Lyspersin regulates endosomal positioning. (A) Deletion of lyspersin results in the accumulation of late endosomes/lysosomes in the perinuclear region, and the distribution can be restored by reconstitution of the lyspersin KO cells with a tagged version of the protein. Indirect IF images of HeLa WT, lyspersin KO, and rescue cell line. Representative single-channel images of endogenous lysosomal marker LAMP1 are shown. Bars, 10 µm. (B) Deletion of lyspersin leads to an increase in the number of perinuclear LAMP1-positive endosomes. Images were taken on a confocal microscope and analyzed using Huygens and Imaris software. Number of cells per genotype, n ≥ 15. Depicted is the percentage of late endosomes found at a given distance from the center of nucleus. (C) The mean distance of late endosomes/lysosomes to the center of nucleus is significantly smaller in lyspersin KO cells than in WT or rescued cells. Images were analyzed as in B. Mean ± SD. ***, P ≤ 0.001. n ≥ 15 cells. WT/lyspersin KO, P = 3.0 × 10^-12; lyspersin KO HS-lyspersin/lyspersin KO, P = 1.2 × 10^-6. (D) LAMTOR1 localizes to late endosomes in a lyspersin independent manner. Control and lyspersin depleted cells were analyzed. Shown are representative, single plane confocal images of indirect IF of endogenous LAMTOR1 (red) and endogenous LAMP1 (green). Bars: 10 µm; (inset) 1 µm.