Journal: bioRxiv
Article Title: Damage-sensing recruitment of a lipid phosphatase couples lysosomal membrane repair to proteostatic adaptation
doi: 10.64898/2026.04.04.716461
Figure Lengend Snippet: (a) Representative confocal images of U2OS cells co-expressing YFP-MTMR14 WT with mCherry-MTMR14 WT, mCherry-MTMR14 ΔN-term, mCherry-MTMR14 ΔC-term, mCherry-MTMR14 Δ620-650, or mCherry-MTMR14 Δ586-650 after LLOMe treatment. Scale bar, 10 µm. Lower panel: cartoons illustrating the deletion mutants with N-terminus in yellow, C-terminus in blue, and phosphatase domain in pink. (b) Left: representative confocal images of U2OS cells stained with Fluo4-AM after 40 minutes treatment with control (DMSO), LLOMe, 25 µM MSDH, 200 nM GPN, 5 µg/ml BAC, or 5 µM Ionomycin. Scale bar, 20 µm. Right: Fluo4-AM intensity quantification in images. one-way ANOVA, control (n = 17 fields of view), LLOMe (n = 18 fields of view), MSDH (n = 14 fields of view), GPN (n = 20 fields of view), BAC (n = 18 fields of view), Ionomycin (n = 17 fields of view), one field of view containing 10-20 cells, with a size of 1664 × 1664 µm. (c) Representative confocal images of U2OS cells in DMEM/FBS growth medium containing Ca + and Ca + depletion medium (2 mM EDTA in DMEM/FBS + 100 µM BAPTA-AM) stained with Fluo4-AM after 35 minutes treatment with LLOMe. Scale bar, 10 µm. (d) Representative confocal images of U2OS cells expressing YFP-MTMR14 treated with control (DMSO), LLOMe, 25 µM MSDH, 200 nM GPN, 5 µg/ml BAC, or 5 µM Ionomycin. Scale bar, 10µm. (e) Coomassie blue staining of purified mCherry-MTMR14. (f) Total lipids were extracted from WT or SMS1/2 DKO U2OS cells metabolically labeled with a clickable sphingosine analogue, click-reacted with 3-azido-7-hydroxycoumarin, separated by TLC, and analyzed by fluorescence detection. cCer, coumarin-labeled ceramide; cPC, coumarin-labeled phosphatidylcholine; cSM, coumarin-labeled sphingomyelin. (g) Levels of sphingomyelin (SM; left) and hexosylceramide (HexCer; right) in total lipid extracts from wildtype (WT) and SMS1/2 DKO cells determined by LC-MS/MS and expressed in pmol per 100 pmol of total phospholipid analyzed. t test (n = 4 independent experiments). (h) Left: number of eGFP-EqtSM puncta/cell area quantified from confocal images of U2OS WT and SMS-DKO cells after 1 h LLOMe treatment, t test (n = 3 independent experiments, total number of cells is 102 for WT and 125 for SMS-DKO). Right: number of MTMR14 puncta/cell area in confocal images of U2OS WT and SMS-DKO cells after 1 h LLOMe treatment, t test (n = 3 independent experiments, total number of cells is 117 for WT and 113 for SMS-DKO). Statistical analyses were performed using GraphPad Prism. Two-tailed unpaired t-test, paired t-test or one-sample t-tests were conducted using column statistics to compare the sample means to a hypothetical value of 1 or one-way ANOVA with Tukey’s multiple comparisons test. All bar graphs represent mean ± SD unless otherwise stated. ***p < 0.001, **p < 0.01, *p < 0.05.
Article Snippet: SUVs were prepared from synthetic lipid mixtures containing either 60 mol% dioleoyl-phosphatidylcholine (DOPC; Avanti Polar Lipids, #850375), 20 mol% cholesterol (Avanti Polar Lipids, #700000), and 20 mol% porcine brain sphingomyelin (SM; Avanti Polar Lipids, #860062), or 80 mol% DOPC and 20 mol% cholesterol.
Techniques: Expressing, Staining, Control, Purification, Metabolic Labelling, Labeling, Fluorescence, Liquid Chromatography with Mass Spectroscopy, Two Tailed Test
![(A) Schematic illustrating the time course of lysosomal membrane damage-induced changes in ESCRTs, lysophagy, PI(4)P, PI(3)P, canonical mTORC1 signalling, and protein translation. Curves were modelled according to experimental data shown in [PI(3)P], (mTORC1), and (Lysophagy), [PI(4)P], (translation), (ESCRT). PI(3)P, mTORC1, and translations curves were normalized between 0 and 1 to allow for comparison of the kinetics. The ESCRT curve represents an average of CHMP4B, IST1, and Alix data . (b) Volcano plot illustrating the enrichment of proteasome subunits and ubiquitylation machinery on lysosomes immunoprecipitated from control or 1 h 1mM LLOMe-treated HEK293-TMEM192-3xHA cells determined by quantitative proteomics. (c) Representative confocal images of HMC3 and U2OS cells co-stained with DAPI and PSMB5 at indicated time points after 1mM LLOMe treatment. Scale bar, 10 µm. (d) Representative confocal images of 3 h 1mM LLOMe-treated HMC3 cells co-stained for the proteasome subunit PSBM5, Lamp1, and DAPI. Scale bar, 2 µm. (e) Left: representative immunoblot of U2OS-mScarlet-MTMR14 KI cells treated with DMSO control, 1 h 1mM LLOMe, or 5 h 1mM LLOMe. Right: densitometric quantification of mScarlet-MTMR14 relative to GAPDH in control, 1 h and 5 h LLOMe treatment from immunoblots as shown in the left panel. one-way ANOVA (n = 3 independent experiments). (f) Immunoblot of global ubiquitinated protein probed by FK2 antibodies in immunoprecipitation fractions collected from control or 2 h 1mM LLOMe-treated U2OS-mScarlet-MTMR14 KI cells with RFP-trap beads to enrich for mScarlet-MTMR14. (g) Representative confocal images of 2 h LLOMe-treated U2OS cells co-expressing mCherry-MTMR14 and eGFP-Ubiqutin. Scale bar, 10 µm. (h) Left: representative immunoblot of U2OS-mScarlet-MTMR14 KI cells treated with control (DMSO) or 5 h LLOMe with or without 3 µM TAK-243 co-incubation. Right: densitometric quantification of mScarlet-MTMR14 intensity/ ß-actin from immunoblots as shown in the left panel. t test (n = 4 independent experiments). Dotted line denotes mScarlet-MTMR14 level in controls set to 1. (i) Left: representative confocal live cell images of U2OS cells expressing GFP-2xFYVE imaged after treatment with 5 h LLOMe with or without 3 µM TAK-243. Scale bar, 10 µm. Right: number of PI(3)P puncta/cell area quantified in cells as shown in the left panel, fold change over control. t test (n = 3 independent experiments, total number of cells is 108 in LLOMe and 115 in LLOMe + TAK-243 conditions. (j) Quantification of mean LysoTracker intensity/field of view in U2OS cells treated with LLOMe for 5 h with or without 3 µM TAK-243 co-incubation, fold change over control. t test (n = 3 independent experiments, each data point represents 30 fields of view, one field of view containing 35-50 cells, with a size of 3328 × 3328 µm). (k) Left: representative immunoblot of U2OS-mScarlet-MTMR14 KI cells treated with DMSO control, 5 h LLOMe, or 5 h LLOMe with 200 nM Baf A1 and 2 µM Bortezomib. Right: densitometric quantification of mScarlet-MTMR14 relative to GAPDH from immunoblots as shown on the left panel. one-way ANOVA (n = 3 independent experiments). (l) Schematic illustration of MTMR14 recruitment to damaged lysosomes via calcium and sphingomyelin leading to local hydrolysis of PI(3)P during early lysosome damage. Upon prolonged lysosome damage, MTMR14 becomes ubiquitinated, and PI(3)P levels are restored. Statistical analyses were performed using GraphPad Prism. Two-tailed unpaired t-test, paired t-test or one-sample t-tests were conducted using column statistics to compare the sample means to a hypothetical value of 1 or one-way ANOVA with Tukey’s multiple comparisons test. All bar graphs represent mean ± SD unless otherwise stated. ***p < 0.001, **p < 0.01, *p < 0.05. See also .](https://bio-rxiv-images-cdn.bioz.com/dois_ending_with_61/10__64898_slash_2026__04__04__716461/10__64898_slash_2026__04__04__716461___F7.large.jpg)
