Exploring the structural dynamics of the vesicle priming machinery.

Various cell types release neurotransmitters, hormones and many other compounds that are stored in secretory vesicles by exocytosis via the formation of a fusion pore traversing the vesicular membrane and the plasma membrane. This process of membrane fusion is mediated by the Soluble N-ethylmaleimide-Sensitive Factor Attachment Proteins REceptor (SNARE) protein complex, which in neurons and neuroendocrine cells is composed of the vesicular SNARE protein Synaptobrevin and the plasma membrane proteins Syntaxin and SNAP25 (Synaptosomal-Associated Protein of 25 kDa). Before a vesicle can undergo fusion and release of its contents, it must dock at the plasma membrane and undergo a process named 'priming', which makes it ready for release. The primed vesicles form the readily releasable pool, from which they can be rapidly released in response to stimulation. The stimulus is an increase in Ca2+ concentration near the fusion site, which is sensed primarily by the vesicular Ca2+ sensor Synaptotagmin. Vesicle priming involves at least the SNARE proteins as well as Synaptotagmin and the accessory proteins Munc18, Munc13, and Complexin but additional proteins may also participate in this process. This review discusses the current views of the interactions and the structural changes that occur among the proteins of the vesicle priming machinery.

All SNAP25 molecules in the vesicle-plasma membrane contact zone change conformation during vesicle priming.

In neuronal cell types, vesicular exocytosis is governed by the SNARE (soluble NSF attachment receptor) complex consisting of synaptobrevin2, SNAP25, and syntaxin1. These proteins are required for vesicle priming and fusion. We generated an improved SNAP25-based SNARE COmplex Reporter (SCORE2) incorporating mCeruelan3 and Venus and overexpressed it in SNAP25 knockout embryonic mouse chromaffin cells. This construct rescues vesicle fusion with properties indistinguishable from fusion in wild-type cells. Combining electrochemical imaging of individual release events using electrochemical detector arrays with total internal reflection fluorescence resonance energy transfer (TIR-FRET) imaging reveals a rapid FRET increase preceding individual fusion events by 65 ms. The experiments are performed under conditions of a steady-state cycle of docking, priming, and fusion, and the delay suggests that the FRET change reflects tight docking and priming of the vesicle, followed by fusion after ~65 ms. Given the absence of wt SNAP25, SCORE2 allows determination of the number of molecules at fusion sites and the number that changes conformation. The number of SNAP25 molecules changing conformation in the priming step increases with vesicle size and SNAP25 density in the plasma membrane and equals the number of copies present in the vesicle-plasma membrane contact zone. We estimate that in wt cells, 6 to 7 copies of SNAP25 change conformation during the priming step.

Patch Amperometry and Intracellular Patch Electrochemistry.

Both patch amperometry (PA) and intracellular patch electrochemistry (IPE) take advantage of a recording configuration where an electrochemical detector-carbon fiber electrode (CFE)-is housed inside a patch pipette. PA, which is employed in cell-attached or excised inside-out patch clamp configuration, offers high-resolution patch capacitance measurements with simultaneous amperometric detection of catecholamines released during exocytosis. The method provides precise information on single-vesicle size and quantal content, fusion pore conductance, and permeability of the pore for catecholamines. IPE, on the other hand, measures cytosolic catecholamines that diffuse into the patch pipette following membrane rupture to achieve the whole-cell configuration. In amperometric mode, IPE detects total catechols, whereas in cyclic voltammetric mode, it provides more specific information on the nature of the detected molecules and may selectively quantify catecholamines, providing a direct approach to determine cytosolic concentrations of catecholaminergic transmitters and their metabolites. Here, we provide detailed instructions on setting up PA and IPE, performing experiments and analyzing the data.

Fusion pores with low conductance are cation selective.

Many neurotransmitters are organic ions that carry a net charge, and their release from secretory vesicles is therefore an electrodiffusion process. The selectivity of early exocytotic fusion pores is investigated by combining electrodiffusion theory, measurements of amperometric foot signals from chromaffin cells with anion substitution, and molecular dynamics simulation. The results reveal that very narrow fusion pores are cation selective, but more dilated fusion pores become anion permeable. The transition occurs around a fusion pore conductance of ∼300 pS. The cation selectivity of a narrow fusion pore accelerates the release of positively charged transmitters such as dopamine, noradrenaline, adrenaline, serotonin, and acetylcholine, while glutamate release may require a more dilated fusion pore.

On-Chip Cyclic Voltammetry Measurements Using a Compact 1024-Electrode CMOS IC.

Complementary metal-oxide-semiconductor (CMOS) microelectrode arrays integrate amplifier arrays with on-chip electrodes, offering high-throughput platforms for electrochemical sensing with high spatial and temporal resolution. Such devices have been developed for highly parallel constant voltage amperometric detection of transmitter release from multiple cells with single-vesicle resolution. Cyclic voltammetry (CV) is an electrochemical method that applies voltage waveforms, which provides additional information about electrode properties and about the nature of analytes. A 16-channel, 64-electrode-per-channel CMOS integrated circuit (IC) fabricated in a 0.5 µm CMOS process for CV is demonstrated. Each detector consists of only 11 transistors and an integration capacitor with a unit dimension of 0.0015 mm2. The device was postfabricated using Pt as the working electrode material with a shifted electrode design, which makes it possible to redefine the size and the location of working electrodes. The system incorporating cell-sized (8 µm radius) microelectrodes was validated with dopamine injection tests and CV measurements of potassium ferricyanide at a 1 V/s scanning rate. The cyclic voltammograms were in excellent agreement with theoretical predictions. The technology enables rigorous characterization of electrode performance for the application of CMOS microelectrode arrays in low-noise amperometric measurements of quantal transmitter release as well as other biosensing applications.

Drug testing complementary metal-oxide-semiconductor chip reveals drug modulation of transmitter release for potential therapeutic applications.

Neurodegenerative diseases, such as Parkinson's disease, Alzheimer's disease, and Huntington's disease, are considered incurable and significantly reduce the quality of life of the patients. A variety of drugs that modulate neurotransmitter levels have been used for the treatment of the neurodegenerative diseases but with limited efficacy. In this work, an amperometric complementary metal-oxide-semiconductor (CMOS) chip is used for high-throughput drug testing with respect to the modulation of transmitter release from single vesicles using chromaffin cells prepared from bovine adrenal glands as a model system. Single chromaffin cell amperometry was performed with high efficiency on the surface-modified CMOS chip and follow-up whole-cell patch-clamp experiments were performed to determine the readily releasable pool sizes. We show that the antidepressant drug bupropion significantly increases the amount of neurotransmitter released in individual quantal release events. The antidepressant drug citalopram accelerates rapid neurotransmitter release following stimulation and follow-up patch-clamp experiments reveal that this is because of the increase in the pool of readily releasable vesicles. These results shed light on the mechanisms by which bupropion and citalopram may be potentially effective in the treatment of neurodegenerative diseases. These results demonstrate that the CMOS amperometry chip technology is an excellent tool for drug testing to determine the specific mechanisms by which they modulate neurotransmitter release.

Precise Time Superresolution by Event Correlation Microscopy.

Fluorescence imaging is often used to monitor dynamic cellular functions under conditions of very low light intensities to avoid photodamage to the cell and rapid photobleaching. Determination of the time of a fluorescence change relative to a rapid high time-resolution event, such as an action potential or pulse stimulation, is challenged by the low photon rate and the need to use imaging frame durations that limit the time resolution. To overcome these limitations, we developed a time superresolution method named event correlation microscopy that aligns repetitive events with respect to the high time-resolution events. We describe the algorithm of the method, its step response function, and a theoretical, computational, and experimental analysis of its precision, providing guidelines for camera exposure time settings depending on imaging signal properties and camera parameters for optimal time resolution. We also demonstrate the utility of the method to recover rapid nonstepwise kinetics by deconvolution fits. The event correlation microscopy method provides time superresolution beyond the photon rate limit and imaging frame duration with well-defined precision.

Molecular mechanism of fusion pore formation driven by the neuronal SNARE complex.

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.

A Bidirectional-Current CMOS Potentiostat for Fast-Scan Cyclic Voltammetry Detector Arrays.

A potentiostat circuit for the application of bipolar electrode voltages and detection of bidirectional currents using a microelectrode array is presented. The potentiostat operates as a regulated-cascode amplifier for positive input currents, and as an active-input regulated-cascode mirror for negative input currents. This topology enables constant-potential amperometry and fast-scan cyclic voltammetry (FSCV) at microelectrode arrays for parallel recording of quantal release events, electrode impedance characterization, and high-throughput drug screening. A 64-channel FSCV detector array, fabricated in a 0.5-$\mu$m, 5-V CMOS process, is also demonstrated. Each detector occupies an area of 45  $\mu$m $\times$ 30 $\mu$m and consists of only 14 transistors and a 50-fF integrating capacitor. The system was validated using prerecorded input stimuli from actual FSCV measurements at a carbon-fiber microelectrode.

Single-Cell Recording of Vesicle Release From Human Neuroblastoma Cells Using 1024-ch Monolithic CMOS Bioelectronics.

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 [Formula: see text], 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.

The fusion pore, 60 years after the first cartoon.

Neurotransmitter release occurs in the form of quantal events by fusion of secretory vesicles with the plasma membrane, and begins with the formation of a fusion pore that has a conductance similar to that of a large ion channel or gap junction. In this review, we propose mechanisms of fusion pore formation and discuss their implications for fusion pore structure and function. Accumulating evidence indicates a direct role of soluble N-ethylmaleimide-sensitive-factor attachment receptor proteins in the opening of fusion pores. Fusion pores are likely neither protein channels nor purely lipid, but are of proteolipidic composition. Future perspectives to gain better insight into the molecular structure of fusion pores are discussed.

Surface-modified CMOS IC electrochemical sensor array targeting single chromaffin cells for highly parallel amperometry measurements.

Amperometry is a powerful method to record quantal release events from chromaffin cells and is widely used to assess how specific drugs modify quantal size, kinetics of release, and early fusion pore properties. Surface-modified CMOS-based electrochemical sensor arrays allow simultaneous recordings from multiple cells. A reliable, low-cost technique is presented here for efficient targeting of single cells specifically to the electrode sites. An SU-8 microwell structure is patterned on the chip surface to provide insulation for the circuitry as well as cell trapping at the electrode sites. A shifted electrode design is also incorporated to increase the flexibility of the dimension and shape of the microwells. The sensitivity of the electrodes is validated by a dopamine injection experiment. Microwells with dimensions slightly larger than the cells to be trapped ensure excellent single-cell targeting efficiency, increasing the reliability and efficiency for on-chip single-cell amperometry measurements. The surface-modified device was validated with parallel recordings of live chromaffin cells trapped in the microwells. Rapid amperometric spikes with no diffusional broadening were observed, indicating that the trapped and recorded cells were in very close contact with the electrodes. The live cell recording confirms in a single experiment that spike parameters vary significantly from cell to cell but the large number of cells recorded simultaneously provides the statistical significance.

t-SNARE Transmembrane Domain Clustering Modulates Lipid Organization and Membrane Curvature.

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.

AFM/TIRF force clamp measurements of neurosecretory vesicle tethers reveal characteristic unfolding steps.

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 (<10 nm) tether extension events was markedly higher when a fluorescent vesicle was present at the cantilever tip and increased in the presence of GTPγS, indicating that these events reflect specifically the properties of vesicle-plasma membrane tethers. The magnitude of the short tether extension events is consistent with extension lengths expected from progressive unfolding of individual helices of the exocyst complex, supporting its direct role in forming the physical vesicle-plasma membrane link.

v-SNARE transmembrane domains function as catalysts for vesicle fusion.

Vesicle fusion is mediated by an assembly of SNARE proteins between opposing membranes, but it is unknown whether transmembrane domains (TMDs) of SNARE proteins serve mechanistic functions that go beyond passive anchoring of the force-generating SNAREpin to the fusing membranes. Here, we show that conformational flexibility of synaptobrevin-2 TMD is essential for efficient Ca(2+)-triggered exocytosis and actively promotes membrane fusion as well as fusion pore expansion. Specifically, the introduction of helix-stabilizing leucine residues within the TMD region spanning the vesicle's outer leaflet strongly impairs exocytosis and decelerates fusion pore dilation. In contrast, increasing the number of helix-destabilizing, ß-branched valine or isoleucine residues within the TMD restores normal secretion but accelerates fusion pore expansion beyond the rate found for the wildtype protein. These observations provide evidence that the synaptobrevin-2 TMD catalyzes the fusion process by its structural flexibility, actively setting the pace of fusion pore expansion.

Prostaglandin E1 inhibits endocytosis in the ß-cell endocytosis.

Prostaglandins inhibit insulin secretion in a manner similar to that of norepinephrine (NE) and somatostatin. As NE inhibits endocytosis as well as exocytosis, we have now examined the modulation of endocytosis by prostaglandin E1 (PGE1). Endocytosis following exocytosis was recorded by whole-cell patch clamp capacitance measurements in INS-832/13 cells. Prolonged depolarizing pulses producing a high level of Ca(2+) influx were used to stimulate maximal exocytosis and to deplete the readily releasable pool (RRP) of granules. This high Ca(2+) influx eliminates the inhibitory effect of PGE1 on exocytosis and allows specific characterization of the inhibitory effect of PGE1 on the subsequent compensatory endocytosis. After stimulating exocytosis, endocytosis was apparent under control conditions but was inhibited by PGE1 in a Pertussis toxin-sensitive (PTX)-insensitive manner. Dialyzing a synthetic peptide mimicking the C-terminus of the α-subunit of the heterotrimeric G-protein Gz into the cells blocked the inhibition of endocytosis by PGE1, whereas a control-randomized peptide was without effect. These results demonstrate that PGE1 inhibits endocytosis and Gz mediates the inhibition.

A Wireless FSCV Monitoring IC With Analog Background Subtraction and UWB Telemetry.

A 30-µW wireless fast-scan cyclic voltammetry monitoring integrated circuit for ultra-wideband (UWB) transmission of dopamine release events in freely-behaving small animals is presented. On-chip integration of analog background subtraction and UWB telemetry yields a 32-fold increase in resolution versus standard Nyquist-rate conversion alone, near a four-fold decrease in the volume of uplink data versus single-bit, third-order, delta-sigma modulation, and more than a 20-fold reduction in transmit power versus narrowband transmission for low data rates. The 1.5- mm(2) chip, which was fabricated in 65-nm CMOS technology, consists of a low-noise potentiostat frontend, a two-step analog-to-digital converter (ADC), and an impulse-radio UWB transmitter (TX). The duty-cycled frontend and ADC/UWB-TX blocks draw 4 µA and 15 µA from 3-V and 1.2-V supplies, respectively. The chip achieves an input-referred current noise of 92 pA(rms) and an input current range of ±430 nA at a conversion rate of 10 kHz. The packaged device operates from a 3-V coin-cell battery, measures 4.7 × 1.9 cm(2), weighs 4.3 g (including the battery and antenna), and can be carried by small animals. The system was validated by wirelessly recording flow-injection of dopamine with concentrations in the range of 250 nM to 1 µM with a carbon-fiber microelectrode (CFM) using 300-V/s FSCV.

A Coarse Grained Model for a Lipid Membrane with Physiological Composition and Leaflet Asymmetry.

The resemblance of lipid membrane models to physiological membranes determines how well molecular dynamics (MD) simulations imitate the dynamic behavior of cell membranes and membrane proteins. Physiological lipid membranes are composed of multiple types of phospholipids, and the leaflet compositions are generally asymmetric. Here we describe an approach for self-assembly of a Coarse-Grained (CG) membrane model with physiological composition and leaflet asymmetry using the MARTINI force field. An initial set-up of two boxes with different types of lipids according to the leaflet asymmetry of mammalian cell membranes stacked with 0.5 nm overlap, reliably resulted in the self-assembly of bilayer membranes with leaflet asymmetry resembling that of physiological mammalian cell membranes. Self-assembly in the presence of a fragment of the plasma membrane protein syntaxin 1A led to spontaneous specific positioning of phosphatidylionositol(4,5)bisphosphate at a positively charged stretch of syntaxin consistent with experimental data. An analogous approach choosing an initial set-up with two concentric shells filled with different lipid types results in successful assembly of a spherical vesicle with asymmetric leaflet composition. Self-assembly of the vesicle in the presence of the synaptic vesicle protein synaptobrevin 2 revealed the correct position of the synaptobrevin transmembrane domain. This is the first CG MD method to form a membrane with physiological lipid composition as well as leaflet asymmetry by self-assembly and will enable unbiased studies of the incorporation and dynamics of membrane proteins in more realistic CG membrane models.