Lysosomal exocytosis occurs in the vicinity of FAs. (A) Left: Color-coded representation of the 3D distribution of lysosomes in HeLa cells. Images are acquired by CLSM at 0.5 µm intervals on the z-axis. The depth information was encoded in the color scheme indicated at the bottom of the images. Bar, 10 µm. Right: Quantitative results of the lysosome z-position in WT and MYO18B-KO HeLa cells (mean ± SD; n = 4 image stacks; N = 2 biological replicates; unpaired two-tailed t test; **P ≤ 0.01). (B) Representative CLSM image of U2OS cells expressing EB1-EGFP stained with vinculin and LAMP1. Bar, 10 µm in the original image and 3 µm in the magnified image. Mander’s coefficient is 0.993 (vinculin to EB1-EGFP) and 0.863 (EB1-EGFP to vinculin). (C) Colocalization of LAMP1 (lysosomes) and vinculin (FA) in U2OS cells. Left: Representative CLSM images of U2OS cells co-stained with vinculin and LAMP1. Bar, 10 µm. Right: Quantification of the distance between every lysosome and its nearest FA, measured by DiAna (mean ± SD; n = 10 images; N = 2 biological replicates; unpaired two-tailed t test; **P ≤ 0.01). See also Fig. S4 B. (D) Distance between lysosomal exocytosis and FA. Left: Dual-color live imaging with paxillin-mCherry and VAMP7-pHluorin acquired using TIRF microscopy. The grayscale image is the paxillin-mCherry channel, magenta dots are lysosome–plasma membrane fusion events, and green dots result from randomly simulated coordinates. Right: Distance between every experimental fusion event and its nearest FA, measured by DiAna and plotted in a cumulative frequency plot (magenta curve). Simulated coordinates were generated by the shuffling function in DiAna, and the distance was also measured and plotted in the same plot. A total of 100 rounds simulations were performed, and two extremes are represented in the green curve, whereas the average is represented in the black curve. See also Video 5.