Independent Research Fund Denmark - Technology and Production Sciences
8020-00228A
Denmark
Lundbeckfonden
R277-2018-802
Denmark
Citation
Journal: EMBO Rep / Year: 2023 Title: Morphofunctional changes at the active zone during synaptic vesicle exocytosis. Authors: Julika Radecke / Raphaela Seeger / Anna Kádková / Ulrike Laugks / Amin Khosrozadeh / Kenneth N Goldie / Vladan Lučić / Jakob B Sørensen / Benoît Zuber / Abstract: Synaptic vesicle (SV) fusion with the plasma membrane (PM) proceeds through intermediate steps that remain poorly resolved. The effect of persistent high or low exocytosis activity on intermediate ...Synaptic vesicle (SV) fusion with the plasma membrane (PM) proceeds through intermediate steps that remain poorly resolved. The effect of persistent high or low exocytosis activity on intermediate steps remains unknown. Using spray-mixing plunge-freezing cryo-electron tomography we observe events following synaptic stimulation at nanometer resolution in near-native samples. Our data suggest that during the stage that immediately follows stimulation, termed early fusion, PM and SV membrane curvature changes to establish a point contact. The next stage-late fusion-shows fusion pore opening and SV collapse. During early fusion, proximal tethered SVs form additional tethers with the PM and increase the inter-SV connector number. In the late-fusion stage, PM-proximal SVs lose their interconnections, allowing them to move toward the PM. Two SNAP-25 mutations, one arresting and one disinhibiting spontaneous release, cause connector loss. The disinhibiting mutation causes loss of membrane-proximal multiple-tethered SVs. Overall, tether formation and connector dissolution are triggered by stimulation and respond to spontaneous fusion rate manipulation. These morphological observations likely correspond to SV transition from one functional pool to another.
pH: 7.4 Details: NB medium (Neurobasal with 2% B-27, 1 M HEPES, 0.26% lutamax, 14.3 mM beta-mercaptoethanol, 10000 IU penicillin, 10 mg streptomycin)
Vitrification
Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV Details: After 12 to 14 days of incubation grids with mouse neurons were plunge frozen with a Vitrobot (Thermofisher Scientific, Mark IV) with a blot time of 3 s and a blot force of -10. Wait time ...Details: After 12 to 14 days of incubation grids with mouse neurons were plunge frozen with a Vitrobot (Thermofisher Scientific, Mark IV) with a blot time of 3 s and a blot force of -10. Wait time and drain time were not used. Humidity was set to 100% at 4 degrees C. 4 ul undiluted 10 nm BSA gold tracer (Aurion) was added directly onto the grid prior to plunge freezing..
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