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) Schematic illustration of putative roles of PI(4)P and PI(3)P in lysosome repair and removal pathways. ESCRT, endosomal sorting complex required for transport. PITT, phosphoinositide-initiated membrane tethering and lipid transport pathway. ER, endoplasmic reticulum. mTORC1, mechanistic target of rapamycin complex 1. (b) Biphasic PI(3)P dynamics in 0.5mM LLOMe-treated cells. The same HeLa cell expressing eGFP-2xFYVE was imaged before and after different durations of LLOMe treatment. Scale bar, 10 µm. (c) Left: quantification of (b), number of PI(3)P puncta/cell area before and after 30 minutes of 0.5mM LLOMe was compared by t test (n = 39 cells). Right: time course of PI(3)P change in 0.5mM LLOMe treated HeLa cells expressing eGFP-2xFYVE. Quantification of the number of PI(3)P puncta/cell area, fold change over control. (n = 39 cells). Data are mean ± s.e.m. (d) LLOMe decreases PI(3)P on lysosomes. Left: magnification of the perinuclear region of a U2OS cell co-expressing eGFP-2xFYVE and mCherry-LAMP1 imaged after different durations of 0.5mM LLOMe treatment. Capital letters indicate same vesicles before and after 8 minutes of LLOMe treatment. Scale bar, 5 µm. Middle: time-resolved changes of GFP-2xFYVE on the same mCherry-LAMP1 vesicles as indicated in the left panels by capital letters. Right: Representative time course (mean±s.e.m.) of LMP-induced PI(3)P decline on lysosomes from ten cells. See also and for quantifications. (e) Volcano plot illustrating the enrichment of phosphoinositide kinases, phosphatases, phosphoinositide-binding proteins, and lipid-transport proteins in Lyso-IP samples from LLOMe-treated cells. Lysosomes were immunoprecipitated from control or 30 minutes 1mM LLOMe-treated HEK293 cells expressing TMEM192-3xHA and subjected to quantitative MS/MS analysis. (f) Representative confocal images of mScarlet-MTMR14 CRISPR-Cas9 knockin U2OS cells treated with control (DMSO) or 15 minutes 1mM LLOMe. Cells were co-stained with antibodies specific for mScarlet (MTMR14) and LAMP1. Scale bar, 10 µm. (g) Representative confocal images of HeLa cells co-expressing YFP-MTMR14 with mCherry-Galectin3 imaged live before and after 5 and 15 minutes of 1mM LLOMe treatment. Scale bar, 10 µm. (h) Quantification of the number of MTMR14 puncta/cell from control and 1 h 1mM LLOMe treated HeLa cells. t test (n = 29 cells). (i) Expression of EGFP-Tau P301L recruits MTMR14 to lysosomes. Shown are magnifications of confocal images of HeLa cells co-expressing EGFP-Tau P301L with mCherry-MTMR14 and stained with antibodies against Lamp2a. Scale bar, 2 µm. (j) Left: representative confocal live cell images of wild type and MTMR14 KO U2OS cells co-expressing GFP-2xFYVE with mCherry-LAMP1 imaged before and after 20 minutes of 0.5mM LLOMe treatment. Right: quantification of the Pearson’s Coefficient of GFP-2xFYVE and mCherry-LAMP1 from LLOMe treated U2OS WT and MTMR14 KO cells before and after 20 minutes LLOMe treatment. Dotted line denotes Pearson’s Coefficient of GFP-2xFYVE and mCherry-LAMP1 before LLOMe set to 1. Scale bar, 20 µm. t test, n=43 cells in U2OS WT and 41 cells in U2OS MTMR14 KO cells. 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 and .

Article Snippet: At 24 or 48 hours post-transfection, lysosomal integrity was assessed by monitoring Galectin-3 (Gal3) recruitment, a marker of lysosomal membrane damage.

Techniques: Membrane, Expressing, Control, Binding Assay, Immunoprecipitation, Tandem Mass Spectroscopy, CRISPR, Knock-In, Staining, Two Tailed Test

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) Left: eGFP-2xFYVE cytosolic intensity before and after 24 minutes LLOMe treatment in U2OS cells. Right: eGFP-2xFYVE intensity in the cytosol at different times post-induction of LMP by LLOMe. Scale bar, 10 µm. Data represent mean of 12 cytoplasmic ROIs in 12 cells ± s.e.m. See also . (b) Quantification of the Pearson’s Coefficient of GFP-2xFYVE and mCherry-LAMP1 from U2OS cells before and after 23 minutes treatment with control (DMSO), 0.5mM LLOMe and 0.5mM LLOMe plus 5µM VPS34-IN1. Dotted line denotes Pearson’s Coefficient of GFP-2xFYVE and mCherry-LAMP1 before treatment set to 1. One-way ANOVA, n=10 cells analysed for each condition. (c) Left: time-lapse of single lysosome in U2OS cells co-expressing GFP-OSBP-PH and mCherry-LAMP1 imaged after different durations of LLOMe treatment. Scale bar, 2 µm. Right: quantification of the eGFP-OSBP-PH intensity on lysosomes. n = 30 lysosomes quantified from 14 cells. Data are mean ± s.e.m. (d) PI(3,5)P 2 dynamics after LLOMe treatment. Top: representative U2OS cell expressing GFP-SNXA imaged after different durations of LLOMe treatment. Scale bar, 10 µm. Bottom: the number of SNXA puncta/cell area quantified from images as shown in the top panel. Data represent mean of 3 independent experiment ± s.e.m from 33 cells. (e) Enrichment of myotubularin phosphatases in Lyso-IP fractions of 30 minutes 1mM LLOMe-treated over control (DMSO)-treated HEK293-TMEM192-3×HA cells determined by mass spectrometry. (f) Representative U2OS cells expressing GFP-MTM1, GFP-MTMR1, GFP-MTMR2, GFP-MTMR6, GFP-MTMR7, GFP-MTMR8 or YFP-MTMR14 imaged before and after 2 h LLOMe treatment. Scale bar, 10 µm. (g) Quantification of the number of MTMR14 puncta/cell from control and 1 h 1mM LLOMe treated U2OS cells. t test (n = 40 cells). (h) C2C12 cells co-expressing YFP-MTMR14 and mCherry-Galectin3 imaged after 2 h control (DMSO) or LLOMe treatment. Scale bar, 10 µm. (i) BV2 cells co-expressing YFP-MTMR14 and mCherry-Galectin3 imaged after 1 h LLOMe treatment. Scale bar, 10 µm. (j) HMC3 cells co-expressing YFP-MTMR14 and mCherry-Galectin3 imaged before and after LLOMe treatment. Scale bar, 10 µm. (k) Left: representative HeLa cells expressing YFP-MTMR14 imaged after 30 minutes treatment with different concentrations of LLOMe. Scale bar, 10 µm. Right: quantification of MTMR14 foci per cell in images as shown in the top panel. one-way ANOVA (34 cells in control, 41 cells in 100µM LLOMe, 36 cells in 250µM LLOMe, 32 cells in 500µM LLOMe, 30 cells in 1mM LLOMe). 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. All bar graphs represent mean ± SD unless otherwise stated. ***p < 0.001, **p < 0.01, *p < 0.05.

Article Snippet: At 24 or 48 hours post-transfection, lysosomal integrity was assessed by monitoring Galectin-3 (Gal3) recruitment, a marker of lysosomal membrane damage.

Techniques: Control, Expressing, Mass Spectrometry, Two Tailed Test

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 U2OS cell expressing YFP-MTMR14 stained with LysoTracker and imaged before and after 30 minutes 50 µM MSDH treatment. Scale bar, 10 µm. (b) Representative U2OS cell expressing YFP-MTMR14 stained with LysoTracker and imaged before and after 30 minutes 200 nM GPN treatment. Scale bar, 10 µm. (c) Representative U2OS cells expressing YFP-MTMR14 imaged before and after 5 µg/mL BAC, 0.5 mM H 2 O 2 , 0.25 M D-Mannitol, 50% H 2 O, 5 µM CCCP, 100 nM Bafilomycin A1 or 5 µM Nigericin treatment. Scale bar, 10 µm. (d) Left: representative U2OS cells co-expressing GFP-Tau P301L and mCherry-Galectin3 for 24 h or 48 h. Scale bar, 10 µm. Right: quantification of Galectin3 puncta/cell area after 24 h or 48 h GFP-Tau P301L expression in U2OS cells as shown in the left panel. t test (n = 3 independent experiments, total number of cells is 86 in 24 h and 107 in 48 h conditions). (e) Top: immunoblot of U2OS WT and MTMR14 KO #1 cells. Bottom: immunoblot of U2OS WT and MTMR14 KO #2 cells. (f) Left: WT and MTMR14 KO #1 U2OS cells expressing GFP-2xFYVE imaged before and after 1 h 1mM LLOMe treatment. Scale bar, 10 µm. Right: quantification of PI(3)P puncta/cell area in WT and MTMR14 KO #1 U2OS cells as shown in the left panel. t test (n = 4 independent experiments, total number of cells is 230 for WT and 190 for MTMR14 KO). (g) Impaired LMP-induced PI(3)P dynamics in MTMR14 knockout cells. Quantification of GFP 2xFYVE intensity from confocal images of U2OS WT and U2OS MTMR14 KO cells. Cells were treated with LLOMe for the indicated time points, fixed, permeabilized, and stained with purified GFP-2xFYVE as overlay probe. One-way ANOVA (n = 5 independent experiments, each datapoint represents 15 fields of view, one field of view containing 10-20 cells, with a size of 1664 × 1664 µm). Data are mean ± s.e.m. 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. All bar graphs represent mean ± SD unless otherwise stated. ***p < 0.001, **p < 0.01, *p < 0.05.

Article Snippet: At 24 or 48 hours post-transfection, lysosomal integrity was assessed by monitoring Galectin-3 (Gal3) recruitment, a marker of lysosomal membrane damage.

Techniques: Expressing, Staining, Western Blot, Knock-Out, Purification, Two Tailed Test

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) Left: defective lysosomal recovery in MTMR14 KO cells. Representative confocal images of LysoTracker loaded WT and MTMR14 KO clone #1 U2OS cells imaged before LLOMe treatment, after 30 minutes LLOMe treatment, and 4.5 h after washout of LLOMe. Scale bar, 20 µm. Right: quantification of mean LysoTracker intensity/field of view, fold change over control. t test (n = 6 independent experiments, each datapoint represents 10 fields of view, one field of view containing 35-50 cells, with a size of 3328 × 3328 µm). (b) Quantification of mean LysoTracker intensity/field of view after 5 h chronic LLOMe treatment in U2OS MTMR14 KO clone #1 fold change over WT U2OS cells, t test (n = 5 independent experiments, each datapoint represents 10 fields of view, one field of view containing 35-50 cells, with a size of 3328 × 3328 µm). (c) Left: Loss of MTMR14 causes elevated lysosomal membrane damage monitored by mCherry-Galectin3 in U2OS cells co-expressing mutant eGFP-Tau (P301L) for 24 hours. Confocal images of representative cells. Scale bar, 10 µm. Right: Quantification of mCherry-Galectin3 puncta/cell area in U2OS WT and MTMR14 KO cells after co-expressing mutant eGFP-Tau (P301L) for 24 hours. t test (n = 4 independent experiments, total number of cells is 107 for both WT and MTMR14 KO). (d) Quantification of cytotoxicity using LDH assay in 5 h 4mM LLOMe-treated WT and MTMR14 KO U2OS cells. t test (n = 4 independent experiments, each datapoint represents triplicate measurements). (e) MTMR14 regulates PI(4)P generation in response to LMP. Left: representative confocal images of WT and MTMR14 KO U2OS cells stained with DAPI and antibodies against PI(4)P and Lamp2a in control conditions (DMSO) and after 30 minutes LLOMe treatment. Right: quantification of PI(4)P intensity on Lamp2a detections in images as shown in the left panel. t test (n = 4 independent experiments, total number of fields of view is 109 for WT and 114 for MTMR14 KO, one field of view containing 10-15 cells, with a size of 1664 × 1664 µm). (f) Left: representative colored electron micrographs of lysosomes in WT and MTMR14 KO U2OS cells treated with LLOMe, ER is colored in blue and lysosomes in pink. Scale bar, 200 nm. Right: length of lysosome-ER MCS relative to lysosomal perimeter in WT and MTMR14 KO U2OS cells. t test, WT (n = 31 lysosomes), MTMR14 KO (n = 37 lysosomes). 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 .

Article Snippet: At 24 or 48 hours post-transfection, lysosomal integrity was assessed by monitoring Galectin-3 (Gal3) recruitment, a marker of lysosomal membrane damage.

Techniques: Control, Membrane, Expressing, Mutagenesis, Lactate Dehydrogenase Assay, Staining, Two Tailed Test