Exocytosis Events Research Articles

Monitoring the Effect of Osmotic Stress on Secretory Vesicles and Exocytosis.

Amperometry recording of cells subjected to osmotic shock show that secretory cells respond to this physical stress by reducing the exocytosis activity and the amount of neurotransmitter released from vesicles in single exocytosis events. It has been suggested that the reduction in neurotransmitters expelled is due to alterations in membrane biophysical properties when cells shrink in response to osmotic stress and with assumptions made that secretory vesicles in the cell cytoplasm are not affected by extracellular osmotic stress. Amperometry recording of exocytosis monitors what is released from cells the moment a vesicle fuses with the plasma membrane, but does not provide information on the vesicle content before the vesicle fusion is triggered. Therefore, by combining amperometry recording with other complementary analytical methods that are capable of characterizing the secretory vesicles before exocytosis at cells is triggered offers a broader overview for examining how secretory vesicles and the exocytosis process are affected by osmotic shock. We here describe how complementing amperometry recording with intracellular electrochemical cytometry and transmission electron microscopy (TEM) imaging can be used to characterize alterations in secretory vesicles size and neurotransmitter content at chromaffin cells in relation to exocytosis activity before and after exposure to osmotic stress. By linking the quantitative information gained from experiments using all three analytical methods, conclusions were previously made that secretory vesicles respond to extracellular osmotic stress by shrinking in size and reducing the vesicle quantal size to maintain a constant vesicle neurotransmitter concentration. Hence, this gives some clarification regarding why vesicles, in response to osmotic stress, reduce the amount neurotransmitters released during exocytosis release. The amperometric recordings here indicate this is a reversible process and that vesicle after osmotic shock are refilled with neurotransmitters when placed cells are reverted into an isotonic environment.

Electrochemical Investigation of the Interaction between Catecholamines and ATP.

The study of the colligative properties of adenosine 5'-triphosphate (ATP) and catecholamines has received the attention of scientists for decades, as they could explain the capabilities of secretory vesicles (SVs) to accumulate neurotransmitters. In this Article, we have applied electrochemical methods to detect such interactions in vitro, at the acidic pH of SVs (pH 5.5) and examined the effect of compounds having structural similarities that correlate with functional groups of ATP (adenosine, phosphoric acid and sodium phosphate salts) and catecholamines (catechol). Chronoamperometry and fast scan cyclic voltammetry (FSCV) provide evidence compatible with an interaction of the catechol and adenine rings. This interaction is also reinforced by an electrostatic interaction between the phosphate group of ATP and the protonated ammonium group of catecholamines. Furthermore, chronoamperometry data suggest that the presence of ATP subtlety reduces the apparent diffusion coefficient of epinephrine in aqueous media that adds an additional factor leading to a slower rate of catecholamine exocytosis. This adds another plausible mechanism to regulate individual exocytosis events to alter communication.

Molecules involved in acrosomal exocytosis and cortical granule exocytosis

At fertilization, the acrosome reaction and cortical reaction are crucial process to block polyspermy and the prevention of triploidy. Although molecules involved in trigger secretion are various in different exocytosis events, the two processes may share the similar exocytosis mechanism. Exocytosis is an accurate regulated process that consists of multiple stages such as recruitment, targeting, tethering and docking of secretory vesicles with the plasma membrane, priming of the fusion machinery and calcium-triggered membrane fusion. After fusion, the membrane around the secretory vesicle is incorporated into the plasma membrane and the granule releases its contents. The proteins involved in these processes belong to several highly conserved families: Rab GTPases, SNAREs (soluble NSF-attachment protein receptors), α-SNAP (α-NSF attachment protein), NSF (N-ethylmaleimide-sensitive factor), Munc13, Munc18, complexins and synaptotagmins. This review discussed that molecules believed to participate in the secretory function and their involvement in acrosome and cortical granule exocytosis.

Kv2.1 Clustering Contributes to Exocytosis Hotspots and Rescues Human β-Cell Dysfunction

Background: Kv2.1, which mediates the repolarization of action potentials and interacts with SNARE proteins such as Syntaxin1A, is known to target to large cell surface clusters in neurons. Previously, we confirmed that Kv2.1 form clusters in human β-cells, and play a role as facilitators of insulin exocytosis. It is reported that exocytosis events are clustered at hotspots in insulinoma cells. Thus, we hypothesize that, Kv2.1 clustering may contribute to forming exocytosis hotspots in human β-cells.

(Invited) Novel Carbon-Based Sensors for the Next Generation of Neurochemical Measurements

Carbon-fiber microelectrodes have been the fundamental tool for the electrochemical detection of neurotransmitters since their introduction in the early 1980s. They are biologically compatible and, compared to larger electrodes, microelectrodes offer highly increased diffusional flux to the electrode surface and faster response times to enable high speed measurements. The demonstrated benefit of enhanced mass transport to very small electrodes has led us to develop several new carbon-based microelectrodes for the improved detection of dynamic neurochemical fluctuations. This talk will describe the characterization of a recessed microdisk electrode for enhanced detection of individual exocytosis events at single cells, a nanoscale microelectrode with a conical geometry for fast intracellular voltammetric measurements, and innovative yarn-like sensing substrates engineered entirely of multi-walled carbon nanotubes for enhanced sensitivity when monitoring from individual cells or discrete brain regions. Each of these ultramicroelectrode designs exploits mass transport to either provide maximal diffusional flux to, or to limit diffusional flux from, the electrode surface. These new tools provide significant advantages when coupled with fast-scan cyclic voltammetry, and applications that provide new insight into neurochemical dynamics will be discussed.

Ultrasonic-Aided Fabrication of Nanostructured Au-Ring Microelectrodes for Monitoring Transmitters Released from Single Cells.

We report a novel ultrasonic-aided fast and straightforward approach to fabricate Au microelectrodes by electroless deposition of nanostructured gold films on the rigid outer surface of pulled glass capillaries. Microelectrodes with tip diameters ranging from several hundred nanometers to several micrometers were fabricated within 20 min via three sequential ultrasonication steps. The ultrasonication technique has been validated to be a very effective route in engineering the morphology of Au film surfaces and improves the fabrication efficiency of Au microelectrodes. The nanostructured surfaces of the Au microelectrodes demonstrate excellent sensing activity and antifouling for dopamine oxidation. The microelectrodes were applied for measurement of catecholamines released from exocytosis events from single chromaffin cells and exhibited faster dynamic peak parameters, compared with carbon fiber microelectrodes. This report provides a generally accessible and complementary platform for analyzing catecholamines release events, which should be useful for new electrode designs and neurochemical sensing.

Surface Plasmon Resonance for Measuring Exocytosis from Populations of PC12 Cells: Mechanisms of Signal Formation and Assessment of Analytical Capabilities.

Because of cell to cell variation, it is difficult to obtain statistically significant data on the frequency of exocytosis events (Rexocytosis, t-1 m-2) with traditional single cell electrophysiological or fluorescence microscopy based methods. Here we take the first steps toward a rapid cost-effective surface plasmon resonance (SPR) based method for measuring the Rexocytosis for populations of PC12 cells. The conditions for culturing confluent monolayers on the sensor slides were optimized, and neurotransmitter exocytosis was evoked by injecting solutions with elevated [K+]. Exocytosis caused a shift of the resonance angle (Δθr) that was linearly proportional to Rexocytosis. The Δθr was mainly due to elevated concentration of secretory vesicles close to the cell membrane. The increased vesicle concentration thus acted as a proxy for the Rexocytosis that could not be measured directly. The Δθr was calibrated for Rexocytosis using single cell amperometry on parallel cell cultures. The cell populations were large enough for variation in responses between sensor slides to only reflect actual differences in biological condition. The applicability for drug screening is demonstrated by studying the effects of EGTA, reserpine, and prolonged stimulation by K+.

Curvature-Undulation Coupling as a Basis for Curvature Sensing and Generation in Bilayer Membranes

Cell membranes play an important role in modulating cell signaling pathways by hosting the assembly of protein structures which are capable of initiating downstream signaling events. Understanding how proteins both sense and generate membrane curvature during these processes is therefore necessary to relate specific protein-membrane interactions with larger mesoscale membrane shape changes observed in experiments. We employ coarse-grained molecular dynamics simulations of the epsin N-terminal homology (ENTH) domain bound to phosphatidylinositol 4,5-bisphosphate (PIP2) in a lipid bilayer to demonstrate a formalism for computing the induced curvature field. We formulate an objective function for matching the fluctuations observed in simulations to the deformation fields defined by the theory. This method allows us to recover the curvature fields induced by proteins at varying concentrations near the dilute limit, even when the protein-induced curvature is the same order as the amplitude of thermal undulations. We predict that proteins at dilute concentrations can sense a site of spontaneous curvature at distances much larger than their size. This suggests that increasing protein concentrations can localize or focus the dynamic curvature field to the site of the proteins, and that the protein-induced stabilization of membrane curvature is a cooperative process. This method may be used to predict the influence of protein concentration, assembly, and membrane binding on endocytosis and exocytosis events in cells.

Membrane Deformation Induced by Cation Binding to Negatively Charged Phospholipids

The divalent cation Ca2+ plays a critical role in many cell signaling and membrane trafficking pathways, including exocytosis and membrane fusion. Ca2+ signal transduction is commonly thought to occur through interaction with protein partners; however, it may also interact with negatively charged phospholipids within the plasma membrane leading to changes in lipid behavior.Here we show that an asymmetric concentration of Ca2+ ions across the membrane can generate tubules or protrusions from membranes containing phosphatidylserine (PS) or phosphatidylinositol-4,5-bisphosphate (PIP2). We transferred single vesicles with controlled membrane tension into Ca2+ solution and observed the formation of inward tubules, indicating negative curvature generation by Ca2+. The Ca2+-induced membrane deformation is inhibited by high tension or low cation binding, in a similar manner as we have previously observed for BAR domain proteins. We propose that the membrane deformation is caused by Ca2+-induced clustering of PS and PIP2 creating negative curvature. In confirmation of this we find that Mg2+, which is known to only weakly cluster PS and PIP2, only generates curvature at relatively high concentrations and low tension. Additionally, for PS vesicles the membrane deformation requires high Ca2+ concentration (a few millimolar), while PIP2 vesicles, which are more sensitive to Ca2+-induced clustering, are deformed at lower Ca2+ concentrations. The presence of salt ions (Na+) also influences Ca2+-induced membrane curvature. High salt content can reduce Ca2+-induced curvature by shielding Ca2+ ions from the membrane. An asymmetric salt concentration can influence membrane structure and either enhance or oppose Ca2+-induced membrane curvature. The observed Ca2+-induced membrane deformation represents a new mechanism for Ca2+ signaling events. This mechanism is likely to be especially important for the role of Ca2+ in exocytosis and membrane fusion events.

A CMOS based Sensor Array Platform for Analysis of Exocytosis Events

The release of neurotransmitters into the extracellular space is an important drug target and crucial for molecular manipulation. Amperometric measurements provide details about the amount and kinetics of transmitter release in single quantal events. However, amperometric spikes vary from cell to cell even under the same condition; therefore, a large number of release events must be analyzed to achieve the statistical significance. We present CMOS chips with 100 or 1,024 active electrodes with surface modifications for amperometry measurements. To enable amperometry measurements, polarizable electrodes such as platinum are patterned on the original aluminum metal contact on the chip. To prevent potential current leakage, a new shifted electrode design was used to enhance the reliability of post-fabrication. Platinum electrode material is deposited on the aluminum contact extending onto the periphery area and further insulated to redefine the openings of the active electrodes by lithography and sputtering. The insulation of the surface is performed by lithography using photoresist. The 100 sensor array was validated by simultaneous recording from all electrodes at 1MS/s using dopamine injection and recordings from live chromaffin cells cultured on-chip. A new chip with 1024 working electrodes capable of bi-directional current recordings was developed for fast scan cyclic voltammetry. Positive as well as negative currents are measured in the same working manner, and a current mirror and a biased cascode transistor ensure a gain error <1%. A sign detection function is implemented to report the polarity of the current input. Simulations of the circuit revealed reliable capacitive current recording and the linearity of the gain over a wide range of input with the correct sign-bit output. We anticipate that the chip will serve as a universal platform for characterizing various physiological events.

Simulation-Guided Optimization of Electrode Arrays for Electrochemical Imaging of Quantal Exocytosis

Fluorescence imaging can provide important information about the mechanisms of exocytosis at the single-vesicle level, e.g., conformational changes in SNARE proteins reported via FRET. FRET signals are very small and therefore the time and location of the fusion events are not readily apparent to allow signal analyses. The Lindau lab has pioneered the use of “electrochemical imaging” to identify the time and location of quantal exocytosis by comparing the amperometric signal between 4 electrodes arranged around a cell. Since it is necessary to obtain a measurable signal in at least three electrodes in order to “triangulate” the position, a limitation of this method is that it can only localize exocytosis events that originate near the center of the electrode arrays. We therefore carried out finite-element-array (COMSOL) reaction-diffusion simulations to guide the design of electrode arrays that can maximize the area on the cell surface where a release event can be localized with high precision. The number and placement of electrodes around the cell were varied and the SD of localization precision was simulated. Our results indicate that as the number of electrodes arranged circumferentially around the cell increases, the detectable area further from the center of the array increases, however, the precision of localization in the center decreases because the amperometric charge is divided between more electrodes. Thus the optimal number of electrodes depends on the signal-to-noise ratio of the amperometric recordings and the acceptable location precision. Under our simulation conditions, if a localization precision (SD) less than 0.23 μm is needed, an array of 4 electrodes will give the largest detectable area. However, if slightly less precision is acceptable, 5 or 6 electrodes allows a larger detectable area than 4 or 3 electrodes. Supported by NIH R44MH096650.

Extracellular Osmotic Stress Reduces the Vesicle Size while Keeping a Constant Neurotransmitter Concentration.

Secretory cells respond to hypertonic stress by cell shrinking, which causes a reduction in exocytosis activity and the amount of signaling molecules released from single exocytosis events. These changes in exocytosis have been suggested to result from alterations in biophysical properties of cell cytoplasm and plasma membrane, based on the assumption that osmotic stress does not affect the secretory vesicle content and size prior to exocytosis. To further investigate whether vesicles in secretory cells are affected by the osmolality of the extracellular environment, we used intracellular electrochemical cytometry together with transmission electron microscopy imaging to quantify and determine the catecholamine concentration of dense core vesicles in situ before and after cell exposure to osmotic stress. In addition, single cell amperometry recordings of exocytosis at chromaffin cells were used to monitor the effect on exocytosis activity and quantal release when cells were exposed to osmotic stress. Here we show that hypertonic stress hampers exocytosis secretion after the first pool of readily releasable vesicles have been fused and that extracellular osmotic stress causes catecholamine filled vesicles to shrink, mainly by reducing the volume of the halo solution surrounding the protein matrix in dense core vesicles. In addition, the vesicles demonstrate the ability to perform adjustments in neurotransmitter content during shrinking, and intracellular amperometry measurements in situ suggest that vesicles reduce the catecholamine content to maintain a constant concentration within the vesicle compartment. Hence, the secretory vesicles in the cell cytoplasm are highly affected and respond to extracellular osmotic stress, which gives a new perspective to the cause of reduction in quantal size by these vesicles when undergoing exocytosis.

Single cell amperometry reveals curcuminoids modulate the release of neurotransmitters during exocytosis from PC12 cells

We used single cell amperometry to examine whether curcumin and bisdemethoxycurcumin (BDMC), substances that are suggested to affect learning and memory, can modulate monoamine release from PC12 cells. Our results indicate both curcumin and BDMC need long-term treatment (72h in this study) to influence exocytosis effectively. By analyzing the parameters calculated from single exocytosis events, it can be concluded that curcumin and BDMC affect exocytosis through different mechanisms. Curcumin accelerates the event dynamics with no significant change of the monoamine amount released from single exocytotic events, whereas BDMC attenuates the amount from single exocytotic event with no significant change of the event dynamics. This comparison of the effect of curcumin and BDMC on exocytosis at the single cell level brings insight into their different mechanisms, which might lead to different biological actions. The effect of curcumin and BDMC on the opening and closing of the exocytotic fusion pore were also investigated. These results might be helpful for understanding the improvement of learning and memory and the anti-depression properties of curcuminoids.

Quantitative Chemical Measurements of Vesicular Transmitters with Electrochemical Cytometry.

Electrochemical cytometry adds a new dimension to our ability to study the chemistry and chemical storage of transmitter molecules stored in nanometer vesicles. The approach involves the adsorption and subsequent rupture of vesicles on an electrode surface during which the electroactive contents are quantitatively oxidized (or reduced). The measured current allows us to count the number of molecules in the vesicles using Faraday's law and to correlate this to the amount of molecules released when single exocytosis events take place at communicating cells. The original format for this method involved a capillary electrophoresis separation step to singly address each vesicle, but we have more recently discovered that cellular vesicles tend to adsorb to carbon electrodes and spontaneously as well as stochastically rupture to give mostly single vesicle events. This approach, called impact electrochemical cytometry, even though the impact is perhaps not the important part of this process, has been studied and the vesicle rupture appears to be at the interface between the vesicle and the electrode and is probably driven by electroporation. The pore size and rate of content electrolysis are a function of the pore diameter and the presence of a protein core in the vesicles. In model liposomes with no protein, events appear extremely rapidly as the soft nanoparticles impact the electrode and the contents are oxidized. It appears that the proteins decorating the surface of the vesicle are important in maintaining a gap from the electrode and when this gap is closed electroporation takes place. Models of the event response times suggest the pores formed are small enough so we can carry out these measurements at nanotip electrodes and we have used this to quantify the vesicle content in living cells in a mode we call intracellular impact electrochemical cytometry. The development of electrochemical cytometry allows comparison between vesicle content and vesicular release and we have found that only part of the vesicle content is released in typical exocytotic cases measured by amperometry. This has led to the novel hypothesis that most exocytosis from dense core vesicles is via mechanism where vesicles fuse with the cell membrane, some content is released and then close again to be reloaded and reused. It leaves open the possibility that cells regulate release during individual events. This might be important in learning and memory and be a nonreceptor pharmaceutical target for brain-related disorders. Indeed, the concept of the chemo-brain observed in cisplatin-treated cancer patients appears to be at least in part the result of changing the fraction of transmitter released and we have been able to show this by using the combined amperometric measurement of release and electrochemical cytometry at model cells.

A central role for phosphatidic acid as a lipid mediator of regulated exocytosis in apicomplexa.

Lipids are commonly known for the structural roles they play, however, the specific contribution of different lipid classes to wide-ranging signalling pathways is progressively being unravelled. Signalling lipids and their associated effector proteins are emerging as significant contributors to a vast array of effector functions within cells, including essential processes such as membrane fusion and vesicle exocytosis. Many phospholipids have signalling capacity, however, this review will focus on phosphatidic acid (PA) and the enzymes implicated in its production from diacylglycerol (DAG) and phosphatidylcholine (PC): DGK and PLD respectively. PA is a negatively charged, cone-shaped lipid identified as a key mediator in specific membrane fusion and vesicle exocytosis events in a variety of mammalian cells, and has recently been implicated in specialised secretory organelle exocytosis in apicomplexan parasites. This review summarises the recent work implicating a role for PA regulation in exocytosis in various cell types. We will discuss how these signalling events are linked to pathogenesis in the phylum Apicomplexa.

Fusion Pore Size Limits 5-HT Release From Single Enterochromaffin Cell Vesicles.

Enterochromaffin cells are the major site of serotonin (5-HT) synthesis and secretion providing ∼95% of the body's total 5-HT. 5-HT can act as a neurotransmitter or hormone and has several important endocrine and paracrine roles. We have previously demonstrated that EC cells release small amounts of 5-HT per exocytosis event compared to other endocrine cells. We utilized a recently developed method to purify EC cells to demonstrate the mechanisms underlying 5-HT packaging and release. Using the fluorescent probe FFN511, we demonstrate that EC cells express VMAT and that VMAT plays a functional role in 5-HT loading into vesicles. Carbon fiber amperometry studies illustrate that the amount of 5-HT released per exocytosis event from EC cells is dependent on both VMAT and the H(+)-ATPase pump, as demonstrated with reserpine or bafilomycin, respectively. We also demonstrate that increasing the amount of 5-HT loaded into EC cell vesicles does not result in an increase in quantal release. As this indicates that fusion pore size may be a limiting factor involved, we compared pore diameter in EC and chromaffin cells by assessing the vesicle capture of different-sized fluorescent probes to measure the extent of fusion pore dilation. This identified that EC cells have a reduced fusion pore expansion that does not exceed 9 nm in diameter. These results demonstrate that the small amounts of 5-HT released per fusion event in EC cells can be explained by a smaller fusion pore that limits 5-HT release capacity from individual vesicles.

SUMOylation of Syntaxin1A regulates presynaptic endocytosis.

Neurotransmitter release from the presynaptic terminal is under very precise spatial and temporal control. Following neurotransmitter release, synaptic vesicles are recycled by endocytosis and refilled with neurotransmitter. During the exocytosis event leading to release, SNARE proteins provide most of the mechanical force for membrane fusion. Here, we show one of these proteins, Syntaxin1A, is SUMOylated near its C-terminal transmembrane domain in an activity-dependent manner. Preventing SUMOylation of Syntaxin1A reduces its interaction with other SNARE proteins and disrupts the balance of synaptic vesicle endo/exocytosis, resulting in an increase in endocytosis. These results indicate that SUMOylation regulates the emerging role of Syntaxin1A in vesicle endocytosis, which in turn, modulates neurotransmitter release and synaptic function.

Electrochemical Measurements of Optogenetically Stimulated Quantal Amine Release from Single Nerve Cell Varicosities in Drosophila Larvae.

The nerve terminals found in the body wall of Drosophila melanogaster larvae are readily accessible to experimental manipulation. We used the light-activated ion channel, channelrhodopsin-2, which is expressed by genetic manipulation in Type II varicosities to study octopamine release in Drosophila. We report the development of a method to measure neurotransmitter release from exocytosis events at individual varicosities in the Drosophila larval system by amperometry. A microelectrode was placed in a region of the muscle containing a varicosity and held at a potential sufficient to oxidize octopamine and the terminal stimulated by blue light. Optical stimulation of Type II boutons evokes exocytosis of octopamine, which is detected through oxidization at the electrode surface. We observe 22700±4200 molecules of octopamine released per vesicle. This system provides a genetically accessible platform to study the regulation of amine release at an intact synapse.

Electrochemical Measurements of Optogenetically Stimulated Quantal Amine Release from Single Nerve Cell Varicosities in Drosophila Larvae

AbstractThe nerve terminals found in the body wall of Drosophila melanogaster larvae are readily accessible to experimental manipulation. We used the light‐activated ion channel, channelrhodopsin‐2, which is expressed by genetic manipulation in Type II varicosities to study octopamine release in Drosophila. We report the development of a method to measure neurotransmitter release from exocytosis events at individual varicosities in the Drosophila larval system by amperometry. A microelectrode was placed in a region of the muscle containing a varicosity and held at a potential sufficient to oxidize octopamine and the terminal stimulated by blue light. Optical stimulation of Type II boutons evokes exocytosis of octopamine, which is detected through oxidization at the electrode surface. We observe 22700±4200 molecules of octopamine released per vesicle. This system provides a genetically accessible platform to study the regulation of amine release at an intact synapse.

Extrasynaptic release of GABA and dopamine by retinal dopaminergic neurons.

In the mouse retina, dopaminergic amacrine (DA) cells synthesize both dopamine and GABA. Both transmitters are released extrasynaptically and act on neighbouring and distant retinal neurons by volume transmission. In simultaneous recordings of dopamine and GABA release from isolated perikarya of DA cells, a proportion of the events of dopamine and GABA exocytosis were simultaneous, suggesting co-release. In addition, DA cells establish GABAergic synapses onto AII amacrine cells, the neurons that transfer rod bipolar signals to cone bipolars. GABAA but not dopamine receptors are clustered in the postsynaptic membrane. Therefore, dopamine, irrespective of its site of release-synaptic or extrasynaptic-exclusively acts by volume transmission. Dopamine is released upon illumination and sets the gain of retinal neurons for vision in bright light. The GABA released at DA cells' synapses probably prevents signals from the saturated rods from entering the cone pathway when the dark-adapted retina is exposed to bright illumination. The GABA released extrasynaptically by DA and other amacrine cells may set a 'GABAergic tone' in the inner plexiform layer and thus counteract the effects of a spillover of glutamate released at the bipolar cell synapses of adjacent OFF and ON strata, thus preserving segregation of signals between ON and OFF pathways.