Single-vesicle architecture of synaptobrevin2 in astrocytes

Priyanka Singh, Jernej Jorgačevski, Marko Kreft, Vladimir Grubišić, Randy F. Stout, Maja Potokar, Vladimir Parpura () and Robert Zorec ()
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Priyanka Singh: Laboratory of Neuroendocrinology—Molecular Cell Physiology, Institute of Pathophysiology, Faculty of Medicine, University of Ljubljana
Jernej Jorgačevski: Laboratory of Neuroendocrinology—Molecular Cell Physiology, Institute of Pathophysiology, Faculty of Medicine, University of Ljubljana
Marko Kreft: Laboratory of Neuroendocrinology—Molecular Cell Physiology, Institute of Pathophysiology, Faculty of Medicine, University of Ljubljana
Vladimir Grubišić: Center for Glial Biology in Medicine, University of Alabama
Randy F. Stout: Center for Glial Biology in Medicine, University of Alabama
Maja Potokar: Laboratory of Neuroendocrinology—Molecular Cell Physiology, Institute of Pathophysiology, Faculty of Medicine, University of Ljubljana
Vladimir Parpura: Center for Glial Biology in Medicine, University of Alabama
Robert Zorec: Laboratory of Neuroendocrinology—Molecular Cell Physiology, Institute of Pathophysiology, Faculty of Medicine, University of Ljubljana

Nature Communications, 2014, vol. 5, issue 1, 1-12

Abstract: 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.

Date: 2014
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DOI: 10.1038/ncomms4780

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