The mouth of a dense-core vesicle opens and closes in a concerted action regulated by calcium and amphiphysin

Secretion of hormones and peptides by neuroendocrine cells occurs through fast and slow modes of vesicle fusion but the mechanics of these processes are not understood. We used interference reflection microscopy to monitor deformations of the membrane surface and found that both modes of fusion involve the tightly coupled dilation and constriction of the vesicle. The rate of opening is calcium dependent and occurs rapidly at concentrations <5 μM. The fast mode of fusion is blocked selectively by a truncation mutant of amphiphysin. Vesicles do not collapse when fusion is triggered by strontium, rather they remain locked open and membrane scission is blocked. In contrast, constriction of the vesicle opening continues when endocytosis is blocked by inhibiting the function of dynamin. Thus, fast and slow modes of fusion involve similar membrane deformations and vesicle closure can be uncoupled from membrane scission. Regulation of these processes by calcium and amphiphysin may provide a mechanism for controlling the release of vesicle contents.     

Synaptotagmin C2A loop 2 mediates Ca2+-dependent SNARE interactions essential for Ca2+-triggered vesicle exocytosis

Synaptotagmins contain tandem C2 domains and function as Ca(2+) sensors for vesicle exocytosis but the mechanism for coupling Ca(2+) rises to membrane fusion remains undefined. Synaptotagmins bind SNAREs, essential components of the membrane fusion machinery, but the role of these interactions in Ca(2+)-triggered vesicle exocytosis has not been directly assessed. We identified sites on synaptotagmin-1 that mediate Ca(2+)-dependent SNAP25 binding by zero-length cross-linking. Mutation of these sites in C2A and C2B eliminated Ca(2+)-dependent synaptotagmin-1 binding to SNAREs without affecting Ca(2+)-dependent membrane binding. The mutants failed to confer Ca(2+) regulation on SNARE-dependent liposome fusion and failed to restore Ca(2+)-triggered vesicle exocytosis in synaptotagmin-deficient PC12 cells. The results provide direct evidence that Ca(2+)-dependent SNARE binding by synaptotagmin is essential for Ca(2+)-triggered vesicle exocytosis and that Ca(2+)-dependent membrane binding by itself is insufficient to trigger fusion. A structure-based model of the SNARE-binding surface of C2A provided a new view of how Ca(2+)-dependent SNARE and membrane binding occur simultaneously.     

Palmitoylation of the synaptic vesicle fusion machinery

The fusion of synaptic vesicles with the pre-synaptic plasma membrane mediates the secretion of neurotransmitters at nerve terminals. This pathway is regulated by an array of protein–protein interactions. Of central importance are the soluble NSF ( N -ethylmaleimide-sensitive factor) attachment protein receptor (SNARE) proteins syntaxin 1 and SNAP25, which are associated with the pre-synaptic plasma membrane and vesicle-associated membrane protein (VAMP2), a synaptic vesicle SNARE. Syntaxin 1, SNAP25 and VAMP2 interact to form a tight complex bridging the vesicle and plasma membranes, which has been suggested to represent the minimal membrane fusion machinery. Synaptic vesicle fusion is stimulated by a rise in intraterminal Ca²⁺ levels, and a major Ca²⁺ sensor for vesicle fusion is synaptotagmin I. Synaptotagmin is likely to couple Ca²⁺ entry to vesicle fusion via Ca²⁺-dependent and independent interactions with membrane phospholipids and the SNARE proteins. Intriguingly, syntaxin 1, SNAP25, VAMP2 and synaptotagmin I have all been reported to be modified by palmitoylation in neurons. In this review, we discuss the mechanisms and dynamics of palmitoylation of these proteins and speculate on how palmitoylation might contribute to the regulation of synaptic vesicle fusion.     

Methods for patch clamp capacitance recordings from the calyx

We demonstrate the basic techniques for presynaptic patch clamp recording at the calyx of Held, a mammalian central nervous system nerve terminal. Electrical recordings from the presynaptic terminal allow the measurement of action potentials, calcium channel currents, vesicle fusion (exocytosis) and subsequent membrane uptake (endocytosis). The fusion of vesicles containing neurotransmitter causes the vesicle membrane to be added to the cell membrane of the calyx. This increase in the amount of cell membrane is measured as an increase in capacitance. The subsequent reduction in capacitance indicates endocytosis, the process of membrane uptake or removal from the calyx membrane. Endocytosis, is necessary to maintain the structure of the calyx and it is also necessary to form vesicles that will be filled with neurotransmitter for future exocytosis events. Capacitance recordings at the calyx of Held have made it possible to directly and rapidly measure vesicular release and subsequent endocytosis in a mammalian CNS nerve terminal. In addition, the corresponding postsynaptic activity can be simultaneously measured by using paired recordings. Thus a complete picture of the presynaptic and postsynaptic electrical activity at a central nervous system synapse is achievable using this preparation. Here, the methods for slice preparation, morphological features for identification of calyces of Held, basic patch clamping techniques, and examples of capacitance recordings to measure exocytosis and endocytosis are presented.     

The mouth of a dense-core vesicle opens and closes in a concerted action regulated by calcium and amphiphysin

Secretion of hormones and peptides by neuroendocrine cells occurs through fast and slow modes of vesicle fusion but the mechanics of these processes are not understood. We used interference reflection microscopy to monitor deformations of the membrane surface and found that both modes of fusion involve the tightly coupled dilation and constriction of the vesicle. The rate of opening is calcium dependent and occurs rapidly at concentrations <5 microM. The fast mode of fusion is blocked selectively by a truncation mutant of amphiphysin. Vesicles do not collapse when fusion is triggered by strontium, rather they remain locked open and membrane scission is blocked. In contrast, constriction of the vesicle opening continues when endocytosis is blocked by inhibiting the function of dynamin. Thus, fast and slow modes of fusion involve similar membrane deformations and vesicle closure can be uncoupled from membrane scission. Regulation of these processes by calcium and amphiphysin may provide a mechanism for controlling the release of vesicle contents.     

Hydrophobic ions amplify the capacitive currents used to measure exocytotic fusion.

The detection of exocytotic fusion in patch-clamped secretory cells depends on measuring an increase in the cell membrane capacitance as new membrane is added to the plasma membrane. However, in the majority of secretory cells, secretory vesicles are too small (< 200 nm in diameter) to cause a detectable signal. We have found that incubations of normal mouse mast cells with the hydrophobic anion dipicrylamine (DPA), increases cell membrane capacitance by about three times. The large capacitive current induced by DPA was voltage-dependent, having a maximum value at -10 mV. The DPA-induced charge movement could be described by a single barrier model in which the DPA molecules move between two stable states in the bulk lipid matrix of the membrane. More importantly, the DPA treatment produced a sevenfold increase in the size of the capacitance steps observed upon the exocytotic fusion of single secretory granules. A similar amplification of DPA on the secretory vesicle capacitance was observed in a cell with larger (< or = 5 microns in diameter) or with smaller secretory granules (< 250 nm in diameter). Additionally, the increased granule membrane capacitance enlarged the transient capacitive discharge measured upon formation of a fusion pore in normal mast cell granules. Our results indicate that hydrophobic ions provide an important tool for high resolution studies of membrane capacitance.     

Spatial Regulation of Membrane Fusion Controlled by Modification of Phosphoinositides

Membrane fusion plays a central role in many cell processes from vesicular transport to nuclear envelope reconstitution at mitosis but the mechanisms that underlie fusion of natural membranes are not well understood. Studies with synthetic membranes and theoretical considerations indicate that accumulation of lipids characterised by negative curvature such as diacylglycerol (DAG) facilitate fusion. However, the specific role of lipids in membrane fusion of natural membranes is not well established. Nuclear envelope (NE) assembly was used as a model for membrane fusion. A natural membrane population highly enriched in the enzyme and substrate needed to produce DAG has been isolated and is required for fusions leading to nuclear envelope formation, although it contributes only a small amount of the membrane eventually incorporated into the NE. It was postulated to initiate and regulate membrane fusion. Here we use a multidisciplinary approach including subcellular membrane purification, fluorescence spectroscopy and Förster resonance energy transfer (FRET)/two-photon fluorescence lifetime imaging microscopy (FLIM) to demonstrate that initiation of vesicle fusion arises from two unique sites where these vesicles bind to chromatin. Fusion is subsequently propagated to the endoplasmic reticulum-derived membranes that make up the bulk of the NE to ultimately enclose the chromatin. We show how initiation of multiple vesicle fusions can be controlled by localised production of DAG and propagated bidirectionally. Phospholipase C (PLCγ), GTP hydrolysis and (phosphatidylinsositol-(4,5)-bisphosphate (PtdIns(4,5)P₂) are required for the latter process. We discuss the general implications of membrane fusion regulation and spatial control utilising such a mechanism.     

Capacitance flickers and pseudoflickers of small granules, measured in the cell-attached configuration.

We have studied exocytosis of single small granules from human neutrophils by capacitance recordings in the cell-attached configuration. We found that 2.2% of the exocytotic events were flickers. The flickers always ended with a downward step. This indicates closing of the fusion pore. During flickering, the fusion pore conductance remained below 1 nS, and no net membrane transfer was detectable. After fusion pore expansion beyond 1 nS the pore expanded irreversibly, leading to rapid full incorporation of the granule/vesicle into the plasma membrane. Following exocytosis of single granules, a capacitance decrease directly related to the preceding increase was observed in 7% of the exocytotic events. This decrease followed immediately after irreversible pore expansion, and is presumably triggered by full incorporation of the vesicle into the patch membrane. The capacitance decrease could be interpreted as endocytosis triggered by exocytosis. However, the gradual decrease could also reflect a decrease in the "free" patch area following incorporation of an exocytosed vesicle. We conclude that non-stepwise capacitance changes must be interpreted with caution, since a number of factors go into determining cell or patch admittance.     

Analysis of membrane fusion as a two-state sequential process: evaluation of the stalk model

We propose a model that accounts for the time courses of PEG-induced fusion of membrane vesicles of varying lipid compositions and sizes. The model assumes that fusion proceeds from an initial, aggregated vesicle state ((A) membrane contact) through two sequential intermediate states (I(1) and I(2)) and then on to a fusion pore state (FP). Using this model, we interpreted data on the fusion of seven different vesicle systems. We found that the initial aggregated state involved no lipid or content mixing but did produce leakage. The final state (FP) was not leaky. Lipid mixing normally dominated the first intermediate state (I(1)), but content mixing signal was also observed in this state for most systems. The second intermediate state (I(2)) exhibited both lipid and content mixing signals and leakage, and was sometimes the only leaky state. In some systems, the first and second intermediates were indistinguishable and converted directly to the FP state. Having also tested a parallel, two-intermediate model subject to different assumptions about the nature of the intermediates, we conclude that a sequential, two-intermediate model is the simplest model sufficient to describe PEG-mediated fusion in all vesicle systems studied. We conclude as well that a fusion intermediate "state" should not be thought of as a fixed structure (e.g., "stalk" or "transmembrane contact") of uniform properties. Rather, a fusion "state" describes an ensemble of similar structures that can have different mechanical properties. Thus, a "state" can have varying probabilities of having a given functional property such as content mixing, lipid mixing, or leakage. Our data show that the content mixing signal may occur through two processes, one correlated and one not correlated with leakage. Finally, we consider the implications of our results in terms of the "modified stalk" hypothesis for the mechanism of lipid pore formation. We conclude that our results not only support this hypothesis but also provide a means of analyzing fusion time courses so as to test it and gauge the mechanism of action of fusion proteins in the context of the lipidic hypothesis of fusion.     

For better or for worse: complexins regulate SNARE function and vesicle fusion

In contrast to constitutive secretion, SNARE-mediated synaptic vesicle fusion is controlled by multiple regulatory proteins, which determine the Ca²⁺ sensitivity of the vesicle fusion process and the speed of excitation–secretion coupling. Complexins are among the best characterized SNARE regulators known to date. They operate by binding to trimeric SNARE complexes consisting of the vesicle protein synaptobrevin and the plasma membrane proteins syntaxin and SNAP-25. The question as to whether complexins facilitate or inhibit SNARE-mediated fusion processes is currently a matter of significant controversy. This is mainly because of the fact that biochemical experiments in vitro and studies on vertebrate complexins in vivo have yielded apparently contradictory results. In this review, I provide a summary of available data on the role of complexins in SNARE-mediated vesicle fusion and attempt to define a model of complexin function that incorporates evidence for both facilitatory and inhibitory roles of complexins in SNARE-mediated fusion.     

Putting the clamps on membrane fusion: how complexin sets the stage for calcium-mediated exocytosis

Three recent papers have addressed a long-standing question in exocytosis: how does a sudden calcium influx trigger a coordinated synchronous release in regulated exocytosis [Giraudo, C.G., Eng, W.S., Melia, T.J. and Rothman, J.E. (2006) A clamping mechanism involved in SNARE-dependent exocytosis. Science 313, 676-680; Schaub, J.R., Lu, X., Doneske, B., Shin, Y.K. and McNew, J.A. (2006) Hemifusion arrest by complexin is relieved by Ca(2+)-synaptotagmin I. Nat. Struct. Mol. Biol. 13, 748-750; Tang, J., Maximov, A., Shin, O.H., Dai, H., Rizo, J. and Sudhof, T.C. (2006) A complexin/synaptotagmin 1 switch controls fast synaptic vesicle exocytosis. Cell 126, 1175-1187]? Using diverse approaches that include cell-free reconstitution of the membrane fusion machinery and in vivo manipulation of fusogenic proteins, these groups have established that the complexin proteins are fusion clamps. By arresting vesicle secretion just prior to fusion, complexin primes select vesicles for a fast, synchronous response to calcium.     

The Effect of Hydrophilic Ionic Liquids 1-Ethyl-3-Methylimidazolium Lactate and Choline Lactate on Lipid Vesicle Fusion

Ionic liquids (ILs) are room-temperature molten salts that have applications in both physical sciences and more recently in the purification of proteins and lipids, gene transfection and sample preparation for electron microscopy (EM) studies. Transfection of genes into cells requires membrane fusion between the cell membrane and the transfection reagent, thus, ILs may be induce a membrane fusion event. To clarify the behavior of ILs with cell membranes the effect of ILs on model membranes, i.e., liposomes, were investigated. We used two standard ILs, 1-ethyl-3-methylimidazolium lactate ([EMI][Lac]) and choline lactate ([Ch][Lac]), and focused on whether these ILs can induce lipid vesicle fusion. Fluorescence resonance energy transfer and dynamic light scattering were employed to determine whether the ILs induced vesicle fusion. Vesicle solutions at low IL concentrations showed negligible fusion when compared with the controls in the absence of ILs. At concentrations of 30% (v/v), both types of ILs induced vesicle fusion up to 1.3 and 1.6 times the fluorescence intensity of the control in the presence of [Ch][Lac] and [EMI][Lac], respectively. This is the first demonstration that [EMI][Lac] and [Ch][Lac] induce vesicle fusion at high IL concentrations and this observation should have a significant influence on basic biophysical studies. Conversely, the ability to avoid vesicle fusion at low IL concentrations is clearly advantageous for EM studies of lipid samples and cells. This new information describing IL-lipid membrane interactions should impact EM observations examining cell morphology.     

Synaptobrevin-2 C-Terminal Flexible Region Regulates the Discharge of Catecholamine Molecules

The discharge of neurotransmitters from vesicles is a regulated process. Synaptobrevin-2, a snap receptor (SNARE) protein, participates in this process by interacting with other SNARE and associated proteins. Synaptobrevin-2 transmembrane domain is embedded into the vesicle lipid bilayer except for its last three residues. These residues are hydrophilic and constitute synaptobrevin-2 C-terminal flexible region. The residue Y113 of synaptobrevin-2 flexible region was mutated to lysine and glutamate. The effects of these mutations on the exocytotic process in chromaffin cells were assessed using capacitance measurements combined with amperometry and stimulation by flash photolysis of caged Ca²⁺. Both Y113E and Y113K mutations reduced the number of fusion-competent vesicles and reduced the rates of release of catecholamine molecules in quanta release events. These results exclude any direct interaction of this domain with the catecholamine molecules that are escaping through the fusion pore but favor its interaction with the vesicle membrane as a mean of regulating exocytosis.     

Novel inner monolayer fusion assays reveal differential monolayer mixing associated with cation-dependent membrane fusion

The ability to specifically monitor the behavior of the inner monolayer lipids of membranous vesicles during the membrane fusion process is useful technically and experimentally. In this study, we have identified N-NBD-phosphatidylserine as a reducible probe particularly suitable for inner monolayer fusion assays because of its low rate of membrane translocation after reduction of the outer monolayer probes by dithionite. Data are presented on translocation as a function of temperature, vesicle size, membrane composition, and serum protein concentration. Translocation as a result of the fusion event itself was also characterized. We further show here that a second membrane-localized probe, a long wavelength carbocyanine dye referred to a diI(5)C18ds, appears to form a membrane-bound resonance energy transfer pair with N-NBD-PS, and its outer monolayer fluorescence can also be eliminated by dithionite treatment. Lipid dilution of these probes upon fusion with unlabeled membranes leads to an increase in NBD donor fluorescence, and hence is a new type of inner monolayer fusion assay. These inner monolayer probe mixing assays were compared to random lipid labeling and aqueous contents mixing assays for cation-dependent fusion of liposomes composed of phosphatidylserine and phosphatidylethanolamine. The results showed that the inner monolayer fusion assay eliminates certain artifacts and reflects fairly closely the rate of non-leaky mixing of aqueous contents due to fusion, while outer monolayer mixing always precedes mixing of aqueous contents. In fact, vesicle aggregation and outer monolayer lipid mixing were found to occur over very long periods of time without inner monolayer mixing at low cation concentrations. Externally added lysophosphatidylcholine inhibited vesicle aggregation, outer monolayer mixing and any subsequent fusion. The state of vesicle aggregation and outer monolayer exchange that occurs below the fusion threshold may represent a metastable intermediate state that may be useful for further studies of the mechanism of membrane fusion.     

Studies of synaptic vesicle endocytosis in the nematode C. elegans

After synaptic vesicle exocytosis, synaptic vesicle proteins must be retrieved from the plasma membrane, sorted away from other membrane proteins, and reconstituted into a functional synaptic vesicle. The nematode Caenorhabditis elegans is an organism well suited for a genetic analysis of this process. In particular, three types of genetic studies have contributed to our understanding of synaptic vesicle endocytosis. First, screens for mutants defective in synaptic vesicle recycling have identified new proteins that function specifically in neurons. Second, RNA interference has been used to quickly confirm the roles of known proteins in endocytosis. Third, gene targeting techniques have elucidated the roles of genes thought to play modulatory or subtle roles in synaptic vesicle recycling. We describe a molecular model for synaptic vesicle recycling and discuss how protein disruption experiments in C. elegans have contributed to this model.     

Measuring the Lamellarity of Giant Lipid Vesicles with Differential Interference Contrast Microscopy

Giant unilamellar vesicles are a widely utilized model membrane system, providing free-standing bilayers unaffected by support-induced artifacts. To measure the lamellarity of such vesicles, fluorescence microscopy is one commonly utilized technique, but it has the inherent disadvantages of requiring lipid staining, thereby affecting the intrinsic physical and chemical properties of the vesicles, and it requires a calibration by statistical analysis of a vesicle ensemble. Herein we present what we believe to be a novel label-free optical method to determine the lamellarity of giant vesicles based on quantitative differential interference contrast (qDIC) microscopy. The method is validated by comparison with fluorescence microscopy on a statistically significant number of vesicles, showing correlated quantization of the lamellarity. Importantly, qDIC requires neither sample-dependent calibration nor sample staining, and thus can measure the lamellarity of any giant vesicle without additional preparation or interference with subsequent investigations. Furthermore, qDIC requires only a microscope equipped with differential interference contrast and a digital camera.     

Measuring the Lamellarity of Giant Lipid Vesicles with Differential Interference Contrast Microscopy

Giant unilamellar vesicles are a widely utilized model membrane system, providing free-standing bilayers unaffected by support-induced artifacts. To measure the lamellarity of such vesicles, fluorescence microscopy is one commonly utilized technique, but it has the inherent disadvantages of requiring lipid staining, thereby affecting the intrinsic physical and chemical properties of the vesicles, and it requires a calibration by statistical analysis of a vesicle ensemble. Herein we present what we believe to be a novel label-free optical method to determine the lamellarity of giant vesicles based on quantitative differential interference contrast (qDIC) microscopy. The method is validated by comparison with fluorescence microscopy on a statistically significant number of vesicles, showing correlated quantization of the lamellarity. Importantly, qDIC requires neither sample-dependent calibration nor sample staining, and thus can measure the lamellarity of any giant vesicle without additional preparation or interference with subsequent investigations. Furthermore, qDIC requires only a microscope equipped with differential interference contrast and a digital camera.     

Membrane-embedded synaptotagmin penetrates cis or trans target membranes and clusters via a novel mechanism

The synaptic vesicle protein synaptotagmin I has been proposed to serve as a Ca(2+) sensor for rapid exocytosis. Synaptotagmin spans the vesicle membrane once and possesses a cytoplasmic domain largely comprised of two C2 domains designated C2A and C2B. We have determined how deep the Ca(2+)-binding loops of Ca(2+).C2A penetrate into the lipid bilayer and report mutations in synaptotagmin that can uncouple membrane penetration from Ca(2+)-triggered interactions with the SNARE complex. To determine whether C2A penetrates into the vesicle ("cis") or plasma ("trans") membrane, we reconstituted a fragment of synaptotagmin that includes the membrane-spanning and C2A domain (C2A-TMR) into proteoliposomes. Kinetics experiments revealed that cis interactions are rapid (< or =500 micros). Binding in the trans mode was distinguished by the slow diffusion of trans target vesicles. Both modes of binding were observed, indicating that the linker between the membrane anchor and C2A domain functions as a flexible tether. C2A-TMR assembled into oligomers via a novel N-terminal oligomerization domain suggesting that synaptotagmin may form clusters on the surface of synaptic vesicles. This novel mode of clustering may allow for rapid Ca(2+)-triggered oligomerization of the protein via the membrane distal C2B domain.     

Calcium-induced fusion of sea urchin egg secretory vesicles with planar phospholipid bilayer membranes.

The fusion of sea urchin egg secretory vesicles to planar phospholipid bilayer membranes was studied by differential interference contrast (DIC) and fluorescent microscopy, in combination with electrical recordings of membrane conductance. A strong binding of vesicles to protein-free planar membranes was observed in the absence of calcium. Calcium-induced fusion of vesicles was detected using two independent assays: loss of the contents of individual vesicles visible by DIC microscopy: and vesicle content discharge across the planar membrane detected by an increase in the fluorescence of a dye. In both cases, no increase in the membrane conductance was observed unless vesicles were incubated with either Amphotericin B or digitonin prior to applying them to the planar membrane, an indication that native vesicles are devoid of open channels. Pre-incubation of vesicles with n-ethylmaleimide (NEM) abolished calcium-induced fusion. Fusion was also detected when vesicles were osmotically swollen to the point of lysis. In contrast, no fusion of vesicles to planar bilayers was seen when vesicles on plasma membrane (native cortices) were applied to a phospholipid membrane, despite good binding of vesicles to the planar membrane and fusion of vesicles to plasma membrane. It is suggested that cortical vesicles (CVs) have sufficient calcium-sensitive proteins for fusion to lipid membranes, but in native cortices granular fusion sites are oriented toward the plasma membrane. Removal of vesicles from the plasma membrane may allow fusion sites on vesicles access to new membranes.