FIGURE 1.

Secretion kinetics by counting single-vesicle exocytosis events. (A) A single-vesicle exocytosis, visualized by TIRFM. A nearly diffraction limited fluorescent spot disappears by giving off a puff of light as the released soluble vesicular content marker, NPY-GFP, diffuses away. (B) Cumulative number of exocytosis events as a function of time, in digitonin-permeabilized cells in the presence of 30 μM calcium. Since the delay for successful permeabilization was different from cell to cell, the timing of the initial event for every cell (marking successful permeabilization and calcium rise) was set to t = 0 s. With data from 35 cells pooled together, this procedure causes the 35 initial events to artificially cluster together at t = 0 s. This artifact is removed by excluding the 35 initial events from the plot. Dots are experimental data, while the continuous red curve is a fit to the data, up to t = 45 s (see text). Inset shows data and the fit for short times. (C) Cumulative number of exocytosis events as a function of time from 59 cells from caged-calcium experiments in which eight UV pulses were applied at 10 s intervals to increase the [Ca²⁺]_i abruptly. Peak [Ca²⁺]_i generated per pulse is estimated to be ∼1 μM (see Supplementary Material). The value t = 0 s marks the moment the first pulse was applied. Every pulse caused a burst of activity, but with different efficiency. Pulse 1 only caused four events in one cell. Inset shows the number of exocytosis events as a function of time after a UV pulse was applied, for data from all pulses combined (1138 events). Bins are 0.8 s wide. The maximum rate of exocytosis is reached ∼2 s after a UV pulse. (D) Average number of exocytosis events per pulse. On a per-cell basis, early pulses (pulses 2–4) were approximately two-times more efficient as late ones (pulses 6–8), on average.