| 1. |
- Lovric, Jelena, 1980, et al.
(författare)
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On the mechanism of electrochemical vesicle cytometry: chromaffin cell vesicles and liposomes
- 2016
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Ingår i: Faraday Discussions. - : Royal Society of Chemistry (RSC). - 1359-6640 .- 1364-5498. ; 193, s. 65-79
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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.
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| 2. |
- Majdi, Soodabeh, 1980, et al.
(författare)
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DMSO Chemically Alters Cell Membranes to Slow Exocytosis and Increase the Fraction of Partial Transmitter Released
- 2017
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Ingår i: Chembiochem. - : Wiley. - 1439-4227 .- 1439-7633. ; 18:19, s. 1898-1902
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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.
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| 3. |
- Najafinobar, Neda, 1985, et al.
(författare)
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Excited Fluorophores Enhance the Opening of Vesicles at Electrode Surfaces in Vesicle Electrochemical Cytometry
- 2016
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Ingår i: Angewandte Chemie International Edition. - : Wiley. - 1433-7851 .- 1521-3773. ; 55:48, s. 15081-15085
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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
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| 4. |
- Asadpour, Farzaneh, et al.
(författare)
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Vesicular release dynamics are altered by the interaction between the chemical cargo and vesicle membrane lipids
- 2021
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Ingår i: Chemical Science. - : Royal Society of Chemistry (RSC). - 2041-6520 .- 2041-6539. ; 12:30, s. 10273-10278
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Tidskriftsartikel (refereegranskat)abstract
- The release of the cargo from soft vesicles, an essential process for chemical delivery, is mediated by multiple factors. Among them, the regulation by the interaction between the chemical cargo species and the vesicular membrane, widely existing in all vesicles, has not been investigated to date. Yet, these interactions hold the potential to complicate the release process. We used liposomes loaded with different monoamines, dopamine (DA) and serotonin (5-HT), to simulate vesicular release and to monitor the dynamics of chemical release from isolated vesicles during vesicle impact electrochemical cytometry (VIEC). The release of DA from liposomes presents a longer release time compared to 5-HT. Modelling the release time showed that DA filled vesicles had a higher percentage of events where the time for the peak fall was better fit to a double exponential (DblExp) decay function, suggesting multiple kinetic steps in the release. By fitting to a desorption-release model, where the transmitters adsorbed to the vesicle membrane, the dissociation rates of DA and 5-HT from the liposome membrane were estimated. DA has a lower desorption rate constant, which leads to slower DA release than that observed for 5-HT, whereas there is little difference in pore size. The alteration of vesicular release dynamics due to the interaction between the chemical cargo and vesicle membrane lipids provides an important mechanism to regulate vesicular release in chemical and physiological processes. It is highly possible that this introduces a fundamental chemical regulation difference between transmitters during exocytosis.
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| 5. |
- Dunevall, Johan, 1984, et al.
(författare)
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Vesicle impact electrochemical cytometry compared to amperometric exocytosis measurements
- 2017
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Ingår i: Current Opinion in Electrochemistry. - : Elsevier BV. - 2451-9103 .- 2451-9111. ; 5:1, s. 85-91
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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.
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| 6. |
- Larsson, Anna, et al.
(författare)
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Intracellular Electrochemical Nanomeasurements Reveal that Exocytosis of Molecules at Living Neurons is Subquantal and Complex
- 2020
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Ingår i: Angewandte Chemie-International Edition. - : Wiley. - 1433-7851 .- 1521-3773. ; 59:17, s. 6711-6714
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Tidskriftsartikel (refereegranskat)abstract
- Since the early work of Bernard Katz, the process of cellular chemical communication through exocytosis, quantal release, has been considered to be all or none. Recent evidence has shown exocytosis to be partial or "subquantal" at single-cell model systems, but there is a need to understand this at communicating nerve cells. Partial release allows nerve cells to control the signal at the site of release during individual events, for which the smaller the fraction released, the greater the range of regulation. Herein, we show that the fraction of the vesicular octopamine content released from a living Drosophila larval neuromuscular neuron is very small. The percentage of released molecules was found to be only 4.5 % for simple events and 10.7 % for complex (i.e., oscillating or flickering) events. This large content, combined with partial release controlled by fluctuations of the fusion pore, offers presynaptic plasticity that can be widely regulated.
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| 7. |
- Larsson, Anna, et al.
(författare)
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Vesicular Transmitter Content in Chromaffin Cells Can Be Regulated via Extracellular ATP
- 2019
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Ingår i: ACS Chemical Neuroscience. - : American Chemical Society (ACS). - 1948-7193. ; 10:11, s. 4735-4740
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Tidskriftsartikel (refereegranskat)abstract
- The energy carrying molecule adenosine triphosphate (ATP) has been implicated for its role in modulation of chemical signaling for some time. Despite this, the precise effects and mechanisms of action of ATP on secretory cells are not well-known. Here, bovine chromaffin cells have been used as a model system to study the effects of extracellular ATP in combination with the catecholamine transmitter norepinephrine (NE). Both transmitter storage and exocytotic release were quantified using complementary amperometric techniques. Although incubation with NE alone did not cause any changes to either transmitter storage or release, coincubation with NE and ATP resulted in a significant increase that was concentration dependent. To probe the potential mechanisms of action, a slowly hydrolyzable version of ATP, ATP-gamma-S, was used either alone or together with NE. The result implicates two different behaviors of ATP acting on both the purinergic autoreceptors and as a source of the energy needed to load chromaffin cell vesicles.
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| 8. |
- Li, Xianchan, 1982, et al.
(författare)
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Quantitative Measurement of Transmitters in Individual Vesicles in the Cytoplasm of Single Cells with Nanotip Electrodes
- 2015
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Ingår i: Angewandte Chemie International Edition. - : Wiley. - 1433-7851 .- 1521-3773. ; 54:41, s. 11978-11982
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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.
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| 9. |
- Majdi, Soodabeh, 1980, et al.
(författare)
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Electrochemical Measurements of Optogenetically Stimulated Quantal Amine Release from Single Nerve Cell Varicosities in Drosophila Larvae
- 2015
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Ingår i: Angewandte Chemie International Edition. - : Wiley. - 1433-7851 .- 1521-3773. ; 54:46, s. 13609-13612
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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.
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| 10. |
- Majdi, Soodabeh, 1980, et al.
(författare)
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Electrochemistry in and of the Fly Brain
- 2018
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Ingår i: Electroanalysis. - : Wiley. - 1040-0397 .- 1521-4109. ; 30:6, s. 999-1010
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Tidskriftsartikel (refereegranskat)abstract
- Studying brain function and neuronal communication has been always crucial due to the complexity of these systems. A great deal of technology and model systems have been developed to study this subject. Yet, very small invertebrate systems such as the fruit fly, Drosophila are excellent models and often have better defined and more easily manipulated genetics. This review focuses on invivo, ex vivo, and invitro measurements of biogenic amines by electrochemical techniques on the fly nervous system. The methods include capillary electrophoresis, fast scan cyclic voltammetry, and chronoamperometry have been used to study both larval and adult central nervous systems. Better understanding of brain function in model systems should aid in finding solutions to biological and bioanalytical challenges related to human brain function and also neurodegenerative disease.
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