| 1. |
- Aref, Mohaddeseh A., et al.
(författare)
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Intracellular injection of phospholipids directly alters exocytosis and the fraction of chemical release in chromaffin cells as measured by nano-electrochemistry
- 2020
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Ingår i: Chemical Science. - : Royal Society of Chemistry (RSC). - 2041-6520 .- 2041-6539. ; 11:43, s. 11869-11876
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 2. |
- Ranjbari, Elias, et al.
(författare)
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Direct Measurement of Total Vesicular Catecholamine Content with Electrochemical Microwell Arrays
- 2020
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Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 92:16, s. 11325-11331
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Tidskriftsartikel (refereegranskat)abstract
- We have designed and fabricated a microwell array chip (MWAC) to trap and detect the entire content of individual vesicles after disruption of the vesicular membrane by an applied electrical potential. To understand the mechanism of vesicle impact electrochemical cytometry (VIEC) in microwells, we simulated the rupture of the vesicles and subsequent diffusion of entrapped analytes. Two possibilities were tested: (i) the vesicle opens toward the electrode, and (ii) the vesicle opens away from the electrode. These two possibilities were simulated in the different microwells with varied depth and width. Experimental VIEC measurements of the number of molecules for each vesicle in the MWAC were compared to VIEC on a gold microdisk electrode as a control, and the quantified catecholamines between these two techniques was the same. We observed a prespike foot in a significant number of events (similar to 20%) and argue this supports the hypothesis that the vesicles rupture toward the electrode surface with a more complex mechanism including the formation of a stable pore intermediate. This study not only confirms that in standard VIEC experiments the whole content of the vesicle is oxidized and quantified at the surface of the microdisk electrode but actively verifies that the adsorbed vesicle on the surface of the electrode forms a pore in the vicinity of the electrode rather than away from it. The fabricated MWAC promotes our ability to quantify the content of vesicles accurately, which is fundamentally important in bioanalysis of the vesicles.
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