Screening populations of individual cells for secretory heterogeneity

Many common metabolic and neurological disorders are related to defective regulation of exocytosis at the level of single cells. In exocytosis, vesicles containing the secretory product of a given cell type fuse with the plasma membrane allowing release of the vesicular contents into the extracellular environment where the physiological action can be exerted. The typical secretory vesicle contains between 0.15 and 10 attomoles of material that is released on a millisecond timescale. Hence, detection of this process presents several chemical and analytical challenges. In this work, we utilize the native ATP, stored at high concentrations within the secretory vesicles of most neuroendocrine cells and co-released during exocytosis and during cell lysis, as a universal tracer of cellular secretion events. Organisms studied include pancreatic islets, mast cells, and Escherischia coli. Cellular processes investigated include exocytotic release, stimulated cell lysis, and programmed cell lysis.     

Quantitative Nanoscale Secondary Ion Mass Spectrometry (NanoSIMS) Imaging of Individual Vesicles to Investigate the Relation between Fraction of Chemical Release and Vesicle Size

We used correlative transmission electron microscopy (TEM) and nanoscale secondary ion mass spectrometry (NanoSIMS) imaging to quantify the contents of subvesicular compartments, and to measure the partial release fraction of ¹³C-dopamine in cellular nanovesicles as a function of size. Three modes of exocytosis comprise full release, kiss-and-run, and partial release. The latter has been subject to scientific debate, despite a growing amount of supporting literature. We tailored culturing procedures to alter vesicle size and definitively show no size correlation with the fraction of partial release. In NanoSIMS images, vesicle content was indicated by the presence of isotopic dopamine, while vesicles which underwent partial release were identified by the presence of an ¹²⁷I-labelled drug, to which they were exposed during exocytosis allowing entry into the open vesicle prior to its closing again. Demonstration of similar partial release fractions indicates that this mode of exocytosis is predominant across a wide range of vesicle sizes.     

Zinc Regulates Chemical‐Transmitter Storage in Nanometer Vesicles and Exocytosis Dynamics as Measured by Amperometry

We applied electrochemical techniques with nano‐tip electrodes to show that micromolar concentrations of zinc not only trigger changes in the dynamics of exocytosis, but also vesicle content in a model cell line. The vesicle catecholamine content in PC12 cells is significantly decreased after 100 μm zinc treatment, but, catecholamine release during exocytosis remains nearly the same. This contrasts with the number of molecules stored in the exocytosis vesicles, which decreases, and we find that the amount of catecholamine released from zinc‐treated cells reaches nearly 100 % content expelled. Further investigation shows that zinc slows down exocytotic release. Our results provide the missing link between zinc and the regulation of neurotransmitter release processes, which might be important in memory formation and storage.     

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.     

Combined Amperometry and Electrochemical Cytometry Reveal Differential Effects of Cocaine and Methylphenidate on Exocytosis and the Fraction of Chemical Release

Amperometry with nanotip electrodes has been applied to show cocaine and methylphenidate not only trigger declines in vesicle content and exocytotic catecholamine release in a model cell line but also differentially change the fraction of transmitter released from each individual vesicle. In addition, cocaine accelerates exocytotic release dynamics while they remain unchanged after methylphenidate treatment. The parameters from pre‐spike feet for the two drugs are also in opposition, suggesting this aspect of release is affected differentially. As cocaine and methylphenidate are psychostimulants with similar pharmacologic action but have opposite effects on cognition, these results might provide a missing link between the regulation of exocytosis and vesicles and the effect of this regulation on cognition, learning, and memory. A speculative chemical mechanism of the effect of these drugs on vesicle content and exocytosis is presented.     

Cholesterol effects on vesicle pools in chromaffin cells revealed by carbon-fiber microelectrode amperometry

Cell–cell communication is often achieved via granular exocytosis, as in neurons during synaptic transmission or neuroendocrine cells during blood hormone control. Owing to its critical role in membrane properties and SNARE function, cholesterol is expected to play an important role in the highly conserved process of exocytosis. In this work, membrane cholesterol concentration is systematically varied in primary culture mouse chromaffin cells, and the change in secretion behavior of distinct vesicle pools as well as pool recovery following stimulation is measured using carbon-fiber microelectrode amperometry. Amperometric traces obtained from activation of the younger readily releasable and slowly releasable pool (RRP/SRP) vesicles at depleted cholesterol levels showed fewer sustained fusion pore features (6.1 ± 1.1% of spikes compared with 11.2 ± 1.0% for control), revealing that cholesterol content influences fusion pore formation and stability during exocytosis. Moreover, subsequent stimulation of RRP/SRP vesicles showed that cellular cholesterol level influences both the quantal recovery and kinetics of the later release events. Finally, diverging effects of cholesterol on RRP and the older reserve pool vesicle release suggest two different mechanisms for the release of these two vesicular pools.     

Intracellular injection of phospholipids directly alters exocytosis and the fraction of chemical release in chromaffin cells as measured by nano-electrochemistry

Using a nano-injection method, we introduced phospholipids having different intrinsic geometries into single secretory cells and used single cell amperometry (SCA) and intracellular vesicle impact electrochemical cytometry (IVIEC) with nanotip electrodes to monitor the effects of intracellular incubation on the exocytosis process and vesicular storage. Combining tools, this work provides new information to understand the impact of intracellular membrane lipid engineering on exocytotic release, vesicular content and fraction of chemical release. We also assessed the effect of membrane lipid alteration on catecholamine storage of isolated vesicles by implementing another amperometric technique, vesicle impact electrochemical cytometry (VIEC), outside the cell. Exocytosis analysis reveals that the intracellular nano-injection of phosphatidylcholine and lysophosphatidylcholine decreases the number of released catecholamines, whereas phosphatidylethanolamine shows the opposite effect. These observations support the emerging hypothesis that lipid curvature results in membrane remodeling through secretory pathways, and also provide new evidence for a critical role of the lipid localization in modulating the release process. Interestingly, the IVIEC data imply that total vesicular content is also affected by in situ supplementation of the cells with some lipids, while, the corresponding VIEC results show that the neurotransmitter content in isolated vesicles is not affected by altering the vesicle membrane lipids. This suggests that the intervention of phospholipids inside the cell has its effect on the cellular machinery for vesicle release rather than vesicle structure, and leads to the somewhat surprising conclusion that modulating release has a direct effect on vesicle structure, which is likely due to the vesicles opening and closing again during exocytosis. These findings could lead to a novel regulatory mechanism for the exocytotic or synaptic strength based on lipid heterogeneity across the cell membrane.

Amperometry and intracellular vesicle impact electrochemical cytometry with nanotip electrodes were used to monitor the effects on exocytosis and vesicular storage after nano-injection of phospholipids with different geometries into secretory cells.     

Cisplatin Inhibits Neurotransmitter Release during Exocytosis from Single Chromaffin Cells Monitored with Single Cell Amperometry

Chemotherapy with cisplatin induces side effects such as memory loss, confusion of thinking, and difficulties with multi-tasking. However, the mechanism of cisplatin inducing nervous dysfunction is still unknown. Herein, we examine whether and how cisplatin regulates the release of neurotransmitter during exocytosis in single chromaffin cells using single cell amperometry. The results show that cisplatin reduces the amount of transmitter released during exocytosis by reducing the duration of the exocytotic events, including the opening and closing time of the fusion pore. Furthermore, the stability of the initial fusion pore formed during exocytosis is also reduced by cisplatin. Our study holds the promise for understanding the side effects of cisplatin on the nervous system at single cell level.     

Fully automated microchip system for the detection of quantal exocytosis from single and small ensembles of cells

A lab-on-a-chip device that enables positioning of single or small ensembles of cells on an aperture in close proximity to a mercaptopropionic acid (MPA) modified sensing electrode has been developed and characterized. The microchip was used for the detection of Ca(2+)-dependent quantal catecholamine exocytosis from single as well as small assemblies of rat pheochromocytoma (PC12) cells. The frequency of events increased considerably upon depolarization of the PC12 cell membrane using a high extracelluar concentration of potassium. The number of recorded events could be correlated with the number of cells immobilized on the electrode. Quantal characteristics, such as the number of released molecules per recorded event, are equivalent to data obtained using conventional carbon fiber microelectrodes. The detection sensitivity of the device allows for the detection of less than 10 000 dopamine molecules in a quantal release. The distribution of peak rise-time and full width at half maximum was constant during measurement periods of several minutes demonstrating the stability of the MPA modified surface.     

Using Single‐Cell Amperometry To Reveal How Cisplatin Treatment Modulates the Release of Catecholamine Transmitters during Exocytosis

The pretreatment of cultured pheochromocytoma (PC12) cells with cis‐diamminedichloroplatinum (cisplatin), an anti‐cancer drug, influences the exocytotic ability of the cells in a dose‐dependent manner. Low concentrations of cisplatin stimulate catecholamine release whereas high concentrations inhibit it. Single‐cell amperometry reflects that 2 μm cisplatin treatment increases the frequency of exocytotic events and reduces their duration, whereas 100 μm cisplatin treatment decreases the frequency of exocytotic events and increases their duration. Furthermore, the stability of the initial fusion pore that is formed in the lipid membrane during exocytosis is also regulated differentially by different cisplatin concentrations. This study thus suggests that cisplatin influences exocytosis by multiple mechanisms.     

Analytical tools to monitor exocytosis: a focus on new fluorescent probes and methods

A great deal of research has been focused on unraveling the processes governing the exocytotic pathway and the extent of release during the process. Arguments abound for and against both the occurrence and significance of full release during exocytosis and partial release including kiss-and-run events. Several optical methods to directly observe the exocytosis process have been developed and here we focus on fluorescence methods and probes for this work. Although fluorescence imaging has been used for cell experiments for decades, in the last two decades a plethora of new approaches have arrived on the scene. These include application of new microscopy techniques, like total internal reflectance and stimulated emission depletion that are offering new ways to circumvent the limits of far field microscopy with a diffraction limit of 200 nm, and allow tracking of single synaptic vesicles. For selective imaging of synaptic vesicles the introduction of methods to stain the vesicular compartment has involved developing probes of the vesicular membrane and intravesicular solution, nanoparticle quantum dots that can be observed during exocytosis but not via the fusion pore, and fluorescent false neurotransmitters.     

Tunable Molecular Logic Gates Designed for Imaging Released Neurotransmitters

Tunable dual‐analyte fluorescent molecular logic gates (ExoSensors) were designed for the purpose of imaging select vesicular primary‐amine neurotransmitters that are released from secretory vesicles upon exocytosis. ExoSensors are based on the coumarin‐3‐aldehyde scaffold and rely on both neurotransmitter binding and the change in environmental pH associated with exocytosis to afford a unique turn‐on fluorescence output. A pH‐functionality was directly integrated into the fluorophore π‐system of the scaffold, thereby allowing for an enhanced fluorescence output upon the release of labeled neurotransmitters. By altering the pH‐sensitive unit with various electron‐donating and ‐withdrawing sulfonamide substituents, we identified a correlation between the pK_a of the pH‐sensitive group and the fluorescence output from the activated fluorophore. In doing so, we achieved a twelvefold fluorescence enhancement upon evaluating the ExoSensors under conditions that mimic exocytosis. ExoSensors are aptly suited to serve as molecular imaging tools that allow for the direct visualization of only the neurotransmitters that are released from secretory vesicles upon exocytosis.     

Quantitative Measurement of Transmitters in Individual Vesicles in the Cytoplasm of Single Cells with Nanotip Electrodes

The quantification of vesicular transmitter content is important for studying the mechanisms of neurotransmission and malfunction in disease, and yet it is incredibly difficult to measure the tiny amounts of neurotransmitters in the attoliter volume of a single vesicle, especially in the cell environment. We introduce a novel method, intracellular vesicle electrochemical cytometry. A nanotip conical carbon‐fiber microelectrode was used to electrochemically measure the total content of electroactive neurotransmitters in individual nanoscale vesicles in single PC12 cells as these vesicles lysed on the electrode inside the living cell. The results demonstrate that only a fraction of the quantal neurotransmitter content is released during exocytosis. These data support the intriguing hypothesis that the vesicle does not open all the way during the normal exocytosis process, thus resulting in incomplete expulsion of the vesicular contents.     

Examining changes in cellular communication in neuroendocrine cells after noble metal nanoparticle exposure

As nanoparticles enjoy increasingly widespread use in commercial applications, the potential for unintentional exposure has become much more likely during any given day. Researchers in the field of nanotoxicity are working to determine the physicochemical nanoparticle properties that lead to toxicity in an effort to establish safe design rules. This work explores the effects of noble metal nanoparticle exposure in murine chromaffin cells, focusing on examining the effects of size and surface functionality (coating) in silver and gold, respectively. Carbon-fibre microelectrode amperometry was utilized to examine the effect of exposure on exocytosis function, at the single cell level, and provided new insights into the compromised functions of cells. Silver nanoparticles of varied size, between 15 and 60 nm diameter, were exposed to cells and found to alter the release kinetics of exocytosis for those cells exposed to the smallest examined size. Effects of gold were examined after modification with two commonly used 'bio-friendly' polymers, either heparin or poly (ethylene glycol), and gold nanoparticles were found to induce altered cellular adhesion or the number of chemical messenger molecules released, respectively. These results support the body of work suggesting that noble metal nanoparticles perturb exocytosis, typically altering the number of molecules and kinetics of release, and supports a direct disruption of the vesicle matrix by the nanoparticle. Overall, it is clear that various nanoparticle physicochemical properties, including size and surface coating, do modulate changes in cellular communication via exocytosis.     

Highlights of 20?years of electrochemical measurements of exocytosis at cells and artificial cells

Advances in electrochemical methodology over the past 30?years have allowed chemical measurements to be made with decreasing amounts of analyte and at smaller spatial dimensions. This has allowed the investigation of single cells and single vesicles in cells either during release of chemical transmitter or separately. The cellular event called exocytosis can be measured with amperometry or cyclic voltammetry as discovered by Wightman and first published in 1990. In addition, the measurement of vesicle contents with electrochemistry is a new approach we have termed electrochemical cytometry. This involves isolation of intact vesicles, separation of the vesicles, and then lysing followed by coulometric analysis of the electroactive vesicle content. In this review, we will highlight work done by us and by others to discuss measurements of exocytosis at single cells and measurements at artificial cell models for studying the biophysical properties of vesicle membrane dynamics and lipid nanotubes connecting artificial cells using electrochemical methods.     

Real-time detection of<Emphasis Type="SmallCaps"> L</Emphasis>-glutamate released from C6 glioma cells using a modified enzyme-luminescence method

There is an increasing interest in new strategies to detect neurotransmitters released from nerve cells in real time for brain science, drug assessment, and so on. Previously we reported real-time monitoring of dopamine release from nerve model cells by enzyme-catalyzed luminescence measurement with tyramine oxidase and peroxidase. In the present study, the system was modified with glutamate oxidase instead of tyramine oxidase to detect L-glutamate sensitively (≈ 10 nM) and rapidly with high temporal resolution (<1 s). We applied this modified method successfully to perform real-time monitoring of L-glutamate release from brain model cell (C6 glioma cell) using a luminescence plate reader upon stimulation with high concentration of KCl (>10 mM) or 5-hydroxytryptamine (>1 μM). The measurement solution was not toxic and therefore the L-glutamate release from the cell was measured by the second stimulation after exchanging the measurement solution. We conclude that the developed monitoring system is suitable for real-time detection of dynamic L-glutamate release from nerve cells in vitro and will be suitable for application in assessment of drugs acting on the nervous system. Figure Enzyme luminescence detection of L-glutamate released from cells     

Simultaneous detection of vesicular content and exocytotic release with two electrodes in and at a single cell

We developed a technique employing two electrodes to simultaneously and dynamically monitor vesicular neurotransmitter storage and vesicular transmitter release in and at the same cell. To do this, two electrochemical techniques, single-cell amperometry (SCA) and intracellular vesicle impact electrochemical cytometry (IVIEC), were applied using two nanotip electrodes. With one electrode being placed on top of a cell measuring exocytotic release and the other electrode being inserted into the cytoplasm measuring vesicular transmitter storage, upon chemical stimulation, exocytosis is triggered and the amount of release and storage can be quantified simultaneously and compared. By using this technique, we made direct comparison between exocytotic release and vesicular storage, and investigated the dynamic changes of vesicular transmitter content before, during, and after chemical stimulation of PC12 cells, a neuroendocrine cell line. While confirming that exocytosis is partial, we suggest that chemical stimulation either induces a replenishment of the releasable pool with a subpool of vesicles having higher amount of transmitter storage, or triggers the vesicles within the same subpool to load more transiently at approximately 10–20 s. Thus, a time scale for vesicle reloading is determined. The effect of l-3,4-dihydroxyphenylalanine (l-DOPA), the precursor to dopamine, on the dynamic alteration of vesicular storage upon chemical stimulation for exocytosis was also studied. We found that l-DOPA incubation reduces the observed changes of vesicular storage in regular PC12 cells, which might be due to an increased capacity of vesicular transmitter loading caused by l-DOPA. Our data provide another mechanism for plasticity after stimulation via quantitative and dynamic changes in the exocytotic machinery.

Simultaneous measurements of IVIEC and SCA by two nanotip electrodes allows direct and dynamic comparison between vesicular transmitter content and vesicular transmitter release to shed light on stimulation-induced plasticity.     

Modeling Ca-Polyanion Crosslinking in Secretory Networks. Assessment of Charge Density and Bond Affinity in Polyanionic Secretory Networks

Materials released by secretory cells are stored inside intracellular membrane-bound vesicles. These moieties are not freely diffusible in the vesicle but remain immobilized in a Ca²⁺-crosslinked condensed-phase polyanionic polymer matrix. During exocytosis a Na⁺/Ca²⁺ ion exchange process triggers a volume phase transition resulting in massive swelling and release of the materials to the extracellular space. Here we formulate a simple model to assess Ca²⁺-ion binding from the swelling kinetics of polymer networks. We found the diffusivity of the networks (D) exhibits a power-law dependency on the Ca²⁺ concentration where D ∝ [Ca²⁺]^−2/3. The model yields an estimate of charge density and ionic affinity of the polymer chains. Studies of post-exocytic swelling kinetics in airway mucin granules, mast cell granules and granules from the microalga (Phaeocystis globosa) were used to validate predictions from our model. These results suggest that independent of the cell type, from animal to plant cells, a single polyelectrolyte interaction mechanism appear to be responsible for product release in exocytosis.     

Vesicular exocytosis under hypotonic conditions shows two distinct populations of dense core vesicles in bovine chromaffin cells.

Several previous reports have discussed the effects of external osmolarity on vesicular exocytotic processes. However, few of these studies considered hypotonic conditions on chromaffin cells. Herein, the exocytosis of catecholamines by chromaffin cells was investigated in a medium of low osmolarity (200 mOsm) by amperometry at carbon fiber microelectrodes. It is observed that the frequency of the exocytotic events is significantly higher under hypotonic conditions than under physiological conditions (315 mOsm). This further confirms that the swelling of the polyelectrolytic matrix (which follows ionic exchanges) contained in dense core vesicles is the energetic driving force of the exocytotic phenomenon, being favored by a lower osmolarity. The mean amount of catecholamines released during secretory events also increases importantly under the hypotonic condition. This may be rationalized by the coexistence of two distinct populations of dense core vesicles with a relative content ratio of 4.7. The larger content population is favored under hypotonic conditions but plays only a side role under isotonic conditions.     

Monitoring of vesicular exocytosis from single cells using micrometer and nanometer-sized electrochemical sensors

Communication between cells by release of specific chemical messengers via exocytosis plays crucial roles in biological process. Electrochemical detection based on ultramicroelectrodes (UMEs) has become one of the most powerful techniques in real-time monitoring of an extremely small number of released molecules during very short time scales, owing to its intrinsic advantages such as fast response, excellent sensitivity, and high spatiotemporal resolution. Great successes have been achieved in the use of UME methods to obtain quantitative and kinetic information about released chemical messengers and to reveal the molecular mechanism in vesicular exocytosis. In this paper, we review recent developments in monitoring exocytosis by use of UMEs-electrochemical-based techniques including electrochemical detection using micrometer and nanometer-sized sensors, scanning electrochemical microscopy (SECM), and UMEs implemented in lab-on-a-chip (LOC) microsystems. These advances are of great significance in obtaining a better understanding of vesicular exocytosis and chemical communications between cells, and will facilitate developments in many fields, including analytical chemistry, biological science, and medicine. Furthermore, future developments in electrochemical probing of exocytosis are also proposed. Figure In this paper, we review recent developments in monitoring the exocytosis by use of UMEs-electrochemical-based techniques including electrochemical detection using micrometer and nanometer-sized sensors, Scanning Electrochemical Microscopy (SECM) and UMEs implemented in lab-on-a-chip (LOC) microsystems. These advances are of great significance in obtaining a better understanding of vesicular exocytosis and chemical communications between cells, and will facilitate developments in many fields including analytical chemistry, biological science and medicine. Furthermore, future developments in electrochemical probing of exocytosis are proposed.