| 1. |
- Dunevall, Johan, 1984, et al.
(författare)
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Characterizing the Catecholamine Content of Single Mammalian Vesicles by Collision-Adsorption Events at an Electrode
- 2015
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Ingår i: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 0002-7863 .- 1520-5126. ; 137:13, s. 4344-4346
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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.
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| 2. |
- 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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| 3. |
- Mashadi Fathali, Hoda, 1983, et al.
(författare)
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Extracellular Osmotic Stress Reduces the Vesicle Size while Keeping a Constant Neurotransmitter Concentration
- 2017
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Ingår i: Acs Chemical Neuroscience. - : American Chemical Society (ACS). - 1948-7193. ; 8:2, s. 368-375
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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.
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| 4. |
- Mashadi Fathali, Hoda, 1983, et al.
(författare)
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Monitoring the Effect of Osmotic Stress on Secretory Vesicles and Exocytosis
- 2018
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Ingår i: Journal of Visualized Experiments. - : MyJove Corporation. - 1940-087X. ; :132, s. 1-10
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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.
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