Fig. 3: AEP deficiency promotes lysosome perinuclear clustering and fusion and impairs lysosomal degradation.

A Lysosome positioning in cells in complete culture medium, revealed by Lysotracker labeling and chasing. WT and AEPKD MDA-MB-231 cells were labeled with 2.5 µM Lysotracker for 1 min, washed with cold PBS, and chased for 30 min. B Lysosome positioning in cells in complete culture medium, revealed by LAMP2 staining. C Elevated lysosome perinuclear clustering and fusion in serum-starved AEPKD cells, revealed by Lysotracker labeling and chasing. D Elevated lysosome perinuclear clustering and fusion in serum-starved AEPKD cells, revealed by LAMP2 staining. Cells were double stained with LAMP2 and AEP and examined by fluorescence microscopy. E Quantification of cells with predominant perinuclear lysosome clustering in (D). Data are mean ± s.d.; n = 150 cells from representative experiments. F Close examination of lysosome perinuclear localization by confocal microscopy in cells treated as in D. Quantification of LAMP2⁺ lysosomes (≥1 µm) (G) and total LAMP2⁺ lysosomes (H) per cell in (F). Data are mean ± s.d.; n = 50 cells from representative experiments. I Elevated lysosome perinuclear clustering observed in AEPKD cells expressing LAMP-1-GFP. J Autophagosomes fused with lysosomes in AEPKD cells. Cells were labeled with Lysotracker, fixed, and stained with LC3 antibody. K Analysis of lysosomes in WT and AEPKD cells by transmission electronic microscopy (TEM). Arrows indicate electron-dense lysosomes in WT cells, arrowheads indicate lysosomes/multivesicular bodies with undegraded contents. L analysis of LAMP-1 and LAMP-2 in WT and AEPKD cells by immunoblotting. Results shown are from representative experiments. ***p < 0.001 by two-tailed t-test.