Extended Data Fig. 9: MTM1 controls lysosomal PI3P and PI(3,5)P₂ through ER–lysosome membrane contact sites.

a,b, Representative confocal images showing colocalization of PI3P (GST–HRS probe) and PI(3,5)P₂ (GST–SnxA probe) with lysosomes (Lamp2) in MTM1-KO^Cas9^ myotubes expressing lysosome-targeted (TMEM192–MTM1) or ER-targeted (MOSPD2–MTM1) constructs, either wild-type (FL) or phosphatase-dead (C375S). c, Quantification of PI3P- and PI(3,5)P₂-positive lysosomes from a and b. Data are shown as mean ± s.d., n = 35 myotubes per condition from three biologically independent experiments. One-way ANOVA followed by Dunnett’s multiple-comparisons test. Scale bar, 20 µm. d, Representative confocal images showing ER–lysosome contacts visualized by lysosomal TMEM192–3×HA and ER marker Nogo in CTRL and MTM1-KO^cas9 myotubes. The number of ER–lysosome contacts per region of interest (ROI) was quantified. Data are shown as mean ± s.d., n = 123 ROIs per condition from three biologically independent experiments. Two-sided Mann–Whitney test. Scale bar, 100 µm. e, Quantification of ER–lysosome contact sites using proximity ligation assay (PLA) with anti-HA (lysosome) and anti-Nogo (ER) antibodies. Data are shown as mean ± s.d., n = 16 myotube areas per condition from three biologically independent experiments. One-way ANOVA followed by Dunnett’s multiple-comparisons test. Scale bar, 20 µm. f, MTM1 interacts with mTOR and ER–lysosome contact site proteins (VAPA/B and OSBP). Representative immunoblots from co-immunoprecipitation assays (three biologically independent experiments). MTM1 association with mTOR via ER–lysosome contacts are altered upon ER stress induction (24 h tunicamycin treatment) or OSW1 treatment. g, MTM1-KO^cas9 myotubes exhibit sustained ER stress during differentiation, particularly at the myotube stage. Representative immunoblots showing activation of unfolded protein response (UPR) pathways, including the p-PERK–p-eIF2α and p-IRE1α axes (two biologically independent experiments). h, Loss of MTM1 or its phosphoinositide phosphatase activity disrupts ER stress–mediated repression of mTORC1 signaling in myotubes. Representative immunoblots of MTM1 and mTORC1 activity (pS6K/S6K) in CTRL, MTM1-KO^cas9, and MTM1-KO^cas9 myotubes re-expressing wild-type (FL) or phosphatase-dead (C375S) MTM1, compared with empty vector (EV). Data are shown as mean ± s.d. from three biologically independent experiments. Two-sided Mann–Whitney test. Protein molecular weights in f–h are indicated in kDa.

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