2026-10-06T09:26:26Z https://api.figshare.com/v2/oai

oai:figshare.com:article/33154970 2026-09-28T17:23:12Z category_24715 category_24730 category_24163 item_type_2 month_year_09_2026

Raw movie of a typical single-molecule TIRF experiment. These data fully demonstrate stochastic transitions of the V-ATPase between active and inactive modes and their regulation by specific inhibitor Bafilomycin A1. Eleftherios Kosmidis (24513937) Maria Fokaeos (24513791) Christopher G. Shuttle (24513941) Michael Isselstein (24513945) Alessandra Narducci (24513946) Aleksander Cvjetkovic (8998085) Peter J. Johnson (24513948) Jesper L. Pedersen (24514126) Reinhard Jahn (24513960) Julia Preobraschenski (24513963) Dimitrios Stamou (24513966) Human biophysics Cellular nervous system Receptors and membrane biology Single molecule biophysics Transporters in the nervous system v-atpases synaptic vesicle acidification TIRF microscopy proton pumping activity <h4 dir="ltr">This video shows single-molecule acidification kinetics of native vacuolar-type ATPase (V-ATPase) in individual synaptic vesicles (SVs) purified from rat brain, labeled with the pH-sensitive lipid pHrodo-DOPE and imaged by total internal reflection fluorescence (TIRF) microscopy (Olympus TIRF system, 532 nm excitation, iXon 897 EMCCD camera, 500 ms exposure per frame, 3 s cycle time).</h4><h4 dir="ltr">The recording comprises three sequential phases: (1) 5 minutes of baseline fluorescence in activity buffer (300 mM glycine, 2 mM MOPS, 2 mM MgSO₄, pH 7.1–7.15) prior to ATP addition; (2) 10 minutes following addition of 1 mM MgATP and 30 mM choline chloride, triggering ATP-dependent proton pumping and acidification of the vesicle lumen; and (3) 15 minutes following addition of the orthosteric V-ATPase inhibitor bafilomycin A1 (50 pM), during which single V-ATPases exhibit stochastic transitions between active (proton-pumping) and inactive modes.</h4><h4 dir="ltr">This raw image stack is representative of the kinetic single-molecule data underlying Figs. 1d and 4a of the associated manuscript, "Orthosteric and allosteric inhibition of the brain V-ATPase outside the alternating-access cycle" (Kosmidis, Fokaeos et al.).</h4><p></p> 2026-09-28T17:23:12Z Dataset Media 10.6084/m9.figshare.33154970.v1 https://figshare.com/articles/media/Raw_movie_of_a_typical_single-molecule_TIRF_experiment_These_data_fully_demonstrate_stochastic_transitions_of_the_V-ATPase_between_active_and_inactive_modes_and_their_regulation_by_specific_inhibitor_Bafilomycin_A1_/33154970 CC BY 4.0