Fig. 6: Hydrolysis of cytosolic SM promotes lysosomal repair in ESCRT-compromised cells.
From: Ca2+-activated sphingomyelin scrambling and turnover mediate ESCRT-independent lysosomal repair
a Bacterial SMase (bSMase, magenta) was fused to the C-terminus of lysosomal membrane protein LAMP1, enabling an efficient metabolic turnover of SM translocated to the cytosolic surface of LLOMe-damaged lysosomes. b Time-lapse images of HeLa cells co-expressing GFP/V5-tagged LAMP1-bSMase or LAMP1-bSMasedead (magenta) and mKate-tagged EqtSM (green) treated with 1 mM LLOMe for the indicated time. Scale bar, 10 µm. c Time-course plotting EqtSM-positive puncta per 100 μm2 cell area in cells treated as in (b). Data are means ± SD. n = 14 cells per condition over two independent experiments. d Time-course plotting LysoTracker-positive puncta in HeLa cells co-expressing GFP/V5-tagged LAMP1-bSMase or LAMP1-bSMasedead and mKate-tagged EqtSM during and after a 2-min pulse of GPN, normalized to the initial number of puncta. Data are means ± SD. n = 16 cells for LAMP1-bSMase and 20 cells for LAMP1-bSMasedead over three independent experiments. P values were calculated by unpaired two-tailed t test. e Time-course plotting LysoTracker-positive puncta in HeLa cells pre-treated with siRNAs targeting ALIX/TSG101 (72 h) and expressing LAMP1-bSMase or LAMP1-bSMasedead during and after a 2-min pulse of GPN. Data are means ± SD. n = 23 cells for siALIX/TSG101 + LAMP1-bSMase and 29 cells for siALIX/TSG101 + LAMP1-bSMasedead over three independent experiments. P values were calculated by unpaired two-tailed t test.
