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1.	Taleat, Zahra, 1982, et al. (författare) Electrochemical Investigation of the Interaction between Catecholamines and ATP 2018 Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 90:3, s. 1601-1607 Tidskriftsartikel (refereegranskat)abstract 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.
2.	Dunevall, Johan, 1984, et al. (författare) Characterizing the Catecholamine Content of Single Mammalian Vesicles by Collision-Adsorption Events at an Electrode 2015 Ingår i: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 0002-7863 .- 1520-5126. ; 137:13, s. 4344-4346 Tidskriftsartikel (refereegranskat)abstract We present the electrochemical response to single adrenal chromaffin vesicles filled with catecholamine hormones as they are adsorbed and rupture on a 33 mu m diameter disk-shaped carbon electrode. The vesicles adsorb onto the electrode surface and sequentially spread out over the electrode surface, trapping their contents against the electrode. These contents are then oxidized, and a current (or amperometric) peak results from each vesicle that bursts. A large number of current transients associated with rupture of single vesicles (86%) are observed under the experimental conditions used, allowing us to quantify the vesicular catecholamine content.
3.	Dunevall, Johan, 1984 (författare) New Approaches for Chemical Analysis of Single Cells and Vesicles 2018 Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract Exocytosis is the major cell-to-cell communication process in the nervous system, involving the conversion of an electrical signal (e.g. action potential) to a chemical one. Signaling molecules like neurotransmitters, hormones and/or peptides are stored in vesicles inside the cell. During exocytosis, calcium triggers the release of the vesicular cargo through SNARE-complex mediated fusion of the vesicles with the plasma (outer-membrane) of the cell. Consequently, a transient pore is formed through which the vesicular cargo is released into the extracellular space and is there able to interact with receptors of target cells. Most often, the pore closes again only allowing a fraction of the cargo to be released, so called partial release or kiss-and-run exocytosis. The extent of partial release is modulated by the intracellular calcium concentration, which can be regulated by the strength of the stimuli or with pharmaceuticals. Despite the importance of this process and the efforts that have been done to resolve the fundamental regulatory mechanisms of exocytosis, much remains unknown. In order to gain understanding of how the amount of vesicular cargo released is regulated, information about the total vesicular cargo (quantal content) has to be obtained. Until recently, no method aiming for quantification of the full quantal content existed. Our group has successfully developed an electrochemical method called vesicle impact electrochemical cytometry (VIEC) that allows direct quantification of the vesicular content in secretory granules as demonstrated with large dense-core granules from chromaffin and pheochromocytoma (PC12) cells. Chromaffin cells of the adrenal medulla are the body´s stress response output, and the best studied model system for exocytosis. The large-dense core vesicles of chromaffin cells contain a very important group of neurotransmitters and hormones, namely the catecholamines (e.g. dopamine, norepinephrine and epinephrine). The catecholamines are electroactive, and can readily be oxidized at the surface of a polarized electrode to give away two electrons per molecule. By counting the number of electrons passed through the system (charge) and knowing the charge of one mole of electrons (Faraday´s constant) the number of molecules can be quantified. In the intracellular or IVIEC method a conical nanotip carbon electrode is used to pierce into the cytosol of a living cell, allowing the vesicles to adsorb onto its surface. The vesicles burst open due to the electric field at the polarized electrode and the vesicular cargo is released towards the electrode surface and is oxidized, which allows the full content to be detected. VIEC is an electrochemical method that potentially can be applied to study the quantal content of the electroactive vesicular content of other cell types like mast cells and blood platelets, that contain both histamine and serotonin.
4.	Dunevall, Johan, 1984, et al. (författare) Vesicle impact electrochemical cytometry compared to amperometric exocytosis measurements 2017 Ingår i: Current Opinion in Electrochemistry. - : Elsevier BV. - 2451-9103 .- 2451-9111. ; 5:1, s. 85-91 Tidskriftsartikel (refereegranskat)abstract Three new tools are discussed for understanding chemical communication between cells and primarily to delve into the content and structure of nanometer transmitter vesicles. These are amperometric measurements of exocytosis, vesicle impact electrochemical cytometry, and intracellular vesicle impact electrochemical cytometry. These are combining in the end nanoscale mass spectrometry imaging to begin determination of vesicle structure. These methods have provided solid evidence for the concept of open and closed exocytosis leading to partial release of the vesicle content during normal exocytosis. They have also been used to discover cases where the fraction of transmitter released is not changed, and other cases where the vesicle transmitter fraction released is altered, as with zinc, thought to alter cognition. Overall, the combination of these methods is showing us details of vesicular processes that would not be measureable without these micro and nano electrochemical methods.
5.	Kaya, Ibrahim, et al. (författare) Spatial Lipidomics Reveals Region and Long Chain Base Specific Accumulations of Monosialogangliosides in Amyloid Plaques in Familial Alzheimer's Disease Mice (5xFAD) Brain 2020 Ingår i: ACS Chemical Neuroscience. - : American Chemical Society (ACS). - 1948-7193. ; 11:1, s. 14-24 Tidskriftsartikel (refereegranskat)abstract Ganglioside metabolism is significantly altered in Alzheimer's disease (AD), which is a progressive neuro-degenerative disease prominently characterized by one of its pathological hallmarks, amyloid deposits or "senile plaques". While the plaques mainly consist of aggregated variants of amyloid-beta protein (A beta), recent studies have revealed a number of lipid species including gangliosides in amyloid plaques along with A beta peptides. It has been widely suggested that long chain (sphingosine) base (LCBs), C18:1-LCB and C20:1-LCB, containing gangliosides might play different roles in neuronal function in vivo. In order to elucidate region-specific aspects of amyloid-plaque associated C18:1-LCB and C20:1-LCB ganglioside accumulations, high spatial resolution (10 mu m per pixel) matrix assisted laser desorption ionization imaging mass spectrometry (MALDI-IMS) of gangliosides in amyloid plaques was performed in hippocampal and adjacent cortical regions of 12 month old 5xFAD mouse coronal brain sections from two different stereotaxic coordinates (bregma points, -2.2 and -2.7 mm). MALDI-IMS uncovered brain-region (2 and 3D) and/or LCB specific accumulations of monosialogangliosides (GMs): GM1, GM2, and GM3 in the hippocampal and cortical amyloid plaques. The results reveal monosialogangliosides to be an important component of amyloid plaques and the accumulation of different gangliosides is region and LCB specific in 12 month old 5xFAD mouse brain. This is discussed in relation to amyloid-associated AD pathogenesis such as lipid related immune changes in amyloid plaques, AD specific ganglioside metabolism, and, notably, AD-associated impaired neurogenesis in the subgranular zone.
6.	Li, Xianchan, 1982, et al. (författare) Mechanistic Aspects of Vesicle Opening during Analysis with Vesicle Impact Electrochemical Cytometry 2017 Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 89:17, s. 9416-9423 Tidskriftsartikel (refereegranskat)abstract Vesicle impact electrochemical cytometry (VIEC) has been used to quantify the vesicular transmitter content in mammalian vesicles. In the present study, we studied the mechanism of VIEC by quantifying the catecholamine content in single vesicles isolated from pheochromocytoma (PC12) cells. These vesicles contain about one tenth of the catecholamine compared with adrenal chromaffin vesicles. The existence of a prespike foot for many events suggests the formation of an initial transiently stable pore at the beginning of vesicle rupture. Increasing the detection temperature from 6 to 30 degrees C increases the possibility of vesicle rupture on the electrode, implying that there is a temperature-dependent process that facilitates electroporation. Natively larger vesicles are shown to rupture earlier and more frequently than smaller ones in VIEC. Likewise, manipulating vesicle content and size with drugs leads to similar trends. These data support the hypothesis that electroporation is the primary force for pore opening in VIEC. We further hypothesize that a critical step for initiating vesicle opening by electroporation is diffusion of membrane proteins away from the membrane region of contact with the electrode to allow closer contact, increasing the lateral potential field and thus facilitating electroporation.
7.	Li, Xianchan, 1982, et al. (författare) Nanopore Opening at Flat and Nanotip Conical Electrodes during Vesicle Impact Electrochemical Cytometry 2018 Ingår i: ACS Nano. - : American Chemical Society (ACS). - 1936-0851 .- 1936-086X. ; 12:3, s. 3010-3019 Tidskriftsartikel (refereegranskat)abstract The oxidation of catecholamine at a microelectrode, following its release from individual vesicles, allows interrogation of the content of single nanometer vesicles with vesicle impact electrochemical cytometry (VIEC). Previous to this development, there were no methods available to quantify the chemical load of single vesicles. However, accurate quantification of the content is hampered by uncertainty in the proportion of substituent molecules reaching the electrode surface (collection efficiency). In this work, we use quantitative modeling to calculate this collection efficiency. For all vesicles except those at the very edge of the electrode, modeling shows that ∼100% oxidation efficiency is achieved when employing a 33 μm diameter disk microelectrode for VIEC, independent of the location of the vesicle release pore. We use this to experimentally determine a precise distribution of catecholamine in individual vesicles extracted from PC12 cells. In contrast, we calculate that when a nanotip conical electrode (∼4 μm length, ∼1.5 μm diameter at the base) is employed, as in intracellular VIEC (IVIEC), the current-time response depends strongly on the position of the catecholamine-releasing pore in the vesicle membrane. When vesicle release occurs with the pore opening occurring far from the electrode, lower currents and partial oxidation (∼75%) of the catecholamine are predicted, as compared to higher currents and ∼100% oxidation, when the pore is close to/at the electrode surface. As close agreement is observed between the experimentally measured vesicular content in intracellular and extracted vesicles from the same cell line using nanotip and disk electrodes, respectively, we conclude that pores open at the electrode surface. Not only does this suggest that electroporation of the vesicle membrane is the primary driving force for catecholamine release from vesicles at polarized electrodes, but it also indicates that IVIEC with nanotip electrodes can directly assess vesicular content without correction. © 2018 American Chemical Society.
8.	Li, Xianchan, 1982, et al. (författare) Quantitative Chemical Measurements of Vesicular Transmitters with Electrochemical Cytometry 2016 Ingår i: Accounts of Chemical Research. - : American Chemical Society (ACS). - 0001-4842 .- 1520-4898. ; 49:10, s. 2347-2354 Tidskriftsartikel (refereegranskat)abstract 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.
9.	Li, Xianchan, 1982, et al. (författare) Quantitative Measurement of Transmitters in Individual Vesicles in the Cytoplasm of Single Cells with Nanotip Electrodes 2015 Ingår i: Angewandte Chemie International Edition. - : Wiley. - 1433-7851 .- 1521-3773. ; 54:41, s. 11978-11982 Tidskriftsartikel (refereegranskat)abstract 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.
10.	Li, Xianchan, 1982, et al. (författare) Using Single-Cell Amperometry To Reveal How Cisplatin Treatment Modulates the Release of Catecholamine Transmitters during Exocytosis 2016 Ingår i: Angewandte Chemie-International Edition. - : Wiley. - 1433-7851 .- 1521-3773. ; 55:31, s. 9041-9044 Tidskriftsartikel (refereegranskat)abstract 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 mm cisplatin treatment increases the frequency of exocytotic events and reduces their duration, whereas 100 mm 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.
11.	Lovric, Jelena, 1980, et al. (författare) Nano Secondary Ion Mass Spectrometry Imaging of Dopamine Distribution Across Nanometer Vesicles 2017 Ingår i: ACS Nano. - : American Chemical Society (ACS). - 1936-086X .- 1936-0851. ; 11:4, s. 3446-3455 Tidskriftsartikel (refereegranskat)abstract We report an approach to spatially resolve the content across nanometer neuroendocrine vesicles in nerve-like cells by correlating super high-resolution mass spectrometry imaging, NanoSIMS, with transmission electron microscopy (TEM). Furthermore, intracellular electrochemical cytometry at nanotip electrodes is used to count the number of molecules in individual vesicles to compare to imaged amounts in vesicles. Correlation between the NanoSIMS and TEM provides nanometer resolution of the inner structure of these organelles. Moreover, correlation with electrochemical methods provides a means to quantify and relate vesicle neurotransmitter content and release, which is used to explain the slow transfer of dopamine between vesicular compartments. These nanoanalytical tools reveal that dopamine loading/unloading between vesicular compartments, dense core and halo solution, is a kinetically limited process. The combination of NanoSIMS and TEM has been used to show the distribution profile of newly synthesized dopamine across individual vesicles. Our findings suggest that the vesicle inner morphology might regulate the neurotransmitter release event during open and closed exocytosis from dense core vesicles with hours of equilibrium needed to move significant amounts of catecholamine from the protein dense core despite its nanometer size.
12.	Lovric, Jelena, 1980, et al. (författare) On the mechanism of electrochemical vesicle cytometry: chromaffin cell vesicles and liposomes 2016 Ingår i: Faraday Discussions. - : Royal Society of Chemistry (RSC). - 1359-6640 .- 1364-5498. ; 193, s. 65-79 Tidskriftsartikel (refereegranskat)abstract The mechanism of mammalian vesicle rupture onto the surface of a polarized carbon fiber microelectrode during electrochemical vesicle cytometry is investigated. It appears that following adsorption to the surface of the polarized electrode, electroporation leads to the formation of a pore at the interface between a vesicle and the electrode and this is shown to be potential dependent. The chemical cargo is then released through this pore to be oxidized at the electrode surface. This makes it possible to quantify the contents as it restricts diffusion away from the electrode and coulometric oxidation takes place. Using a bottom up approach, lipid-only transmitter-loaded liposomes were used to mimic native vesicles and the rupture events occurred much faster in comparison with native vesicles. Liposomes with added peptide in the membrane result in rupture events with a lower duration than that of liposomes and faster in comparison to native vesicles. Diffusional models have been developed and suggest that the trend in pore size is dependent on soft nanoparticle size and diffusion of the content in the nanometer vesicle. In addition, it appears that proteins form a barrier for the membrane to reach the electrode and need to move out of the way to allow close contact and electroporation. The protein dense core in vesicles matrixes is also important in the dynamics of the events in that it significantly slows diffusion through the vesicle.
13.	Majdi, Soodabeh, 1980, et al. (författare) DMSO Chemically Alters Cell Membranes to Slow Exocytosis and Increase the Fraction of Partial Transmitter Released 2017 Ingår i: Chembiochem. - : Wiley. - 1439-4227 .- 1439-7633. ; 18:19, s. 1898-1902 Tidskriftsartikel (refereegranskat)abstract Dimethyl sulfoxide (DMSO) is frequently used as a solvent in biological studies and as a vehicle for drug therapy; but the side effects of DMSO, especially on the cell environment, are not well understood, and controls with DMSO are not neutral at higher concentrations. Herein, electrochemical measurement techniques are applied to show that DMSO increases exocytotic neurotransmitter release, while leaving vesicular contents unchanged. In addition, the kinetics of release from DMSO-treated cells are faster than that of untreated ones. The results suggest that DMSO has a significant influence on the chemistry of the cell membrane, leading to alteration of exocytosis. A speculative chemical mechanism of the effect on the fusion pore during exocytosis is presented.
14.	Majdi, Soodabeh, 1980, et al. (författare) Electrochemical Measurements of Optogenetically Stimulated Quantal Amine Release from Single Nerve Cell Varicosities in Drosophila Larvae 2015 Ingår i: Angewandte Chemie International Edition. - : Wiley. - 1433-7851 .- 1521-3773. ; 54:46, s. 13609-13612 Tidskriftsartikel (refereegranskat)abstract 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 TypeII 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 TypeII 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.
15.	Mashadi Fathali, Hoda, 1983, et al. (författare) Extracellular Osmotic Stress Reduces the Vesicle Size while Keeping a Constant Neurotransmitter Concentration 2017 Ingår i: Acs Chemical Neuroscience. - : American Chemical Society (ACS). - 1948-7193. ; 8:2, s. 368-375 Tidskriftsartikel (refereegranskat)abstract 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.
16.	Mashadi Fathali, Hoda, 1983, et al. (författare) Monitoring the Effect of Osmotic Stress on Secretory Vesicles and Exocytosis 2018 Ingår i: Journal of Visualized Experiments. - : MyJove Corporation. - 1940-087X. ; :132, s. 1-10 Tidskriftsartikel (refereegranskat)abstract 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.
17.	Mellander, Lisa J., et al. (författare) Two modes of exocytosis in an artificial cell 2014 Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 4 Tidskriftsartikel (refereegranskat)abstract The details of exocytosis, the vital cell process of neuronal communication, are still under debate with two generally accepted scenarios. The first mode of release involves secretory vesicles distending into the cell membrane to release the complete vesicle contents. The second involves partial release of the vesicle content through an intermittent fusion pore, or an opened or partially distended fusion pore. Here we show that both full and partial release can be mimicked with a single large-scale cell model for exocytosis composed of material from blebbing cell plasma membrane. The apparent switching mechanism for determining the mode of release is demonstrated to be related to membrane tension that can be differentially induced during artificial exocytosis. These results suggest that the partial distension mode might correspond to an extended kiss-and-run mechanism of release from secretory cells, which has been proposed as a major pathway of exocytosis in neurons and neuroendocrine cells.
18.	Najafinobar, Neda, 1985, et al. (författare) Cholesterol Alters the Dynamics of Release in Protein Independent Cell Models for Exocytosis 2016 Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 6:Article number: 33702 Tidskriftsartikel (refereegranskat)abstract Neurons communicate via an essential process called exocytosis. Cholesterol, an abundant lipid in both secretory vesicles and cell plasma membrane can affect this process. In this study, amperometric recordings of vesicular dopamine release from two different artificial cell models created from a giant unilamellar liposome and a bleb cell plasma membrane, show that with higher membrane cholesterol the kinetics for vesicular release are decelerated in a concentration dependent manner. This reduction in exocytotic speed was consistent for two observed modes of exocytosis, full and partial release. Partial release events, which only occurred in the bleb cell model due to the higher tension in the system, exhibited amperometric spikes with three distinct shapes. In addition to the classic transient, some spikes displayed a current ramp or plateau following the maximum peak current. These post spike features represent neurotransmitter release from a dilated pore before constriction and show that enhancing membrane rigidity via cholesterol adds resistance to a dilated pore to re-close. This implies that the cholesterol dependent biophysical properties of the membrane directly affect the exocytosis kinetics and that membrane tension along with membrane rigidity can influence the fusion pore dynamics and stabilization which is central to regulation of neurochemical release.
19.	Najafinobar, Neda, 1985, et al. (författare) Excited Fluorophores Enhance the Opening of Vesicles at Electrode Surfaces in Vesicle Electrochemical Cytometry 2016 Ingår i: Angewandte Chemie International Edition. - : Wiley. - 1433-7851 .- 1521-3773. ; 55:48, s. 15081-15085 Tidskriftsartikel (refereegranskat)abstract Electrochemical cytometry is a method developed recently to determine the content of an individual cell vesicle. The mechanism of vesicle rupture at the electrode surface involves the formation of a pore at the interface between a vesicle and the electrode through electroporation, which leads to the release and oxidation of the vesicle's chemical cargo. We have manipulated the membrane properties using excited fluorophores conjugated to lipids, which appears to make the membrane more susceptible to electroporation. We propose that by having excited fluorophores in close contact with the membrane, membrane lipids (and perhaps proteins) are oxidized upon production of reactive oxygen species, which then leads to changes in membrane properties and the formation of water defects. This is supported by experiments in which the fluorophores were placed on the lipid tail instead of the headgroup, which leads to a more rapid onset of vesicle opening. Additionally, application of DMSO to the vesicles, which increases the membrane area per lipid, and decreasing the membrane thickness result in the same enhancement in vesicle opening, which confirms the mechanism of vesicle opening with excited fluorophores in the membrane. Light-induced manipulation of membrane vesicle pore opening might be an attractive means of controlling cell activity and exocytosis. Additionally, our data confirm that in experiments in which cells or vesicle membranes are labeled for fluorescence monitoring, the properties of the excited membrane change substantially. © 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
20.	Najafinobar, Neda, 1985, et al. (författare) The Effect of Excited Fluorophore on Vesicle Fusion at the Surface of the Electrode 2015 Ingår i: Biophysical Journal. - 0006-3495 .- 1542-0086. ; 108:2, s. 239A-239A Konferensbidrag (övrigt vetenskapligt/konstnärligt)
21.	Stagkourakis, Stefanos, et al. (författare) Dopamine Release Dynamics in the Tuberoinfundibular Dopamine System 2019 Ingår i: Journal of Neuroscience. - 0270-6474 .- 1529-2401. ; 39:21, s. 4009-4022 Tidskriftsartikel (refereegranskat)abstract The relationship between neuronal impulse activity and neurotransmitter release remains elusive. This issue is especially poorly understood in the neuroendocrine system, with its particular demands on periodically voluminous release of neurohormones at the interface of axon terminals and vasculature. Ashortage of techniques with sufficient temporal resolution has hindered real-time monitoring of the secretion of the peptides that dominate among the neurohormones. The lactotropic axis provides an important exception in neurochemical identity, however, as pituitary prolactin secretion is primarily under monoaminergic control, via tuberoinfundibular dopamine (TIDA) neurons projecting to the median eminence (ME). Here, we combined electrical or optogenetic stimulation and fast-scan cyclic voltammetry to address dopamine release dynamics in the male mouse TIDA system. Imposing different discharge frequencies during brief (3 s) stimulation of TIDA terminals in the ME revealed that dopamine output is maximal at 10 Hz, which was found to parallel the TIDA neuron action potential frequency distribution during phasic discharge. Over more sustained stimulation periods (150 s), maximal output occurred at 5 Hz, similar to the average action potential firing frequency of tonically active TIDA neurons. Application of the dopamine transporter blocker, methylphenidate, significantly increased dopamine levels in the ME, supporting a functional role of the transporter at the neurons' terminals. Lastly, TIDA neuron stimulation at the cell body yielded perisomatic release of dopamine, which may contribute to an ultrafast negative feedback mechanism to constrain TIDA electrical activity. Together, these data shed light on how spiking patterns in the neuroendocrine system translate to vesicular release toward the pituitary and identify how dopamine dynamics are controlled in the TIDA system at different cellular compartments.
22.	Wahlgren, Weixiao Yuan, 1970, et al. (författare) Substrate-bound outward-open structure of a Na+-coupled sialic acid symporter reveals a new Na+ site. 2018 Ingår i: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 9:1 Tidskriftsartikel (refereegranskat)abstract Many pathogenic bacteria utilise sialic acids as an energy source or use them as an external coating to evade immune detection. As such, bacteria that colonise sialylated environments deploy specific transporters to mediate import of scavenged sialic acids. Here, we report a substrate-bound 1.95Å resolution structure and subsequent characterisation of SiaT, a sialic acid transporter from Proteus mirabilis. SiaT is a secondary active transporter of the sodium solute symporter (SSS) family, which use Na+ gradients to drive the uptake of extracellular substrates. SiaT adopts the LeuT-fold and is in an outward-open conformation in complex with the sialic acid N-acetylneuraminic acid and two Na+ ions. One Na+ binds to the conserved Na2 site, while the second Na+ binds to a new position, termed Na3, which is conserved in many SSS family members. Functional and molecular dynamics studies validate the substrate-binding site and demonstrate that both Na+ sites regulate N-acetylneuraminic acid transport.
23.	Wang, Jun, 1976, et al. (författare) Spatial resolution of exocytosis across a single cell by a microwell-based individually addressable thin film ultra-microelectrode array 2013 Ingår i: 17th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2013; Freiburg; Germany; 27 October 2013 through 31 October 2013. - 9781632666246 ; 1, s. 392-394 Konferensbidrag (refereegranskat)abstract We report the fabrication and characterization of micro well-based individually addressable ultra-microelectrode arrays (MEAs) and their application to spatially and temporally resolved detection of neurotransmitter release across a single pheochromocytoma (PC 12) cell. The MEAs consist of sixteen 4-μm-width square microelectrodes, or twenty-five 3-μm-width square microelectrodes, or thirty-six 2-jim-width square microelectrodes. Each MEA is tightly defined in a 30×30 μm square area, which is further encased inside a 40×40 μm SU-8 microwell. We demonstrate the excellent stability and reproducibility of these microelectrodes by using cyclic voltammetry and we have performed recording of spatially resolution of single cell exocytosis with multiple ultra-microelectrodes in 2-μm resolution.
24.	Wang, Jun, 1976, et al. (författare) Spatial Resolution of Single-Cell Exocytosis by Microwell-Based Individually Addressable Thin Film Ultramicroelectrode Arrays 2014 Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 86:9, s. 4515-4520 Tidskriftsartikel (refereegranskat)abstract We report the fabrication and characterization of microwell-based individually addressable microelectrode arrays (MEAs) and their application to spatially and temporally resolved detection of neurotransmitter release across a single pheochromocytoma (PC12) cell. The microwell-based MEAs consist of 16 4-μm-width square ultramicroelectrodes, 25 3-μm-width square ultramicroelectrodes, or 36 2-μm-width square ultramicroelectrodes, all inside a 40 × 40 μm square SU-8 microwell. MEAs were fabricated on glass substrates by photolithography, thin film deposition, and reactive ion etching. The ultramicroelectrodes in each MEA are tightly defined in a 30 × 30 μm square area, which is further encased inside the SU-8 microwell. With this method, we demonstrate that these microelectrodes are stable, reproducible, and demonstrate good electrochemical properties using cyclic voltammetry. Effective targeting and culture of a single cell is achieved by combining cell-sized microwell trapping and cell-picking micropipet techniques. The surface of the microelectrodes in the MEA was coated with collagen IV to promote cell adhesion and further single-cell culture, as good adhesion between the cell membrane and the electrode surface is critical for the quality of the measurements. Imaging the spatial distribution of exocytosis at the surface of a single PC12 cell has also been demonstrated with this system. Exocytotic signals have been successfully recorded from eight independent 2-μm-wide ultramicroelectrodes from a single PC12 cell showing that the subcellular heterogeneity in single-cell exocytosis can be precisely analyzed with these microwell-based MEAs.
25.	Wigström, Joakim, 1971, et al. (författare) Lithographic Microfabrication of a 16-Electrode Array on a Probe Tip for High Spatial Resolution Electrochemical Localization of Exocytosis 2016 Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 88:4, s. 2080-2087 Tidskriftsartikel (refereegranskat)abstract We report the lithographic microfabrication of a movable thin film microelectrode array (MEA) probe consisting of 16 platinum band electrodes placed on top of a supporting borosilicate glass substrate. These 1.2 mu m wide electrodes were tightly packed and positioned parallel in two opposite rows within a 20 mu m x 25 mu m square area and with a distance less than 10 mu m from the edge of the glass substrate. We demonstrate the ability to control and place the probe in close proximity to the surface of adherent bovine chromaffin cells and to amperometrically record single exocytosis release events with high spatiotemporal resolution. The two-dimensional position of single exocytotic events occurring in the center gap area separating the two rows of MEA band electrodes and that were codetected by electrodes in both rows was determined by analysis of the fractional detection of catecholamine released between electrodes and exploiting random walk simulations. Hence, two-dimensional electrochemical imaging recording of exocytosis release between the electrodes within this area was achieved. Similarly, by modeling the current spikes codetected by parallel adjacent band electrodes positioned in the same electrode row, a one-dimensional imaging of exocytosis with submicrometer resolution was accomplished within the area. The one- and twodimensional electrochemical imaging using the MEA probe allowed for high spatial resolution of exocytosis activity and revealed heterogeneous release of catecholamine at the chromaffin cell surface.
26.	Xianchan, Li, 1982, et al. (författare) Electrochemical quantification of transmitter concentration in single nanoscale vesicles isolated from PC12 cells 2018 Ingår i: Faraday Discussions. - : Royal Society of Chemistry (RSC). - 1359-6640 .- 1364-5498. ; 210, s. 353-364 Tidskriftsartikel (refereegranskat)abstract We use an electrochemical platform, nanoparticle tracking analysis, and differential centrifugation of single catecholamine vesicles to study the properties of nanometer transmitter vesicles, including the number of molecules, size, and catecholamine concentration inside. Vesicle impact electrochemical cytometry (VIEC) was used to quantify the catecholamine content of single vesicles in different batches isolated from pheochromocytoma (PC12) cells with different ultracentrifugation speeds. We show that, vesicles containing less catecholamine are obtained at subsequent centrifugation steps with higher speed (force). Important to quantification, the cumulative content after subsequent centrifugation steps is equivalent to that of one-step centrifugation at the highest speed, 70,000g. Moreover, as we count molecules in the vesicles, we compared molecular numbers from VIEC, flow VIEC, and intracellular VIEC to corresponding vesicle size measured by nanoparticle tracking analysis to evaluate catecholamine concentration in vesicles. The data suggest that vesicular catecholamine concentration is relatively constant and independent of the vesicular size, indicating vesicular transmitter content as a main factor regulating the vesicle size.
27.	Zhu, Wanying, et al. (författare) Combined Amperometry and Electrochemical Cytometry Reveal Differential Effects of Cocaine and Methyphenidate on Exocytosis and the Fraction of Chemical Release 2019 Ingår i: Angewandte Chemie - International Edition. - : Wiley. - 1433-7851 .- 1521-3773. ; 58:13, s. 4238-4242 Tidskriftsartikel (refereegranskat)abstract 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.

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