title: Dominant negative SNARE peptides stabilize the fusion pore in a narrow, release-unproductive state.
abstract: Key support for vesicle-based release of gliotransmitters comes from studies of transgenic mice with astrocyte-specific expression of a dominant-negative domain of synaptobrevin 2 protein (dnSNARE). To determine how this peptide affects exocytosis, we used super-resolution stimulated emission depletion microscopy and structured illumination microscopy to study the anatomy of single vesicles in astrocytes. Smaller vesicles contained amino acid and peptidergic transmitters and larger vesicles contained ATP. Discrete increases in membrane capacitance, indicating single-vesicle fusion, revealed that astrocyte stimulation increases the frequency of predominantly transient fusion events in smaller vesicles, whereas larger vesicles transitioned to full fusion. To determine whether this reflects a lower density of SNARE proteins in larger vesicles, we treated astrocytes with botulinum neurotoxins D and E, which reduced exocytotic events of both vesicle types. dnSNARE peptide stabilized the fusion-pore diameter to narrow, release-unproductive diameters in both vesicle types, regardless of vesicle diameter.
authors.affiliation: Laboratory of Neuroendocrinology-Molecular Cell Physiology, Faculty of Medicine, University of Ljubljana, 1000, Ljubljana, Slovenia.
authors.forename: Alenka
authors.initials: A
authors.lastname: Guček
authors.affiliation: Laboratory of Neuroendocrinology-Molecular Cell Physiology, Faculty of Medicine, University of Ljubljana, 1000, Ljubljana, Slovenia.|Celica BIOMEDICAL, 1000, Ljubljana, Slovenia.
authors.forename: Jernej
authors.initials: J
authors.lastname: Jorgačevski
authors.affiliation: Laboratory of Neuroendocrinology-Molecular Cell Physiology, Faculty of Medicine, University of Ljubljana, 1000, Ljubljana, Slovenia.
authors.forename: Priyanka
authors.initials: P
authors.lastname: Singh
authors.affiliation: Department of Optical Nanoscopy, Laser-Laboratory Göttingen e.V., 37077, Göttingen, Germany.
authors.forename: Claudia
authors.initials: C
authors.lastname: Geisler
authors.affiliation: Laboratory of Neuroendocrinology-Molecular Cell Physiology, Faculty of Medicine, University of Ljubljana, 1000, Ljubljana, Slovenia.
authors.forename: Marjeta
authors.initials: M
authors.lastname: Lisjak
authors.affiliation: Laboratory of Neuroendocrinology-Molecular Cell Physiology, Faculty of Medicine, University of Ljubljana, 1000, Ljubljana, Slovenia.|Celica BIOMEDICAL, 1000, Ljubljana, Slovenia.
authors.forename: Nina
authors.initials: N
authors.lastname: Vardjan
authors.affiliation: Laboratory of Neuroendocrinology-Molecular Cell Physiology, Faculty of Medicine, University of Ljubljana, 1000, Ljubljana, Slovenia.|Celica BIOMEDICAL, 1000, Ljubljana, Slovenia.|Department of Biology, Biotechnical Faculty, University of Ljubljana, 1000, Ljubljana, Slovenia.
authors.forename: Marko
authors.initials: M
authors.lastname: Kreft
authors.affiliation: Department of Optical Nanoscopy, Laser-Laboratory Göttingen e.V., 37077, Göttingen, Germany.
authors.forename: Alexander
authors.initials: A
authors.lastname: Egner
authors.affiliation: Laboratory of Neuroendocrinology-Molecular Cell Physiology, Faculty of Medicine, University of Ljubljana, 1000, Ljubljana, Slovenia. robert.zorec@mf.uni-lj.si.|Celica BIOMEDICAL, 1000, Ljubljana, Slovenia. robert.zorec@mf.uni-lj.si.
authors.forename: Robert
authors.initials: R
authors.lastname: Zorec
mesh_terms: D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D001253:Astrocytes / Q000187:drug effects / Q000378:metabolism; D005089:Exocytosis / Q000187:drug effects; D000067128:Extracellular Vesicles / Q000187:drug effects / Q000378:metabolism; D005260:Female; D008561:Membrane Fusion / Q000187:drug effects; D008853:Microscopy; D008954:Models, Biological; D010455:Peptides / Q000378:metabolism*; D017208:Rats, Wistar; D050600:SNARE Proteins / Q000378:metabolism*; D013997:Time Factors
chemical_list: D010455:Peptides; D050600:SNARE Proteins; D000255:Adenosine Triphosphate
keywords: Astrocytes; Capacitance measurements; Fusion pore; Gliotransmitters; Regulated exocytosis; STED microscopy