Impairment of lytic granule movement caused by LAMP1 deficiency. YTS cells, stably transduced with control (CTRL) or LAMP1 RNAi, were labeled with LysoTracker Red. The cells were visualized using spinning disk confocal microscopy, and the movement of LysoTracker-labeled vesicles was recorded in 3 dimensions (x-y-z plane) for 180 seconds. The characteristics of vesicle trajectories were derived from analysis of 10 (LAMP1 RNAi) or 11 (CTRL RNAi) cells in 3 experiments. (A) Histograms of frequency distributions of the length (top), displacement (the distance in straight line between the beginning and end of the track; middle), and mean velocity (bottom) of granule trajectories in CTRL and LAMP1 RNAi cells. (B) Graphs show the median values and interquartile range of the length, displacement, and velocity of granules from CTRL and LAMP1 RNAi cells. ***P < .001; Mann-Whitney U test. Granules in LAMP1 RNAi cells formed shorter tracks, with the majority (75%) in the range of 0.96 to 4.7 μm, whereas in CTRL RNAi cells the majority of tracks were in the range of 1.8 to 6.6 μm. The median track length in LAMP1 RNAi cells was 2.43 μm, and 3.74 μm in CTRL RNAi cells. The track displacement was also decreased in LAMP1 RNAi cells: 0.3 to 1.4 μm vs 0.65 to 2.2 μm in CTRL RNAi cells; the median displacement value was 0.72 μm and 1.24 μm in LAMP1 and CTRL RNAi cells, respectively. The majority of vesicles in LAMP1 RNAi cells moved at 0.12 to 0.33 μm/s, whereas in CTRL RNAi cells they moved in the range of 0.17 to 0.42 μm/s (in agreement with Mentlik et al²⁸ ); the median speed was 0.2 μm/s in LAMP1 RNAi cells, compared with 0.29 μm/s in CTRL RNAi cells. (C) LAMP1 is required for proper recruitment of p150^glued/dynactin to lytic granules. YTS cells, transduced with either CTRL or LAMP1 RNAi, were homogenized, and their lytic granules were purified on a 10% to 40% discontinuous gradient of iodoxanol. The presence of proteins in the total cell lysate (TCL), CLF, cytoplasmic fraction (cyto), and purified lysosomal fraction (PLF) was assessed by immunoblotting with the Ab’s specific for the indicated proteins; granzyme B and LAMP2 were used as loading controls. The result is representative of 2 separate experiments. The changes in p150^glued levels, normalized to LAMP2 levels, were calculated as the ratio between p150^glued band intensity from LAMP1 and CTRL RNAi cells in the appropriate samples (eg, p150^glued band intensity from LAMP1 RNAi cell PLF sample divided by the band intensity from CTRL RNAi cell PLF sample).