Docking of munc13-4 granules at the plasma membrane requires rab27. (A) TIRFM view of YFP-munc13-4 in resting and activated RBL-2H3 stably expressing Cherry-rab27a and YFP-munc13-4. White trajectories represent tracks of individual granules. Scale bar indicates 2 μm. (B) RBL-2H3 cells expressing YFP-munc13-4 were imaged with a confocal microscope using 0.35-μm slices. The smaller granules (closed arrowheads) that are observed in the TIRF view are predominantly found at the basal side of the cell (z = 0 μm), whereas the signal for the out-of-focus, larger granules in the background is indicated with open arrowheads. Note that the optical sections are ∼ 3 times thicker than the TIRF zone. The larger granules with diameter > 0.3 μm are distributed to the upper layers of the z-stack. Scale bar indicates 5 μm. (C) MSD versus Δtime plots of the tracks analyzed per cell (n = 15) in resting (●) or activated (■) Cherry-rab27a YFP-munc13-4–expressing cells of a representative experiment. Black lines indicate the slope of the first 8 seconds. (D) Same analysis as in panel C for Cherry-rab27a YFP-munc13-4(FQL > AAA) and Cherry-rab27a YFP-munc13-4Δ (280-285) cell lines. (E) Decrease in mobile vesicles after activation was analyzed per cell for 15 cells in 3 experiments and compared between wild-type munc13-4 and mutants that do not bind rab27.