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.

<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>