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Nat Commun. 2014 May 7;5:3780. doi: 10.1038/ncomms4780.

Single-vesicle architecture of synaptobrevin2 in astrocytes.

Author information

1
Laboratory of Neuroendocrinology-Molecular Cell Physiology, Institute of Pathophysiology, Faculty of Medicine, University of Ljubljana, 1000 Ljubljana, Slovenia.
2
1] Laboratory of Neuroendocrinology-Molecular Cell Physiology, Institute of Pathophysiology, Faculty of Medicine, University of Ljubljana, 1000 Ljubljana, Slovenia [2] Celica Biomedical Center, 1000 Ljubljana, Slovenia.
3
1] Laboratory of Neuroendocrinology-Molecular Cell Physiology, Institute of Pathophysiology, Faculty of Medicine, University of Ljubljana, 1000 Ljubljana, Slovenia [2] Celica Biomedical Center, 1000 Ljubljana, Slovenia [3] Department of Biology, Biotechnical Faculty, University of Ljubljana, 1000 Ljubljana, Slovenia.
4
Department of Neurobiology, Center for Glial Biology in Medicine, University of Alabama, Birmingham, Alabama 35294, USA.
5
1] Department of Neurobiology, Center for Glial Biology in Medicine, University of Alabama, Birmingham, Alabama 35294, USA [2] The Dominick P. Purpura Department of Neuroscience, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
6
1] Department of Neurobiology, Center for Glial Biology in Medicine, University of Alabama, Birmingham, Alabama 35294, USA [2] Department of Biotechnology, University of Rijeka, 51000 Rijeka, Croatia.

Abstract

Exocytic transmitter release is regulated by the SNARE complex, which contains a vesicular protein, synaptobrevin2 (Sb2). However, Sb2 vesicular arrangement is unclear. Here we use super-resolution fluorescence microscopy to study the prevalence and distribution of endogenous and exogenous Sb2 in single vesicles of astrocytes, the most abundant glial cells in the brain. We tag Sb2 protein at C- and N termini with a pair of fluorophores, which allows us to determine the Sb2 length and geometry. To estimate total number of Sb2 proteins per vesicle and the quantity necessary for the formation of fusion pores, we treat cells with ATP to stimulate Ca2+-dependent exocytosis, increase intracellular alkalinity to enhance the fluorescence presentation of yellow-shifted pHluorin (YpH), appended to the vesicle lumen domain of Sb2, and perform photobleaching of YpH fluorophores. Fluorescence intensity analysis reveals that the total number of endogenous Sb2 units or molecules per vesicle is ≤25.

PMID:
24807050
PMCID:
PMC4344122
DOI:
10.1038/ncomms4780
[Indexed for MEDLINE]
Free PMC Article

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