Direct monitoring reveals impaired lysosomal exocytosis in MYO18B-KO cells. (A) Schematic diagram of fluorescence changes for VAMP7-pHluorin during lysosomal exocytosis. The fluorescence of pHluorin is quenched in the lysosome lumen (acidic pH), elevated by lysosome fusion with the plasma membrane (neutral pH), and rapidly diffused on the plasma membrane after membrane fusion. (B) Representative image of lysosomal exocytosis detected by TIRF microscopy using VAMP7-pHluorin. Bar, 5 µm in the original image (left) and 1 µm in the magnified montage (right). (C) Representative CLSM image of U2OS cells expressing VAMP7-pHluorin stained with LAMP1. Bar, 10 µm in original image (left) and 2 µm in magnified images (right). The Pearson correlation coefficient between LAMP1 and VAMP7-pHluorin is 0.753 in the magnified image. (D) Lysosomal exocytosis detected by TIRF microscopy in WT and MYO18B-KO U2OS cells. Lysosome-plasma membrane fusion events are represented as magenta dots. Bar, 5 µm. (E) Quantification results of lysosome-plasma membrane fusion events during 30 s live imaging in WT and MYO18B-KO U2OS cells (mean ± SD; n = 31 cells; N = 6 biological replicates; unpaired two-tailed t test; ****P ≤ 0.0001). See also Video 1.