Dynamic Visualization and Quantification of Single Vesicle Opening and Content by Coupling Vesicle Impact Electrochemical Cytometry with Confocal Microscopy.

Autor:	Zheng YN; Department of Chemistry and Molecular Biology, University of Gothenburg, Kemivägen 10, 41296 Gothenburg, Sweden., Nguyen TDK; Department of Chemistry and Molecular Biology, University of Gothenburg, Kemivägen 10, 41296 Gothenburg, Sweden., Dunevall J; Department of Chemistry and Molecular Biology, University of Gothenburg, Kemivägen 10, 41296 Gothenburg, Sweden., Phan NTN; Department of Chemistry and Molecular Biology, University of Gothenburg, Kemivägen 10, 41296 Gothenburg, Sweden., Ewing AG; Department of Chemistry and Molecular Biology, University of Gothenburg, Kemivägen 10, 41296 Gothenburg, Sweden.
Jazyk:	angličtina
Zdroj:	ACS measurement science au [ACS Meas Sci Au] 2021 Dec 15; Vol. 1 (3), pp. 131-138. Date of Electronic Publication: 2021 Aug 09.
DOI:	10.1021/acsmeasuresciau.1c00021
Abstrakt:	In this work, we introduce a novel method for visualization and quantitative measurement of the vesicle opening process by correlation of vesicle impact electrochemical cytometry (VIEC) with confocal microscopy. We have used a fluorophore conjugated to lipids to label the vesicle membrane and manipulate the membrane properties, which appears to make the membrane more susceptible to electroporation. The neurotransmitters inside the vesicles were visualized by use of a fluorescence false neurotransmitter 511 (FFN 511) through accumulation inside the vesicle via the neuronal vesicular monoamine transporter 2 (VMAT 2). Optical and electrochemical measurements of single vesicle electroporation were carried out using an in-house, disk-shaped, gold-modified ITO (Au/ITO) microelectrode device (5 nm thick, 33 μm diameter), which simultaneously acted as an electrode surface for VIEC and an optically transparent surface for confocal microscopy. As a result, the processes of adsorption, electroporation, and opening of single vesicles followed by neurotransmitter release on the Au/ITO surface have been simultaneously visualized and measured. Three opening patterns of single isolated vesicles were frequently observed. Comparing the vesicle opening patterns with their corresponding VIEC spikes, we propose that the behavior of the vesicular membrane on the electrode surface, including the adsorption time, residence time before vesicle opening, and the retention time after vesicle opening, are closely related to the vesicle content and size. Large vesicles with high content tend to adsorb to the electrode faster with higher frequency, followed by a shorter residence time before releasing their content, and their membrane remains on the electrode surface longer compared to the small vesicles with low content. With this approach, we start to unravel the vesicle opening process and to examine the fundamentals of exocytosis, supporting the proposed mechanism of partial or subquantal release in exocytosis. Competing Interests: The authors declare no competing financial interest. (© 2021 The Authors. Published by American Chemical Society.)
Databáze:	MEDLINE
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