Granules migrate in response to [Ca²⁺]_i rise. Before each experiment, cells were preincubated with 1 μM ionomycin for at least 5 min in Ca²⁺-free solution (0 Ca²⁺), and all test solutions contained 1 μM ionomycin in the Ca²⁺-free solution. (A) TIRFM images obtained just before (a) and during (b) 2 mM Ca²⁺ (2 Ca²⁺). Bars, 4 μm. (B) Trajectory in the x–y plane of the two granules indicated in A before (black) and during (red) 2 mM Ca²⁺ treatment. Scale bars are in units of micrometers. (C) Cumulative distance traveled in the x–y plane. (D) Speed in the x–y plane. (E) Change of fluorescence intensity of two representative granules in an arbitrary unit (A.U.). Increase of fluorescence intensity represents migration of the granules to the plasma membrane. (F) Intensity of 26 randomly chosen granules from A. All data are sampled at 2-s intervals. For fluorescence intensity of individual granules in E and F, background fluorescence from a granule-free region was subtracted.