Fast Exocytosis Research Articles

The release of inhibition model reproduces kinetics and plasticity of neurotransmitter release in central synapses

Calcium-evoked release of neurotransmitters from synaptic vesicles (SVs) is catalysed by SNARE proteins. The predominant view is that, at rest, complete assembly of SNARE complexes is inhibited (‘clamped’) by synaptotagmin and complexin molecules. Calcium binding by synaptotagmins releases this fusion clamp and triggers fast SV exocytosis. However, this model has not been quantitatively tested over physiological timescales. Here we describe an experimentally constrained computational modelling framework to quantitatively assess how the molecular architecture of the fusion clamp affects SV exocytosis. Our results argue that the ‘release-of-inhibition’ model can indeed account for fast calcium-activated SV fusion, and that dual binding of synaptotagmin-1 and synaptotagmin-7 to the same SNARE complex enables synergistic regulation of the kinetics and plasticity of neurotransmitter release. The developed framework provides a powerful and adaptable tool to link the molecular biochemistry of presynaptic proteins to physiological data and efficiently test the plausibility of calcium-activated neurotransmitter release models.

Involvement of CaV 2.2 channels and α2 δ-1 in homeostatic synaptic plasticity in cultured hippocampal neurons.

In the mammalian brain, presynaptic CaV 2 channels play a pivotal role in synaptic transmission by mediating fast neurotransmitter exocytosis via influx of Ca2+ into the active zone of presynaptic terminals. However, the distribution and modulation of CaV 2.2 channels at plastic hippocampal synapses remains to be elucidated. Here, we assess CaV 2.2 channels during homeostatic synaptic plasticity, a compensatory form of homeostatic control preventing excessive or insufficient neuronal activity during which extensive active zone remodelling has been described. We show that chronic silencing of neuronal activity in mature hippocampal cultures resulted in elevated presynaptic Ca2+ transients, mediated by increased levels of CaV 2.2 channels at the presynaptic site. This work focused further on the role of α2 δ-1subunits, important regulators of synaptic transmission and CaV 2.2 channel abundance at the presynaptic membrane. We found that α2 δ-1 overexpression reduces the contribution of CaV 2.2 channels to total Ca2+ flux without altering the amplitude of the Ca2+ transients. Levels of endogenous α2 δ-1 decreased during homeostatic synaptic plasticity, whereas the overexpression of α2 δ-1 prevented homeostatic synaptic plasticity in hippocampal neurons. Together, this study reveals a key role for CaV 2.2 channels and novel roles for α2 δ-1 during synaptic plastic adaptation. KEY POINTS: The roles of CaV 2.2 channels and α2 δ-1 in homeostatic synaptic plasticity in hippocampal neurons in culture were examined. Chronic silencing of neuronal activity resulted in elevated presynaptic Ca2+ transients, mediated by increased levels of CaV 2.2 channels at presynaptic sites. The level of endogenous α2 δ-1 decreased during homeostatic synaptic plasticity, whereas overexpression of α2 δ-1 prevented homeostatic synaptic plasticity in hippocampal neurons. Together, this study reveals a key role for CaV 2.2 channels and novel roles for α2 δ-1 during synaptic plastic adaptation.

Allosteric stabilization of calcium and phosphoinositide dual binding engages several synaptotagmins in fast exocytosis.

Synaptic communication relies on the fusion of synaptic vesicles with the plasma membrane, which leads to neurotransmitter release. This exocytosis is triggered by brief and local elevations of intracellular Ca2+ with remarkably high sensitivity. How this is molecularly achieved is unknown. While synaptotagmins confer the Ca2+ sensitivity of neurotransmitter exocytosis, biochemical measurements reported Ca2+ affinities too low to account for synaptic function. However, synaptotagmin's Ca2+ affinity increases upon binding the plasma membrane phospholipid PI(4,5)P2 and, vice versa, Ca2+ binding increases synaptotagmin's PI(4,5)P2 affinity, indicating a stabilization of the Ca2+/PI(4,5)P2 dual-bound state. Here, we devise a molecular exocytosis model based on this positive allosteric stabilization and the assumptions that (1.) synaptotagmin Ca2+/PI(4,5)P2 dual binding lowers the energy barrier for vesicle fusion and that (2.) the effect of multiple synaptotagmins on the energy barrier is additive. The model, which relies on biochemically measured Ca2+/PI(4,5)P2 affinities and protein copy numbers, reproduced the steep Ca2+ dependency of neurotransmitter release. Our results indicate that each synaptotagmin engaging in Ca2+/PI(4,5)P2 dual-binding lowers the energy barrier for vesicle fusion by ~5 kBT and that allosteric stabilization of this state enables the synchronized engagement of several (typically three) synaptotagmins for fast exocytosis. Furthermore, we show that mutations altering synaptotagmin's allosteric properties may show dominant-negative effects, even though synaptotagmins act independently on the energy barrier, and that dynamic changes of local PI(4,5)P2 (e.g. upon vesicle movement) dramatically impact synaptic responses. We conclude that allosterically stabilized Ca2+/PI(4,5)P2 dual binding enables synaptotagmins to exert their coordinated function in neurotransmission.

Stability profile of the neuronal SNARE complex reflects its potency to drive fast membrane fusion

Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) form the SNARE complex to mediate most fusion events of the secretory pathway. The neuronal SNARE complex is featured by its high stability and half-zippered conformation required for driving robust and fast synaptic exocytosis. However, these two features seem to be thermodynamically mutually exclusive. In this study, we have employed temperature-dependent disassociation assays and single-molecule Förster resonance energy transfer (FRET) experiments to analyze the stability and conformation of the neuronal SNARE complex. We reclassified the amino acids of the SNARE motif into four sub-groups (core, core-side I and II, and non-contact). Our data showed that the core residues predominantly contribute to the complex stability to meet a basal requirement for SNARE-mediated membrane fusion, while the core-side residues exert an unbalanced effect on the N- and C-half bundle stability that determines the half-zippered conformation of the neuronal SNARE complex, which would accommodate essential regulations by complexins and synaptotagmins for fast Ca2+-triggered membrane fusion. Furthermore, our data confirmed a strong coupling of folding energy between the N- and C-half assembly of the neuronal SNARE complex, which rationalizes the strong potency of the half-zippered conformation to conduct robust and fast fusion. Overall, these results uncovered that the stability profile of the neuronal SNARE complex reflects its potency to drive fast and robust membrane fusion. Based on these results, we also developed a new parameter, the stability factor (Fs), to characterize the overall stability of the neuronal SNARE complex and resolved a linear correlation between the stability and inter-residue coulombic interactions of the neuronal SNARE complex, which would help rationally design artificial SNARE complexes and remold functional SNARE complexes with desirable stability.

Molecular and functional architecture of striatal dopamine release sites

Despite the importance of dopamine for striatal circuit function, mechanistic understanding of dopamine transmission remains incomplete. We recently showed that dopamine secretion relies on the presynaptic scaffolding protein RIM, indicating that it occurs at active zone-like sites similar to classical synaptic vesicle exocytosis. Here, we establish using a systematic gene knockout approach that Munc13 and Liprin-α, active zone proteins for vesicle priming and release site organization, are important for dopamine secretion. Furthermore, RIM zinc finger and C2B domains, which bind to Munc13 and Liprin-α, respectively, are needed to restore dopamine release after RIM ablation. In contrast, and different from typical synapses, the active zone scaffolds RIM-BP and ELKS, and RIM domains that bind to them, are expendable. Hence, dopamine release necessitates priming and release site scaffolding by RIM, Munc13, and Liprin-α, but other active zone proteins are dispensable. Our work establishes that efficient release site architecture mediates fast dopamine exocytosis.

Regulation of MRI contrast and cellular retention time by cellular interfacial distribution of Gd agents: Implications for stem cell tracking

Human mesenchymal stem cells (hMSCs) were labeled with Dotarem or (Gd-DOTA)2- EM7 (EM7Gd2) via electroporation (EP). Cellular transmission electron microscopy (TEM) reveals free distribution of Gd agents and formation of EM7Gd2 clusters in the cytosol. Cellular magnetic resonance imaging (MRI) reveals that the free Gd agents induce MRI signal enhancement effect due to its fast exocytosis and subsequent interaction with intercellular water molecules. The EM7Gd2 clusters exhibits a longer intracellular retention time and induce a persistent MRI signal reduction effect. The cellular MRI results are interpreted by taking into account both T1 and T2 relaxation rates and their correlation with cellular binding structures of Dotarem and EM7Gd2.

Glucose-induced [Ca2+]i oscillations in β cells are composed of trains of spikes within a subplasmalemmal microdomain

Electrical activity and oscillations of cytosolic Ca2+ concentrations ([Ca2+]i) that trigger insulin release in response to glucose are key functions of pancreatic β cells. Although oscillatory Ca2+ signals have been intensively studied in β cells, their lower frequency did not match that of electrical activity. In addition, the measured peak [Ca2+]i did not reach levels that are typically required by synaptotagmins to elicit the release of insulin-containing vesicles in live-cell experiments. We therefore sought to resolve the Ca2+ dynamics in the subplasmalemmal microdomain that is critical for triggering fast exocytosis. Applying total internal reflection fluorescence (TIRF) microscopy in insulin-producing INS-1E and primary mouse β cells, we resolved extraordinary fast trains of Ca2+ spiking (frequency > 3 s−1) in response to glucose exposure. Using a low-affinity [Ca2+]i indicator dye, we provide experimental evidence that Ca2+ spikes reach low micromolar apparent concentrations in the vicinity of the plasma membrane. Analysis of Ca2+ spikes evoked by repeated depolarization for 10 ms closely matched the Ca2+ dynamics observed upon glucose application. To our knowledge, this is the first study that experimentally demonstrates Ca2+ spikes in β cells with velocities that resemble those of bursting or continuously appearing trains of action potentials (APs) in non-patched cells.

Polyamine-Responsive Morphological Transformation of a Supramolecular Peptide for Specific Drug Accumulation and Retention in Cancer Cells.

The precise accumulation and extended retention of nanomedicines in the tumor tissue has been highly desired for cancer therapy. Here a novel supramolecular-peptide derived nanodrug (SPN) that can be transformed to microfibers in response to intracellular polyamine in cancer cells for significantly enhanced tumor specific accumulation and retention is developed. The supramolecular-peptide is constructed via the non-covalent interactions between cucurbit[7]uril (CB[7]) and Phe on Phe-Phe-Val-Leu-Lys-camptothecin conjugates (FFVLK-CPT, PC). The resultant amphiphilic supramolecular complex subsequently self-assembles into nanoparticles with a hydrodynamic diameter of 164.2± 3.7nm. Upon internalization into spermine-overexpressed cancer cells, the CB[7]-Phe host-guest pairs can be competitively dissociated by spermine and can release free PC, which immediately form β-sheet structures and subsequently reorganize into microfibers, leading to dramatically improved accumulation, retention, and sustained release of CPT in tumor cells for highly effective cancer therapy. Accordingly, this SPN exhibit rather low toxicity against non-cancerous cells due to the morphological stability and fast exocytosis of the nanodrugs in those cells without abundant spermine. This study reports the first supramolecular peptide capable of polyamine-responsive "nanoparticle-to-microfiber" transformation for specific tumor therapy with minimal side effects. This work also offers novel insights to the design and development of stimuli-responsive nanomaterials as precision medicine.

Synaptotagmin-7 places dense-core vesicles at the cell membrane to promote Munc13-2- and Ca2+-dependent priming.

Synaptotagmins confer calcium-dependence to the exocytosis of secretory vesicles, but how coexpressed synaptotagmins interact remains unclear. We find that synaptotagmin-1 and synaptotagmin-7 when present alone act as standalone fast and slow Ca2+-sensors for vesicle fusion in mouse chromaffin cells. When present together, synaptotagmin-1 and synaptotagmin-7 are found in largely non-overlapping clusters on dense-core vesicles. Synaptotagmin-7 stimulates Ca2+-dependent vesicle priming and inhibits depriming, and it promotes ubMunc13-2- and phorbolester-dependent priming, especially at low resting calcium concentrations. The priming effect of synaptotagmin-7 increases the number of vesicles fusing via synaptotagmin-1, while negatively affecting their fusion speed, indicating both synergistic and competitive interactions between synaptotagmins. Synaptotagmin-7 places vesicles in close membrane apposition (<6 nm); without it, vesicles accumulate out of reach of the fusion complex (20-40 nm). We suggest that a synaptotagmin-7-dependent movement toward the membrane is involved in Munc13-2/phorbolester/Ca2+-dependent priming as a prelude to fast and slow exocytosis triggering.

Electrodeposited Gold on Carbon-Fiber Microelectrodes for Enhancing Amperometric Detection of Dopamine Release from Pheochromocytoma Cells.

Exocytosis is an ultrafast cellular process which facilitates neuron-neuron communication in the brain. Microelectrode electrochemistry has been an essential tool for measuring fast exocytosis events with high temporal resolution and high sensitivity. Due to carbon fiber's irreproducible and inhomogeneous surface conditions, however, it is often desirable to develop simple and reproducible modification schemes to enhance a microelectrode's analytical performance for single-cell analysis. Here we present carbon-fiber microelectrodes (CFEs) modified with a thin film of electrodeposited gold for the detection of exocytosis from rat pheochromocytoma cells (PC12), a model cell line for neurosecretion. These new probes are made by a novel voltage-pulsing deposition procedure and demonstrate improved electron-transfer characteristics for catecholamine oxidation, and their fabrication is tractable for many different probe designs. When we applied the probes to the detection of catecholamine release, we found that they outperformed unmodified CFEs. Further, the improved performance was conserved at cells incubated with L-DOPA (l-3,4-dihydroxyphenylalanine), a precursor to dopamine that increases the quantal size of the release events. Future use of this method may allow nanoelectrodes to be modified for highly sensitive detection of exocytosis from chemical synapses.

Search for a minimal machinery for Ca2+-triggered millisecond neuroexocytosis

Neurons have the remarkable ability to release a batch of neurotransmitters into the synapse immediately after an action potential. This signature event is made possible by the simultaneous fusion of a number of synaptic vesicles to the plasma membrane upon Ca2+ entry into the active zone. The outcomes of both cellular and in vitro studies suggest that soluble N-ethylmaleimide-sensitive-factor attachment protein receptors (SNAREs) and synaptotagmin 1 (Syt1) constitute the minimal fast exocytosis machinery in the neuron. Syt1 is the major Ca2+-sensor and orchestrates the synchronous start of individual vesicle fusion events while SNAREs are the membrane fusion machinery that dictates the kinetics of each single fusion event. The data also suggest that Ca2+-bound Syt1 is involved in the upstream docking step which leads to an increase in the number of fusion events or the size of the release, leaving the SNARE complex alone to carry out membrane fusion by themselves.

Interactions of Inhaled Nanoparticles with Rat Alveolar Epithelial Cell Monolayers

RationaleTo understand the contribution to lung pathogenesis of ambient ultrafine air pollution particles, knowledge of basic mechanisms underlying how inhaled nanoparticles interact with alveolar epithelial cells (AEC) is needed. We have reported that polystyrene nanoparticles (PNP) at the apical surface of primary rat alveolar AEC monolayers (RAECM) are taken up in a manner dependent on exposure time, dose and particle size via a non‐endocytic ‘diffusional’ process, induce autophagy, and exit via both fast intracellular calcium‐dependent lysosomal exocytosis and a slower (‘diffusional’) process. In this study, we further investigated intracellular fate of PNP.MethodsRAECM were apically exposed to near‐infrared labeled PNP (80 μg/mL; carboxylated; 20 nm). AEC were subjected to confocal imaging throughout the volume of the cells. In each z plane, total intracellular fluorescence intensity as well as vesicular and cytosolic PNP were determined and normalized to total intracellular PNP (PNPic). Volumes of endosomal, autophagosomal and lysosomal compartments, and colocalization of PNP in cytosolic vs vesicular compartments, were assessed using an early endosome marker (Rab5) tagged with green fluorescent protein (GFP), quinacrine, microtubule‐associated protein 1 light chain 3 (LC3B)‐GFP, and Magic Red (a lysosomal marker). [PNPic] was then determined by an isotropic method (cuvette‐based microfluorometry) utilizing AEC lysates as a function of time. [PNP] in vesicles ([PNPv]) and cytosol ([PNPc]) were estimated from the respective fractions of compartmental volumes and co‐localized PNP in each compartment.ResultsAfter 12 hr, [PNPic] reached a plateau of 1.84 mg/mL. Distribution of intracellular PNP was ~80% in intracellular vesicles (primarily lysosomes) and ~20% in cytosol at steady state, equivalent to 8.86 mg/ml and 0.58 mg/mL for [PNPv] and [PNPc], respectively. About 3% of total intracellular PNP was observed in Rab5‐GFP positive early endosomes by 3 hr after PNP exposure, but inhibition of classical endocytosis pathways did not alter PNP uptake kinetics. In contrast, blockade of autophagosome formation by 3‐methyladenine (3MA) markedly reduced PNP uptake and decreased steady state [PNPic] by ~75% with no detectable vesicular PNP. Inhibition of autophagosome‐lysosome fusion by bafilomycin lowered steady state PNPic by ~20% compared to control, with ~81% in cytosol and ~19% in vesicles.SummaryIn AEC exposed to PNP, [PNPv] exceeds both [PNPc] and [PNPic], with [PNPc]&gt;&gt;apical [PNP]. Cytosolic presence of PNP triggers autophagosome formation, leading to PNP accumulation in lysosomes. Inhibition of autophagy leads to decreased PNP entry and [PNPic]. These data suggest that intracellular processing of PNP is regulable, offering possible approaches to amelioration of inhaled nanoparticle‐related cytotoxicity in AEC.Support or Funding InformationFunding: NIH; Will Rogers Institute; Hastings and Whittier FoundationsThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Productive and Non-productive Pathways for Synaptotagmin 1 to Support Ca2+-Triggered Fast Exocytosis.

Ca2+-triggered SNARE-mediated membrane fusion is essential for neuronal communication. The speed of this process is of particular importance because it sets a time limit to cognitive and physical activities. In this work, we expand the proteoliposome-to-supported bilayer (SBL) fusion assay by successfully incorporating synaptotagmin 1 (Syt1), a major Ca2+ sensor. We report that Syt1 and Ca2+ together can elicit more than a 50-fold increase in the number of membrane fusion events when compared with membrane fusion mediated by SNAREs only. What is remarkable is that ~55% of all vesicle fusion events occurs within 20 ms upon vesicle docking. Furthermore, pre-binding of Syt1 to SNAREs prior to Ca2+ inhibits spontaneous fusion, but intriguingly, this leads to a complete loss of the Ca2+ responsiveness. Thus, our results suggest that there is a productive and a non-productive pathway for Syt1, depending on whether there is a premature interaction between Syt1 and SNAREs. Our results show that Ca2+ binding to Syt1 prior to Syt1's binding to SNAREs may be a prerequisite for the productive pathway. The successful reconstitution of Syt1 activities in the physiological time scale provides new opportunities to test the current mechanistic models for Ca2+-triggered exocytosis.

L-type calcium channels in exocytosis and endocytosis of chromaffin cells.

The coexistence of different subtypes of voltage-dependent calcium channels (VDCC) within the same chromaffin cell (CC) and the marked interspecies variability in the proportion of VDCC subtypes that are present in the plasmalemma of the CCs raises the question on their roles in controlling different physiological functions. Particularly relevant seems to be the role of VDCCs in the regulation of the exocytotic neurotransmitter release process, and its tightly coupled membrane retrieval (endocytosis) process since both are Ca2+-dependent processes. This review is focused on the role of Ca2+ influx through L-type VDCC in the regulation of these two processes. It is currently accepted that the different VDCC subtypes (i.e., T, L, N, P/Q, R) contribute to exocytosis proportionally to their density of expression and gating properties. However, the pattern of stimulation defines a preferential role of the different subtypes of VDCC on exocytosis and endocytosis. Thus, L-type channels seem to control catecholamine release induced by prolonged stimuli while fast exocytosis in response to short square depolarizing pulses or action potentials is mediated by Ca2+ entering CCs through P/Q channels. The pattern of stimulation also influences the endocytotic process, and thus, electrophysiological data suggest the sustained Ca2+ entry through slow-inactivating L-type channels could be responsible for the activation of fast endocytosis.

Ca2+ channel clustering with insulin-containing granules is disturbed in type 2 diabetes.

Loss of first-phase insulin secretion is an early sign of developing type 2 diabetes (T2D). Ca2+ entry through voltage-gated L-type Ca2+ channels triggers exocytosis of insulin-containing granules in pancreatic β cells and is required for the postprandial spike in insulin secretion. Using high-resolution microscopy, we have identified a subset of docked insulin granules in human β cells and rat-derived clonal insulin 1 (INS1) cells for which localized Ca2+ influx triggers exocytosis with high probability and minimal latency. This immediately releasable pool (IRP) of granules, identified both structurally and functionally, was absent in β cells from human T2D donors and in INS1 cells cultured in fatty acids that mimic the diabetic state. Upon arrival at the plasma membrane, IRP granules slowly associated with 15 to 20 L-type channels. We determined that recruitment depended on a direct interaction with the synaptic protein Munc13, because expression of the II–III loop of the channel, the C2 domain of Munc13-1, or of Munc13-1 with a mutated C2 domain all disrupted L-type channel clustering at granules and ablated fast exocytosis. Thus, rapid insulin secretion requires Munc13-mediated recruitment of L-type Ca2+ channels in close proximity to insulin granules. Loss of this organization underlies disturbed insulin secretion kinetics in T2D.

Rapid SNARE-Mediated Fusion of Liposomes and Chromaffin Granules with Giant Unilamellar Vesicles

Soluble N-ethylmaleimide-sensitive factor activating protein receptor (SNARE) proteins are the main catalysts for membrane fusion in the secretory pathway of eukaryotic cells. In vitro, SNAREs are sufficient to mediate effective fusion of both native and artificial membranes. Here we have established, to our knowledge, a new platform for monitoring SNARE-mediated docking and fusion between giant unilamellar vesicles (GUVs) and smaller liposomes or purified secretory granules with high temporal and spatial resolution. Analysis of fusion is restricted to the free-standing part of the GUV-membrane exhibiting low curvature and a lack of surface contact, thus avoiding adhesion-mediated interference with the fusion reaction as in fusion with supported bilayers or surface-immobilized small vesicles. Our results show that liposomes and chromaffin granules fuse with GUVs containing activated SNAREs with only few milliseconds delay between docking and fusion. We conclude that after initial contact in trans, SNAREs alone can complete fusion at a rate close to fast neuronal exocytosis.

RIBEYE(B)-domain binds to lipid components of synaptic vesicles in an NAD(H)-dependent, redox-sensitive manner.

Synaptic ribbons are needed for fast and continuous exocytosis in ribbon synapses. RIBEYE is a main protein component of synaptic ribbons and is necessary to build the synaptic ribbon. RIBEYE consists of a unique A-domain and a carboxyterminal B-domain, which binds NAD(H). Within the presynaptic terminal, the synaptic ribbons are in physical contact with large numbers of synaptic vesicle (SV)s. How this physical contact between ribbons and synaptic vesicles is established at a molecular level is not well understood. In the present study, we demonstrate that the RIBEYE(B)-domain can directly interact with lipid components of SVs using two different sedimentation assays with liposomes of defined chemical composition. Similar binding results were obtained with a SV-containing membrane fraction. The binding of liposomes to RIBEYE(B) depends upon the presence of a small amount of lysophospholipids present in the liposomes. Interestingly, binding of liposomes to RIBEYE(B) depends on NAD(H) in a redox-sensitive manner. The binding is enhanced by NADH, the reduced form, and is inhibited by NAD+, the oxidized form. Lipid-mediated attachment of vesicles is probably part of a multi-step process that also involves additional, protein-dependent processes.

C2-domain containing calcium sensors in neuroendocrine secretion.

The molecular mechanisms for calcium-triggered membrane fusion have long been sought for, and detailed models now exist that account for at least some of the functions of the many proteins involved in the process. Key players in the fusion reaction are a group of proteins that, upon binding to calcium, trigger the merger of cargo-filled vesicles with the plasma membrane. Low-affinity, fast-kinetics calcium sensors of the synaptotagmin family - especially synaptotagmin-1 and synaptotagmin-2 - are the main calcium sensors for fast exocytosis triggering in many cell types. Their functions extend beyond fusion triggering itself, having been implicated in the calcium-dependent vesicle recruitment during activity, docking of vesicles to the plasma membrane and priming, and even in post-fusion steps, such as fusion pore expansion and endocytosis. Furthermore, synaptotagmin diversity imparts distinct properties to the release process itself. Other calcium-sensing proteins such as Munc13s and protein kinase C play important, but more indirect roles in calcium-triggered exocytosis. Because of their higher affinity, but intrinsic slower kinetics, they operate on longer temporal and spatial scales to organize assembly of the release machinery. Finally, the high-affinity synaptotagmin-7 and Doc2 (Double C2-domain) proteins are able to trigger membrane fusion invitro, but cellular measurements in different systems show that they may participate in either fusion or vesicle priming. Here, we summarize the properties and possible interplay of (some of) the major C2-domain containing calcium sensors in calcium-triggered exocytosis. This article is part of a mini review series: "Synaptic Function and Dysfunction in Brain Diseases".

Exploring the Formation and the Structure of Synaptobrevin Oligomers in a Model Membrane

SNARE complexes have been shown to act cooperatively to enable the synaptic vesicle fusion in neuronal transmission at millisecond timescale. It has previously been suggested that the oligomerization of SNARE complexes required for cooperative action in fusion is mediated by interactions between transmembrane domains (TMDs). We study the oligomerization of synaptobrevin TMD using ensembles of molecular dynamics (MD) simulations at coarse-grained resolution for both the wild-type (WT) and selected mutants. Trimerization and tetramerization of the sybII WT and mutants displayed distinct kinetics depending both on the rate of dimerization and the availability of alternative binding interfaces. Interestingly, the tetramerization kinetics and propensity for the sybII W89A-W90A mutant was significantly increased as compared with the WT; the tryptophans in WT sybII impose sterical restraints on oligomer packing, thereby maintaining an appropriate plasticity and accessibility of sybII to the binding of its cognate SNARE partners during membrane fusion. Higher-order oligomeric models (ranging from pentamer to octamer), built by incremental addition of peptides to smaller oligomers, revealed substantial stability and high compactness. These larger sybII oligomers may induce membrane deformation, thereby possibly facilitating fast fusion exocytosis.

Functional Nanoscale Imaging of Synaptic Vesicle Cycling with Superfast Fixation.

Functional imaging is the measurement of structural changes during an ongoing physiological process over time. In many cases, functional imaging has been implemented by tracking a fluorescent signal in live imaging sessions. Electron microscopy, however, excludes live imaging which has hampered functional imaging approaches on the ultrastructural level. This barrier was broken with the introduction of superfast fixation. Superfast fixation is capable of stopping and fixing membrane traffic at sufficient speed to capture a physiological process at a distinct functional state. Applying superfast fixation at sequential time points allows tracking of membrane traffic in a step-by-step fashion.This technique has been applied to track labeled endocytic vesicles at central synapses as they pass through the synaptic vesicle cycle. At synapses, neurotransmitter is released from synaptic vesicles (SVs) via fast activity-dependent exocytosis. Exocytosis is coupled to fast endocytosis that retrieves SVs components from the plasma membrane shortly after release. Fluorescent FM dyes that bind to the outer leaflet of the plasma membrane enter the endocytic vesicle during membrane retrieval and remain trapped in endocytic vesicles have been widely used to study SV exo-endocytic cycling in live imaging sessions. FM dyes can also be photoconverted into an electron-dense diaminobenzidine polymer which allows the investigation of SV cycling in the electron microscope. The combination of FM labeling with superfast fixation made it possible to track the fine structure of endocytic vesicles at 1 s intervals. Because this combination is not specialized to SV cycling, many other cellular processes can be studied. Furthermore, the technique is easy to set up and cost effective.This chapter describes activity-dependent FM dye labeling of SVs in cultured hippocampal neurons, superfast microwave-assisted fixation, photoconversion of the fluorescent endocytic vesicles, and the analysis of individual synapses after serial section 3D reconstruction of individual synapses from electron micrographs.