Fig. 1.

Rapid vesicle tethering upon a depolarization pulse and APs at dissociated cMF terminals. (A) An example of transmitted light image of a dissociated cMF terminal. The expanded image was obtained using TIRF microscopy. SVs were labeled by FM1-43. (B) Representative traces of 100-ms depolarization-induced Ca²⁺ current (I_Ca) and capacitance jump (C_m). The slope of the capacitance increase after the depolarization pulse was fitted with a line shown in red. (C) The amount of capacitance increase by a 0-mV depolarization pulse for 100 ms was plotted against peak Ca²⁺ current, basal membrane capacitance of the recorded terminals (n = 58), and the slope of the capacitance increase after the depolarization pulse (n = 56). Data from individual terminals were plotted by open circles. These plots were fitted with a line shown in red. (D) We observed “vanish” and “tethering” types of vesicle movements in TIRF image. (Top) Examples of the two types of events in TIRF image. (Bottom) Averages of the normalized fluorescent intensity were plotted against time for 347-nm-diameter circle centered on the spots (filled squares) and the concentric annulus around the circles (outer diameter of 867 nm; open squares). Error bars show SEM (n = 35, and 31 for vanish and tethering, respectively). “Vanish” events correspond to vesicles moving out from the TIRF field or vesicles undergoing exocytosis. “Tethering” events correspond to vesicles recruiting to the TIRF field. (Scale bars, 500 nm.) (E) Using whole-cell mode, Ca²⁺ current and capacitance change upon a depolarization pulse were recorded. (F) Two types of events were observed by simultaneous TIRF imaging. Peristimulus time histograms for the two types of events and cumulative number of the events from 56 terminals are shown. Fits consist of the sum of a double exponential function and a line for “vanish” events, and the sum of a single exponential function and a line for “tethering” events. According to the result of the different pulse duration experiments (SI Appendix, Fig. S1B), τ₁ for “vanish” events was fixed at 10 ms. The fit has a ratio of the amount of first to second component of 0.06. The 100-ms depolarization pulse was applied from 0 s to 0.1 s. The depolarizing period is indicated by the gray color (the same for subsequent figures). (G) In cell-attached mode, APs were evoked by 2-ms depolarization pulses at 50 Hz for 1 s. (H) Peristimulus time histograms and cumulative number of events detected by simultaneous TIRF imaging from 43 terminals. The time of the train is indicated by the gray color. The cumulative numbers of the events were fitted with a single exponential curve (red).