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Human neuroblastoma cells, SH-SY5Y, are often used as a neuronal model to study Parkinson's disease and dopamine release in the substantia nigra, a midbrain region that plays an important role in motor control. Using amperometric single-cell recordings of single vesicle release events, we can study molecular manipulations of dopamine release and gain a better understanding of the mechanisms of neurological diseases. However, single-cell analysis of neurotransmitter release using traditional techniques yields results with very low throughput. In this paper, we will discuss a monolithically-integrated CMOS sensor array that has the low-noise performance, fine temporal resolution, and 1024 parallel channels to observe dopamine release from many single cells with single-vesicle resolution. The measured noise levels of our transimpedance amplifier are 415, 622, and 1083 ${\text{fA}}_{{\text{RMS}}}$ , at sampling rates of 10, 20, and 30 kS/s, respectively, without additional filtering. Post-CMOS processing is used to monolithically integrate 1024 on-chip gold electrodes, with an individual electrode size of 15 μm × 15 μm, directly on 1024 transimpedance amplifiers in the CMOS device. SU-8 traps are fabricated on individual electrodes to allow single cells to be interrogated and to reject multicellular clumps. Dopamine secretions from 76 cells are simultaneously recorded by loading the CMOS device with SH-SY5Y cells. In the 42-s measurement, a total of 7147 single vesicle release events are monitored. The study shows the CMOS device's capability of recording vesicle secretion at a single-cell level, with 1024 parallel channels, to provide detailed information on the dynamics of dopamine release at a single-vesicle resolution.

Release of neurotransmitters from synaptic vesicles begins with a narrow fusion pore, the structure of which remains unresolved. To obtain a structural model of the fusion pore, we performed coarse-grained molecular dynamics simulations of fusion between a nanodisc and a planar bilayer bridged by four partially unzipped SNARE complexes. The simulations revealed that zipping of SNARE complexes pulls the polar C-terminal residues of the synaptobrevin 2 and syntaxin 1A transmembrane domains to form a hydrophilic core between the two distal leaflets, inducing fusion pore formation. The estimated conductances of these fusion pores are in good agreement with experimental values. Two SNARE protein mutants inhibiting fusion experimentally produced no fusion pore formation. In simulations in which the nanodisc was replaced by a 40-nm vesicle, an extended hemifusion diaphragm formed but a fusion pore did not, indicating that restricted SNARE mobility is required for rapid fusion pore formation. Accordingly, rapid fusion pore formation also occurred in the 40-nm vesicle system when SNARE mobility was restricted by external forces. Removal of the restriction is required for fusion pore expansion.

The t-SNARE complex plays a central role in neuronal fusion. Its components, syntaxin-1 and SNAP25, are largely present in individual clusters and partially colocalize at the presumptive fusion site. How these protein clusters modify local lipid composition and membrane morphology is largely unknown. In this work, using coarse-grained molecular dynamics, the transmembrane domains (TMDs) of t-SNARE complexes are shown to form aggregates leading to formation of lipid nanodomains, which are enriched in cholesterol, phosphatidylinositol 4,5-bisphosphate, and gangliosidic lipids. These nano-domains induce membrane curvature that would promote a closer contact between vesicle and plasma membrane.

SNAP-25 is a Q-SNARE protein mediating exocytosis of neurosecretory vesicles including chromaffin granules. Previous results with a SNAP-25 construct lacking the nine C terminal residues (SNAP-25Δ9) showed changed fusion pore properties (Fang et al., 2008), suggesting a model for fusion pore mechanics that couple C terminal zipping of the SNARE complex to the opening of the fusion pore. The deleted fragment contains the positively charged residues R198 and K201, adjacent to layers 7 and 8 of the SNARE complex. To determine how fusion pore conductance and dynamics depend on these residues, single exocytotic events in bovine chromaffin cells expressing R198Q, R198E, K201Q, or K201E mutants were investigated by carbon fiber amperometry and cell-attached patch capacitance measurements. Coarse grain molecular dynamics simulations revealed spontaneous transitions between a loose and tightly zippered state at the SNARE complex C terminus. The SNAP-25 K201Q mutant showed no changes compared with SNAP-25 wild-type. However, K201E, R198Q, and R198E displayed reduced release frequencies, slower release kinetics, and prolonged fusion pore duration that were correlated with reduced probability to engage in the tightly zippered state. The results show that the positively charged amino acids at the SNAP-25 C terminus promote tight SNARE complex zippering and are required for high release frequency and rapid release in individual fusion events.

Although several proteins have been implicated in secretory vesicle tethering, the identity and mechanical properties of the components forming the physical vesicle-plasma membrane link remain unknown. Here we present the first experimental measurements of nanomechanical properties of secretory vesicle-plasma membrane tethers using combined AFM force clamp and TIRF microscopy on membrane sheets from PC12 cells expressing the vesicle marker ANF-eGFP. Application of pulling forces generated tether extensions composed of multiple steps with variable length. The frequency of short (

The SNARE complex consists of the three proteins synaptobrevin-2, syntaxin, and synaptosomal-associated protein 25 (SNAP25) and is thought to execute a large conformational change as it drives membrane fusion and exocytosis. The relation between changes in the SNARE complex and fusion pore opening is, however, still unknown. We report here a direct measurement relating a change in the SNARE complex to vesicle fusion on the millisecond time scale. In individual chromaffin cells, we tracked conformational changes in SNAP25 by total internal reflection fluorescence resonance energy transfer (FRET) microscopy while exocytotic catecholamine release from single vesicles was simultaneously recorded using a microfabricated electrochemical detector array. A local rapid and transient FRET change occurred precisely where individual vesicles released catecholamine. To overcome the low time resolution of the imaging frames needed to collect sufficient signal intensity, a method named event correlation microscopy was developed, which revealed that the FRET change was abrupt and preceded the opening of an exocytotic fusion pore by ∼90 ms. The FRET change correlated temporally with the opening of the fusion pore and not with its dilation.

The role of Ca2+in synaptic vesicle endocytosis remains uncertain due to the diversity in various preparations where several forms of endocytosis may contribute variably in different conditions. Although recent studies have demonstrated that Ca2+is important for clathrin-mediated endocytosis (CME), the mechanistic role of Ca2+in CME remains to be elucidated. By monitoring CME of single vesicles in mouse chromaffin cells with cell-attached capacitance measurements that offer millisecond time resolution, we demonstrate that the dynamics of vesicle fission during CME is Ca2+dependent but becomes Ca2+independent in synaptotagmin 1 (Syt1) knock-out cells. Our results thus suggest that Syt1 is necessary for the Ca2+dependence of CME.

The modulation of endocytosis following exocytosis by noradrenaline (NA), a physiological inhibitor of insulin secretion, was investigated in INS 832/13 cells using patch-clamp capacitance measurements. Endocytosis was inhibited by NA in a pertussis toxin-insensitive manner. Dialysing a synthetic peptide mimicking the C-terminus of the α-subunit of Gz into the cells blocked the inhibition of endocytosis by NA. Cell-attached capacitance measurements indicated that inhibition by NA was due to a decreased number of endocytotic events without a change in vesicle size. Analysis of fission pore closure kinetics revealed two distinct fission modes, with NA selectively inhibiting the rapid fission pore closure events. Comparison of the actions of NA and deltamethrin, a calcineurin antagonist and potent inhibitor of endocytosis, demonstrated that they inhibit endocytosis by different mechanisms. These findings establish novel actions for NA and Gz in insulin-secreting cells and possibly other cell types.

The molecular mechanisms responsible for the ‘distal’ effect by which noradrenaline (NA) blocks exocytosis in the β-cell were examined by whole-cell and cell-attached patch clamp capacitance measurements in INS 832/13 β-cells. NA inhibited Ca2+-evoked exocytosis by reducing the number of exocytotic events, without modifying vesicle size. Fusion pore properties also were unaffected. NA-induced inhibition of exocytosis was abolished by a high level of Ca2+ influx, by intracellular application of antibodies against the G protein subunit Gβ and was mimicked by the myristoylated βγ-binding/activating peptide mSIRK. NA-induced inhibition was also abolished by treatment with BoNT/A, which cleaves the C-terminal nine amino acids of SNAP-25, and also by a SNAP-25 C-terminal-blocking peptide containing the BoNT/A cleavage site. These data indicate that inhibition of exocytosis by NA is downstream of increased [Ca2+]i and is mediated by an interaction between Gβγ and the C-terminus of SNAP-25, as is the case for inhibition of neurotransmitter release. Remarkably, in the course of this work, a novel effect of NA was discovered. NA induced a marked retardation of the rate of refilling of the readily releasable pool (RRP) of secretory granules. This retardation was specifically abolished by a Gαi1/2 blocking peptide demonstrating that the effect is mediated via activation of Gαi1 and/or Gαi2.

In chromaffin cells, exocytosis of single granules and properties of the fusion pore — the first connection between vesicular lumen and extracellular space1 — can be studied by cell-attached patch amperometry2, 3, which couples patch-clamp capacitance measurements4, 5, 6, 7 with simultaneous amperometric recordings of transmitter release8, 9. Here we have studied exocytosis of single chromaffin granules and endocytosis of single vesicles in cell-free inside-out membrane patches by patch capacitance measurements and patch amperometry. We excised patches from chromaffin cells by using methods developed for studying properties of single ion channels10. With low calcium concentrations in the pipette and bath, the patches showed no spontaneous exocytosis, but exocytosis could be induced in some patches by applying calcium to the cytoplasmic side of the patch. Exocytosis was also stimulated by calcium entry through the patch membrane. Initial conductances of the fusion pore were undistinguishable in cell-attached and excised patch recordings, but the subsequent pore expansion was slower in excised patches. The properties of exocytotic fusion pores in chromaffin cells are very similar to those observed in mast cells and granulocytes. Excised patches provide a tool with which to study the mechanisms of fusion pore formation and endocytosis in vitro.

Nanobiotechnology is a field that utilizes the techniques of nano- and microfabrication to study biosystems or to use biological material and principles to build new devices. As an example we discuss the development of a nanofabricated electrochemical detector array that reveals the spatio-temporal dynamics of exocytosis in single chromaffin cells. In a quantal release event a single vesicle fuses with the plasma membrane releasing its contents through the fusion pore. The time-resolved amperometric currents measured by the individual electrodes detecting different fractions of the released molecules allow determination of the time course as well as localization of quantal events. Such a device may be applicable to study the correlation of exocytotic events with signalling events that could be simultaneously monitored by fluorescence microscopy.

Elevated intracellular Ca2+ concentrations [Ca2+] appear to be a rather universal trigger of massive membrane capacitance increases, presumably reflecting exocytosis of tiny vesicles (Borgonovo et al., 2002). The [Ca2+] required to stimulate this response is very high. In this issue Yaradanakul et al. (see p. 29) report experiments with baby hamster kidney (BHK) cells expressing the Na+/Ca2+ exchanger NCX1. In whole-cell patch clamp experiments, using an intracellular solution with high (40 mM) Na+ concentration and a Na+-free extracellular solution, switching extracellular [Ca2+] from very low (in the presence of 0.5 mM EGTA) to 2 mM is a new trick that makes it work. The massive Ca2+influx that is produced (mediated bv the Na+/Ca2+ exchanger running in reverse) raises intracellular free [Ca2+] to ∼200 μM, which triggers a pronounced capacitance increase. The response is only partially inhibited when ATP is replaced by the nonhydrolyzable analogue AMPPNP. A Ca2+-dependent capacitance increase, distinct from secretory granule exocytosis, was first discovered in rat peritoneal mast cells (Almers and Neher, 1987) and was subsequently reported to occur in many other cell types (Lindau et al., 1993; Coorssen et al., 1996; Oberhauser et al., 1996; Xu et al., 1998; Borgonovo et al., 2002). The very high [Ca2+] increase required to induce the response in BHK cells agrees with the previously reported requirement for intracellular [Ca2+] exceeding 100 μM to induce corresponding capacitance increases in many other cells—though the response in mast cells was apparent already at ∼3 μM free intracellular [Ca2+] (Almers and Neher, 1987). Phosphoinositides have for many years been implicated to play a significant role in regulated exocytosis (Eberhard et al., 1990; Hay et al., 1995; Martin, 2001; Wenk and De Camilli, 2004). Yaradankul et al. (2008) present detailed studies on the role of phosphoinositides in the response that is triggered by high [Ca2+] in BHK cells. The Ca2+ influx activates PI(4,5)P2 breakdown but phosphoinositide metabolism turns out to be neither sufficient nor necessary for the membrane-fusion response. Activation of PI(4,5)P2 breakdown in the absence of a sufficiently high [Ca2+] increase does not stimulate fusion and PLC inhibitors as well as peptides binding PI(4,5)P2 do not interfere with the activation of the Ca2+ influx–induced fusion response. These results indicate that the regulation of this fusion response is quite different from what has been reported for hormone release from neuroendocrine cells, where PI(4,5)P2 appears to have a role in the priming as well as the fusion (Eberhard et al., 1990; Hay et al., 1995; Martin, 2001). What membrane compartment(s) could cause the observed massive capacitance changes? The usual assumption is that these changes reflect an increase in membrane area due to fusion of a large number of small vesicles that are not resolved as individual capacitance steps. Alternatively, for a membrane capacitance increase the plasma membrane thickness would have to decrease or its effective dielectric constant would have to increase dramatically. At present there is no evidence that such changes could occur on the required scale. The size of the response, however, could also not be explained by the vesicle numbers seen in electron micrographs (Yaradanakul et al., 2008). However, in thin sections such small vesicles might be lost, and a mechanism involving vesicle fusion still appears the most likely. Indeed, previous work identified the protein desmoyokin-AHNAK as a marker of the vesicles underlying this exocytotic response and which were named enlargosomes (Borgonovo et al., 2002). In chromaffin cells the fusion of microvesicles that is not associated with catecholamine release is, in contrast to chromaffin granule exocytosis, not sensitive to tetanus toxin (TeTx) as well as Botulinum neurotoxins E, D, A, and C1 (Xu et al., 1998). Fusion of enlargosomes in PC12 cells is also TeTx insensitive (Kasai et al., 1999; Borgonovo et al., 2002). It would be interesting to explore if the response is also toxin insensitive in BHK cells. If that were the case, exocytosis of enlargosomes could be independent of phosphoinositide turnover. In the second paper on the pair of articles, Wang and Hilgemann (see p. 51) extend these studies to the rat basophilic leukemia (RBL) mast cell line. Following serotonin loading, the size of secretory granules increases dramatically in RBL cells (Williams et al., 1999). Exocytosis was stimulated by addition of the Ca2+ ionophore {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187. Extending the recently developed method of patch amperometry (Albillos et al., 1997; Dernick et al., 2005) to giant patches, the authors demonstrate that large capacitance steps, reflecting fusion of these granules with the plasma membrane, are associated with amperometrically detected serotonin release. In addition to these discrete capacitance steps, capacitance changes that cannot be resolved as discrete steps are observed—and which are not associated with serotonin release, consistent with previous observations in peritoneal mast cells (Oberhauser et al., 1996). The authors then proceed to explore the underlying mechanism(s) in excised giant patch capacitance measurements, which were performed on cells pretreated with latrunculin A to disrupt the cytoskeleton. Occasionally, but not reproducibly, exocytosis of large secretory granules associated with serotonin release was recorded by patch amperometry also in this configuration. The basis for the variability most likely is that when the patch is excised, the giant granules that are formed by serotonin loading do not stay docked to the cytoplasmic face of the giant patch but are left behind in the cell, tethered to the cytoskeleton. In giant excised-patch recordings, application of high [Ca2+] from a puffer pipette stimulates massive exocytosis. These experiments were performed on patches from cells that were not preloaded with serotonin. In these cells secretory granules containing mediators such as histamine or hexosaminidase exist but they are small. It is therefore uncertain how much of the response is due to secretory granules and how much is due to microvesicles distinct from the secretory granules. Surprisingly, the authors find almost complete inhibition of the capacitance response following TeTx treatment. Because rat synaptobrevin-1 is not toxin sensitive (Schiavo et al., 2000), the result suggests that the response is mediated by fusion of vesicles containing synaptobrevin-2 or cellubrevin. This is important because, though exocytosis of mast cell secretory granules is thought not to involve TeTx-sensitive synaptobrevin-2 or cellubrevin, recent experiments suggest that different subpopulations of vesicles undergoing regulated exocytosis in mast cells may involve different VAMP family members (Puri and Roche, 2008). The results of Wang and Hilgemann (2008) suggest that, in contrast to other cell types, exocytosis of RBL vesicles that fuse in response to high intracellular [Ca2+] is mediated by a TeTx-sensitive VAMP family member. As for the BHK cells, vesicle fusion in RBL cells appears to be independent of phosphoinositide metabolism. However, experiments with wortmannin/adenosine, which inhibits PI(3) and PI(4) kinases, point to a possible role in priming. Using a technique that inflates cells, such that the plasma membrane detaches from the vesicles bound to the cytoskeleton (Solsona et al., 1998), Wang and Hilgemann further show that the recovery of the capacitance response after reshrinking the cells is markedly reduced following wortmannin/adenosine treatment, suggesting that phosphoinositdes may play a role in priming of these vesicles after making contact with the plasma membrane, consistent with the expected role of PI(4,5)P2 in the priming of secretory vesicles in neuroendocrine cells (Hay et al., 1995; Gong et al., 2005; Milosevic et al., 2005). It still remains to be determined what vesicle type gives rise to the large capacitance changes stimulated by [Ca2+] in the hundreds of micromolar range, and if these are at all similar for all the different cell types. The differential TeTx sensitivity in different cell types suggests a quite complex picture.

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.

article i nfo Background: Neurotransmitters, neuropeptides and hormones are released from secretory vesicles of nerve terminals and neuroendocrine cells by calcium-activated exocytosis. A key step in this process is the forma- tion of a fusion pore between the vesicle membrane and the plasma membrane. Exocytotic fusion leads to an increase in plasma membrane area that can be measured as a proportional increase in plasma membrane capacitance. Scope of review: High resolution capacitance measurements in single cells, nerve terminals and small mem- brane patches have become possible with the development of the patch clamp technique. This review dis- cusses the methods of whole cell patch clamp capacitance measurements and their use in conjunction with voltage clamp pulse stimulation and with stimulation by photorelease of caged calcium. It also discusses patch capacitance measurements for the study of single exocytotic events and fusion pore properties in neu- roendocrine cells and nerve terminals. Major conclusions: Capacitance measurements provide high resolution information on the extent and time course of fusion for the characterization of vesicle pools and the kinetics of exocytosis. They allow the char- acterization of the mode of fusion including distinction of single vesicle full fusion, transient kiss-and-run fu- sion or multivesicular compound exocytosis. Furthermore, measurement of fusion pore conductances and their dynamic behavior has enabled the characterization of fusion pore properties in a way that resembles the characterization of ion channel function through single channel recordings. General significance: The combination of patch clamp capacitance measurements with pharmacological and molecular manipulations of exocytosis is emerging as a powerful approach to investigate the molecular mechanisms of calcium-activated exocytotic fusion pore formation. This article is part of a Special Issue enti- tled Biochemical, biophysical and genetic approaches to intracellular calcium signalling.

Fusion of neurosecretory vesicles with the plasma membrane is mediated by SNARE proteins, which transfer a force to the membranes. However, the mechanism by which this force transfer induces fusion pore formation is still unknown. The neuronal vesicular SNARE protein synaptobrevin 2 (syb2) is anchored in the vesicle membrane by a single C terminal transmembrane (TM) helix. In coarse grain molecular dynamics simulations self-assembly of the membrane occurred with the syb2 TM helix inserted as expected from experimental data. The free energy profile for the position of the TM domain in the membrane was determined applying harmonic potentials to the peptide in its unbiased position, pulling it towards new biased equilibrium positions. The energy profile determined in this way predicts the energy landscapes for pulling syb2 towards the extravesicular side as expected for SNARE complex zippering. Applying a constant pulling force of 160 pN detaches the synaptobrevin C terminus from the vesicle's inner leaflet lipid head groups within ∼100ns and pulls the C terminus deeper into the membrane. This C terminal movement should occur on the physiological millisecond time scale at ∼120 pN force. It is facilitated and hindered by specific mutations in parallel with experimentally observed facilitation and inhibition of fusion. These results suggest a mechanism where fusion pore formation is induced by movement of the charged syb2 C terminus into the hydrophobic core of the membrane in response to the force generated by C terminal zippering of the SNARE complex. This displacement of the charged C terminus is expected to destabilize the membrane providing a plausible pathway to fusion pore formation. Supported by NIH grants R01GM085808, R21NS072577, EDICT Project grant 201924, an MRC fellowship and the Wellcome Trust.

The cytoplasmic face of cell membranes has multiple features, such as cortical actin and tethered vesicles, the nanomechanical properties of which are largely unknown. To study these properties, we prepared plasma membrane sheets from a PC12 cell clone (Lang 2008 Meth.Mol.Biology, 440: 51-9) constitutively expressing ANF-EGFP, GFP-tagged proANF (atrial natriuretic factor) (Han et al 1999 PNAS 96:14577-82) as a secretory vesicle marker. The membrane sheets are firmly attached to the coverslip with their extracellular face while the cytoplasmic face is exposed. We combined a Nikon Ti-E/B total internal reflection fluorescence (TIRF) microscope with an Agilent PicoPlus atomic force microscope (AFM). Soft AFM cantilevers (Olympus Biolevers) were brought into contact with the exposed membrane face, binding spontaneously to the surface and to secretory vesicles. The membrane sheets and the motion of the cantilever tips were imaged by TIRF microscopy while forces of approximately 50pN or more were applied, pulling the tip away from the surface. At 50 ms time resolution, the noise level of the mechanical measurements was ∼1 pN and 0.5 nm (r.m.s.). Dynamic interactions associated with displacements in the 10-40 nm range were recorded. The role of secretory vesicles in this behavior will be investigated.

Fusion of neurosecretory vesicles with the plasma membrane is mediated by SNARE proteins, which transfer a force to the membranes. However, the mechanism by which this force transfer induces fusion pore formation is still unknown. The neuronal vesicular SNARE protein synaptobrevin 2 (syb2) is anchored in the vesicle membrane by a single C-terminal trans- membrane (TM) helix. In coarse-grain molecular-dynamics simulations, self-assembly of the membrane occurred with the syb2 TM domain inserted, as expected from experimental data. The free-energy profile for the position of the syb2 membrane anchor in the membrane was determined using umbrella sampling. To predict the free-energy landscapes for a reaction pathway pulling syb2 toward the extravesicular side of the membrane, which is the direction of the force transfer from the SNARE complex, harmonic potentials were applied to the peptide in its unbiased position, pulling it toward new biased equilibrium positions. Appli- cation of piconewton forces to the extravesicular end of the TM helix in the simulation detached the synaptobrevin C-terminus from the vesicle's inner-leaflet lipid headgroups and pulled it deeper into the membrane. This C-terminal movement was facil- itated and hindered by specific mutations in parallel with experimentally observed facilitation and inhibition of fusion. Direct appli- cation of such forces to the intravesicular end of the TM domain resulted in tilting motion of the TM domain through the membrane with an activation energy of ~70 kJ/mol. The results suggest a mechanism whereby fusion pore formation is induced by movement of the charged syb2 C-terminus within the membrane in response to pulling and tilting forces generated by C-terminal zippering of the SNARE complex.

Formation of a fusion pore between a vesicle and its target membrane is thought to involve the so-called SNARE protein complex. However, there is no mechanistic model explaining how the fusion pore is opened by conformational changes in the SNARE complex. it has been suggested that C-terminal zipping triggers fusion pore opening. A SNAP-25 mutant named SNAP-25 Delta 9 (lacking the last nine C-terminal residues) should lead to a less-tight C-terminal zipping. Single exocytotic events in chromaffin cells expressing this mutant were characterized by carbon fiber amperometry and cell-attached patch capacitance measurements. Cells expressing SNAP-25 Delta 9 displayed smaller amperometric "foot-current" currents, reduced fusion pore conductances, and lower fusion pore expansion rates. We propose that SNARE/lipid complexes form proteolipid fusion pores. Fusion pores involving the SNAP-25 Delta 9 mutant will be less tightly zipped and may lead to a longer fusion pore structure, consistent with the observed decrease of fusion pore conductance.

Fusion of a vesicle with its target membrane is preceded by tethering or docking. However, the physical mechanism of vesicle-tethering is unknown. To study this mechanism, we used eosinophil secretory granules, which undergo stimulated homotypic fusion events inside the cell during degranulation. Using a dual optical trap system, we observed tether formation between isolated eosinophil secretory granules. The results show that secretory granules interact stochastically with a target membrane forming physical tethers linking the vesicle and target membrane, rather than via interactions with the cytoskeleton. The necessary components are membrane-associated, and the addition of cytosolic components is not required. Tether-lifetime measurements as a function of applied mechanical force revealed at least three kinetically distinct tethered states. The tethered-state lifetimes of isolated eosinophil granules match the residence times of chromaffin granules at the plasma membrane in intact cells, suggesting that the tethering mechanisms reported here may represent the physiological mechanisms of vesicle-tethering in the cell.

Release of charged neurotransmitter molecules through a narrow fusion pore requires charge compensation by other ions. It has been proposed that this may occur by ion flow from the cytosol through channels in the vesicle membrane, which would generate a net outward current. This hypothesis was tested in chromaffin cells using cell-attached patch amperometry that simultaneously measured catecholamine release from single vesicles and ionic current across the patch membrane. No detectable current was associated with catecholamine release indicating that

Opening of individual exocytotic fusion pores in chromaffin cells was imaged electrochemically with high time resolution. Electrochemical detector arrays that consist of four platinum microelectrodes were microfabricated on a glass coverslip. Exocytosis of single vesicles containing catecholamines from a cell positioned on top of the array is detected by the individual electrodes as a time-resolved oxidation current, reflecting the time course of arrival of catecholamine molecules at the electrode surfaces. The signals exhibit low noise and reveal foot signals indicating fusion pore formation and expansion. The position of individual release events is determined from the fraction of catecholamines recorded by the individual electrodes. Simultaneous fluorescence imaging of release of acridine orange from individual vesicles confirmed the electrochemical position assignments. This electrochemical camera provides very high time resolution, spatiotemporal localization of individual fusion pore openings and quantitative data on the flux of transmitter from individual vesicles. Analysis of the amperometric currents employing random walk simulations indicates that the time course of amperometric spikes measured near the cell surface is due to a low apparent diffusion coefficient of cat-echolamines near the cell surface and not due to slow dissociation from the granular matrix.

Phosphatidylinositol-4,5-bisphosphate was proposed to be an important regulator of large dense-core vesicle exocytosis from neuroendocrine tissues. Here, we have examined the kinetics of secretion in chromaffin cells from mice lacking phosphatidylinositol phosphate kinase type Iγ, the major neuronal phosphatidylinositol-4-phosphate 5-kinase. Absence of this enzyme caused a reduction of the readily releasable vesicle pool and its refilling rate, with a small increase in morphologically docked vesicles, indicating a defect in vesicle priming. Furthermore, amperometry revealed a delay in fusion pore expansion. These results provide direct genetic evidence for a key role of phosphatidylinositol-4,5-bisphosphate synthesis in the regulation of large dense-core vesicle fusion dynamics.

Recent evidence suggests that endocytosis in neuroendocrine cells and neurons can be tightly coupled to exocytosis, allowing rapid retrieval from the plasma membrane of fused vesicles for future use. This can be a much faster mechanism for membrane recycling than classical clathrin-mediated endocytosis. During a fast exo-endocytotic cycle, the vesicle membrane does not fully collapse into the plasma membrane; nevertheless, it releases the vesicular contents through the fusion pore. Once the vesicle is depleted of transmitter, its membrane is recovered without renouncing its identity. In this report, we show that chromaffin cells contain catecholamine-free granules that retain their ability to fuse with the plasma membrane. These catecholamine-free granules represent 7% of the total population of fused vesicles, but they contributed to 47% of the fusion events when the cells were treated with reserpine for several hours. We propose that rat chromaffin granules that transiently fuse with the plasma membrane preserve their exocytotic machinery, allowing another round of exocytosis.

We investigated the noise levels in cell-attached patch capacitance recordings with a lock-in amplifier. The capacitance noise level decreases with increasing sine wave frequency up to 20-40 kHz. With a 20-mV rms sine wave the rms noise level above 8 kHz is

We studied degranulation of single cord blood-derived mononuclear cells differentiating to eosinophils in cultures containing recombinant human interleukin-5 (rhIL-5) and rhIL-3 by whole-cell patch-clamp capacitance measurements. As in mature cells, degranulation can be stimulated by intracellular application of guanosine-5′-O-(3-thiotriphosphate) (GTP) γS after 10 days in culture, simultaneously with the first morphological appearance of granules. These results demonstrate that the fusion machineries for exocytotic fusion are present and functional as soon as the granules are formed, presumably at the myeloblast stage. In the third week, the total amount of granules exocytosed upon stimulation is similar to that in mature eosinophils from peripheral blood. The capacitance step size distributions in promyelocytes and myelocytes confirm that mature large specific granules are formed by homotypic fusion of unit granules with similar size. Homotypic fusion is facilitated during early stages of differentiation associated with granulogenesis. Between day 10 and day 35 in culture the plasma membrane area of resting cells decreases from ≈700 μm2 to ≈400 μm2, approaching the value of mature cells from peripheral blood. The most prominent decrease occurs between day 25 and day 35 and is accompanied by the appearance of an exocytotic component due to small vesicles. This suggests that a class of small secretory vesicles is formed by endocytosis during a late phase in maturation.

Several proteins involved in exocytosis have been identified recently, but it is still completely unclear which molecules perform the fusion event itself. Although in viral fusion the fusion proteins are known, even there the molecular mechanism remains controversial. Investigation of single fusion events by electrophysiological techniques together with fluorimetric measurements have now provided some insight into the properties of the first aqueous connection, the fusion pore. This pore has an initial size similar to an ion channel and allows movement of lipids only after it has substantially expanded, indicating that it is initially not a purely lipidic structure, but incorporates lipids when it expands. Although neurotransmitter release may occur through narrow transient fusion pores, the fusion pore of synaptic vesicles probably expands vey rapidly, making it unlikely that secretion is performed by rapid exo/endocytosis without full fusion under normal conditions. Recent recordings from small membrane patches have made it possible to resolve fusion events from vesicles as small as synaptic vesicles. Future experiments using excised patches may provide an approach to identify the molecular machinery of exocytotic membrane fusion.

We measured capacitance changes in cell attached patches of human neutrophils using a high frequency lock-in method. With this technique the noise level is reduced to 0.025 fF such that capacitance steps of 0.1 fF are clearly detected corresponding to exo- and endocytosis of single 60 nm vesicles. It is thus possible to detect almost all known exocytotic and endocytotic processes including exocytosis of small neurotransmitter containing vesicles in most cell types as well as endocytosis of coated and uncoated pits. In neutrophils we demonstrate a stepwise capacitance decrease generated by 60-165 nm vesicles as expected for endocytosis of coated and non-coated pits. Following ionomycin stimulation a stepwise capacitance increase is observed consisting of 0.1-5 fF steps corresponding to the different granule types of human neutrophils from secretory vesicles to azurophil granules. The opening of individual fusion pores is resolved during exocytosis of 200 nm vesicles. The initial conductance has a mean value of 150 pS and can be as low as 35 pS which is similar to the conductance of many ion channels suggesting that the initial fusion pore is formed by a protein complex.

We have investigated the granule fusion events associated with exocytosis in degranulating peritoneal guinea pig eosinophils by time-resolved patch-clamp capacitance measurements using the phase detector technique. Intracellular stimulation with micromolar calcium and GTP gamma S induces a 2- to 3-fold capacitance increase. The main phase of the capacitance increase occurs after a delay of 2–7 minutes and is composed of well-resolved capacitance steps. The number of steps is very close to the number of crystalloid granules contained in a resting cell and the step size distribution with a peak at 9 fF is in excellent agreement with the granule size distribution determined by electron microscopy. The individual granules thus fuse sequentially with the plasma membrane. The stepwise capacitance increase is frequently preceded by an apparently continuous capacitance increase which consists of steps smaller than 4 fF, indicating exocytosis of small vesicles as distinct from crystalloid-containing granules. In some cases the time course of the opening of individual fusion pores could be recorded, and this revealed metastable conductance states below 300 pS but random fluctuations at higher conductance levels. This behaviour suggests that the small fusion pore might be a protein structure similar to an ion channel, which becomes a continuously variable lipid pore at higher conductances. In some cells a significant capacitance decrease was observed which is apparently continuous, suggesting a process of membrane uptake by endocytosis of small vesicles.

We have investigated the temporal relationship between depolarization, elevation of [Ca2+]i and exocytosis in single vertebrate neuroendocrine nerve terminals. The change of [Ca2+]i and vasopressin release were measured with a time resolution of less than 1 s in response to K(+)-induced depolarization. Exocytosis was also monitored in the whole-terminal patch-clamp configuration by time resolved capacitance measurements while [Ca2+]i was simultaneously followed by fura-2 fluorescence measurements. In intact as well as patch-clamped nerve terminals sustained depolarization leads to a sustained rise of [Ca2+]i. The rate of vasopressin release from intact nerve terminals rises in parallel with [Ca2+]i but then declines rapidly towards basal (t1/2 approximately 15 s) despite the maintained high [Ca2+]i indicating that only a limited number of exocytotic vesicles can be released. We demonstrate that in nerve terminals exocytosis can be followed during step depolarization by capacitance measurements. The capacitance increase starts instantaneously whereas [Ca2+]i rises with a half time of several hundred milliseconds. An instantaneous steep capacitance increase is followed by a slow increase with a slope of 25–50 fF/s indicating the sequential fusion of predocked and cytoplasmic vesicles. During depolarization the capacitance slope declines to zero with a similar time course as the vasopressin release indicating a decrease in exocytotic activity. Depolarization per se in the absence of a sufficient rise of [Ca2+]i does not induce exocytosis but elevation of [Ca2+]i in the absence of depolarization is as effective as in its presence. The experiments suggest that a rapid rise of [Ca2+]i in a narrow region beneath the plasma membrane induces a burst of exocytotic activity preceding the elevation of bulk [Ca2+]i in the whole nerve terminal.

The secretory process requires many different steps and stages. Vesicles must be formed and transported to the target membrane. They must be tethered or docked at the appropriate sites and must be prepared for fusion (priming). As the last step, a fusion pore is formed and the contents are released. Release of neurotransmitter is an extremely rapid event leading to rise times of the postsynaptic response of less than 100 μs. The release thus occurs during the initial formation of the exocytotic fusion pore. To understand the process of synaptic transmission, it is thus of outstanding importance to understand the molecular structure of the fusion pore, what are the properties of the initial fusion pore, how these properties affect the release process and what other factors may be limiting the kinetics of release. Here we review the techniques currently employed in fusion pore studies and discuss recent data and opinions on exocytotic fusion pore properties.

The number of transmitter molecules released in a quantal event can be regulated, and recent studies suggest that the modulation of quantal size is associated with corresponding changes in vesicle volume (Colliver et al., 2000; Pothos et al., 2002). If so, this could occur either by distension of the vesicle membrane or by incorporation and removal of vesicle membrane. We performed simultaneous measurements of vesicle membrane area and catecholamine release in individual quantal events from chromaffin cells using cell-attached patch amperometry. Cells were treated with reserpine, a vesicular monoamine transport blocker that decreases quantal size, orl-dopa, a catecholamine precursor that increases quantal size. We show that decrease and increase in quantal size are associated with a respective decrease and increase in vesicle membrane area. These results point to a novel mechanism of vesicle membrane dynamics by which vesicles physically change their membrane area in response to changes in transmitter content such that the intravesicular concentration of transmitter is maintained.

In mast cells and granulocytes, exocytosis starts with the formation of a fusion pore. It has been suggested that neurotransmitters may be released through such a narrow pore without full fusion. However, owing to the small size of the secretory vesicles containing neurotransmitter, the properties of the fusion pore formed during Ca2+-dependent exocytosis and its role in transmitter release are still unknown. Here we investigate exocytosis of individual chromaffin granules by using cell-attached capacitance measurements combined with electrochemical detection of catecholamines, achieved by inserting a carbon-fibre electrode into the patch pipette. This allows the simultaneous determination of the opening of individual fusion pores and of the kinetics of catecholamine release from the same vesicle. We found that the fusion-pore diameter stays at

The molecular mechanisms behind the tethering of secretory vesicles to the plasma membrane are not well understood. To study these mechanisms, we use a combined AFM/TIRF method applying forces to GFP-tagged secretory granules tethered to PC-12 cell membrane sheets. In the experiment shown in the figure, the AFM tip captured a granule, and was then lowered onto the sheet (a-b). Pulling forces were applied, ranging from ∼100pN to ∼700pN (c-e). Extension and rupture events are apparent as transient variations in the pulling force, as well as increases in the scanner z position sensor. Change in sensor position is consistent with changes in vesicle height as observed in the TIRF, based on the calibrated excitation decay constant (c-d). Rapid transients with small amplitude reveal partial tether extensions by 10-20nm, while large transients with slow decay reveal more gradual extensions, exceeding 1μm, that appear to be composed of multiple steps. These observations suggest that the dynamics of vesicle tether extensions are much more complex than previously thought. A large data set of recordings like that in the figure is being analyzed. Supported by NIH grant R21NS072577.View Large Image | View Hi-Res Image | Download PowerPoint Slide