suggest that syts trigger fusion via their Ca2+-regulated interactions with t-SNAREs and PIP2, target molecules known to play critical roles in exocytosis.     

High metal concentrations are required for self-association of synaptotagmin II

Several members of the synaptotagmin (syt) family of vesicle proteins have been proposed to act as Ca2+ sensors on synaptic vesicles. The mechanism by which calcium activates this class of proteins has been the subject of controversy, yet relatively few detailed biophysical studies have been reported on how isoforms other than syt I respond to divalent metal ions. Here, we report a series of studies on the response of syt II to a wide range of metal ions. Analytical ultracentrifugation studies demonstrate that Ca2+ induces protein dimerization upon exposure to 5 mM Ca2+. Whereas Ba2+, Mg2+, or Sr2+ do not potentiate self-association as strongly as Ca2+, Pb2+ triggers self-association of syt II at concentrations as low as 10 microM. Partial proteolysis studies suggest that the various divalent metals cause different changes in the conformation of the protein. The high calcium concentrations required for self-association of syt II suggest that the oligomerized state of this protein is not a critical intermediate in vesicle fusion; however, low-affinity calcium sites on syt II may play a critical role in buffering calcium at the presynaptic active zone. In addition, the high propensity of lead to oligomerize syt II offers a possible molecular explanation for how lead interferes with calcium-evoked neurotransmitter release.     

Exocytotic steps in cell-free system after cholesterol deprivation in synaptosomal plasma membranes and synaptic vesicles

Using a cell-free system we investigated a specific role of cholesterol in exocytotic processes. To modulate the cholesterol content in membrane methyl-beta-cyclodextrin was used as a cholesterol binding agent. The experimental conditions for cholesterol depletion from synaptosomal membrane structures were determined and depended on methyl-beta-cyclodextrin concentration, time and mediums temperature. The role of cholesterol was studied on the stages of synaptic vesicles docking and Ca(2+)-stimulated fusion which are the components of multivesicular compound exocytosis. Using dynamic light scattering technique we have found that after cholesterol depletion from synaptic vesicles the process of their aggregation (docking) remains unchanged. It was found that the rate of calcium-triggered fusion of synaptic vesicles depends on the membrane level of cholesterol. The decreasing level of synaptosomal plasma membrane cholesterol by 8% leads to suppression of the Ca(2+)-dependent membrane fusion with synaptic vesicles. But, under 25% reduction of plasma membrane cholesterol the level of membrane merging with synaptic vesicles did not differ from control; probably this is due to changes in physical properties of lipid bilayer and/ or disturbances in function of membrane proteins driving this process. In cholesterol depleted synaptosomes the exocytotic release of glutamate stimulated by calcium was decreased by 32%. Obtained data suggest that the cholesterol concenration in synaptosomal plasma membranes or synaptic vesicles is the crucial determinant for synaptic transmission efficiency in nerve terminals.     

RIM binding proteins (RBPs) couple Rab3-interacting molecules (RIMs) to voltage-gated Ca(2+) channels

Ca(2+) influx through voltage-gated channels initiates the exocytotic fusion of synaptic vesicles to the plasma membrane. Here we show that RIM binding proteins (RBPs), which associate with Ca(2+) channels in hair cells, photoreceptors, and neurons, interact with alpha(1D) (L type) and alpha(1B) (N type) Ca(2+) channel subunits. RBPs contain three Src homology 3 domains that bind to proline-rich motifs in alpha(1) subunits and Rab3-interacting molecules (RIMs). Overexpression in PC12 cells of fusion proteins that suppress the interactions of RBPs with RIMs and alpha(1) augments the exocytosis triggered by depolarization. RBPs may regulate the strength of synaptic transmission by creating a functional link between the synaptic-vesicle tethering apparatus, which includes RIMs and Rab3, and the fusion machinery, which includes Ca(2+) channels and the SNARE complex.     

Synaptotagmin VII as a plasma membrane Ca(2+) sensor in exocytosis

Synaptotagmins I and II are Ca(2+) binding proteins of synaptic vesicles essential for fast Ca(2+)-triggered neurotransmitter release. However, central synapses and neuroendocrine cells lacking these synaptotagmins still exhibit Ca(2+)-evoked exocytosis. We now propose that synaptotagmin VII functions as a plasma membrane Ca(2+) sensor in synaptic exocytosis complementary to vesicular synaptotagmins. We show that alternatively spliced forms of synaptotagmin VII are expressed in a developmentally regulated pattern in brain and are concentrated in presynaptic active zones of central synapses. In neuroendocrine PC12 cells, the C(2)A and C(2)B domains of synaptotagmin VII are potent inhibitors of Ca(2+)-dependent exocytosis, but only when they bind Ca(2+). Our data suggest that in synaptic vesicle exocytosis, distinct synaptotagmins function as independent Ca(2+) sensors on the two fusion partners, the plasma membrane (synaptotagmin VII) versus synaptic vesicles (synaptotagmins I and II).     

Analysis of neurotransmitter release mechanisms by photolysis of caged Ca²⁺ in an autaptic neuron culture system

Neurotransmitter release is triggered by membrane depolarization, Ca(2+) influx and Ca(2+) sensing by the release machinery, causing synaptic vesicle (SV) fusion with the plasma membrane. Interlinked is a complex membrane cycle in which vesicles are tethered to the release site, primed, fused and recycled. As many of these processes are Ca(2+) dependent and simultaneously occurring, it is difficult to dissect them experimentally. This problem can be partially circumvented by controlling synaptic Ca(2+) concentrations via UV photolysis of caged Ca(2+). We developed a culture protocol for Ca(2+) uncaging in small synapses on the basis of the generation of small glia cell islands with single neurons on top, which are sufficiently small to be covered with a UV-light flash. Neurons are loaded with the photolabile Ca(2+)-chelator nitrophenyl-EGTA and Ca(2+) indicators, and a UV flash is used to trigger Ca(2+)-uncaging and SV fusion. The protocol takes three weeks to complete and provides unprecedented insights into the mechanisms of transmitter release.