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Fig. 3 A, B Rapalog-mediated perinuclear clustering of lysosomes. A Lysosomes in WM983B cells stably expressing LAMP1-mCherry-FKBP and BicD2-FRB can be clustered using Rapalog compound (Rap) which induces FKBP-FRB rapid heterodimerization. B Representative images of lysosome perinuclear clustering 24 h after 5 nM Rapalog treatment. C Correlative light and electron microscopy of clustered perinuclear LAMP1 positive compartments. Representative immunofluorescent images and a high magnification overlay of fluorescent and electron microscopy images (LAMP1 in red/magenta, nucleus in blue) are shown for each condition, in duplicate. D Melanoma secretome is altered by lysosome perinuclear clustering. Differential quantitative mass spectrometry analysis of proteins secreted to the cell medium by WM983B (FKBP-FRB) cells in the absence or presence of 5 nM Rapalog. Proteins known to be lysosome-associated are labeled with their name. Magenta = proteins upregulated in cells with spread lysosomes, blue = proteins upregulated in cells with clustered lysosomes (p < 0.05). Differential analysis was done using Limma moderated t test with adaptive Benjamini-Hochberg procedure to adjust the p values. E–I Rapalog-induced perinuclear clustering inhibits lysosome exocytosis. E Lysosome secretion was assessed by TIRF microscopy using VAMP7-pHluorin probe, which is quenched in lysosome acidic environment and brightly fluorescent once exposed to the alkaline pH of the extracellular space. F Representative images of co-cultured WM983B-LAMP1-mCherry cells expressing either single heterodimerizing domain (FKBP only, magenta) or both the domains (FKBP-FRB, blue) treated with 25 nM Rapalog, TIRF microscopy. G TIRF movie was divided into three time-segments (black: 0–25 s, green: 25–50 s, dark red: 50–75 s) and displayed as maximum projection showing the number of events per each time-segment. Two representative examples: ROI 1= spread lysosomes, ROI 2= clustered lysosomes. H, I Quantification of the TIRF movie. H Number of lysosome secretion events in 1 min, one dot= 1 cell. I Event duration, one dot= 1 event, n = 5 cells. Events per cell (Mean ± SD): spread = 27.80 ± 7.19, clustered = 14.40 ± 8.23, p value = 0.0397. Event duration (Mean ± SD): spread = 2.46 ± 2.36, clustered = 2.56 ± 2.20 s, p value = 0.3518. Mann–Whitney test, two-tailed with Dunn’s multiple comparison post hoc test. Source data are provided as a Source Data file.