Amino acid–stimulated lysosomal FYCO1 recruitment and dispersion. (A) RPE-1 cells were starved with EBSS for 4 h and stimulated or not for 2 h with 2× amino acids (AA) in DMEM with or without 0.1% DMSO or 3 µM SAR405, stained with antibodies against FYCO1 and LAMP1, and analyzed by confocal microscopy. Note that upon amino acid stimulation, FYCO1 is recruited to LAMP1-positive lysosomes that spread, and this is prevented by SAR405. (B–D) High-content imaging and automated analysis of cells treated as in A, using the Olympus ScanR system, representing the relative total intensity of FYCO1 (B) or LAMP1 (C) per cell or the relative ratios of peripheral/perinuclear intensity of LAMP1-positive lysosomes (D). Error bars denote ± SEM from independent experiments (n = 3). *, P < 0.05; **, P < 0.01; ***, P < 0.001 (one-sample t test). More than 1,500 cells were analyzed per condition. (E) RPE-1 cells were starved in EBSS for 4 h and stimulated for 2 h with 2× amino acids in DMEM, stained with antibodies against FYCO1 and Protrudin, and analyzed by confocal microscopy.