Fig. 6: MTM1 regulates lysosomal phosphoinositides and mTORC1 through ER–lysosome MCS.
From: Lysosomal phosphoinositide turnover acts upstream of RagGTPase–mTORC1 and controls muscle growth
a, The strategy to target MTM1 to lysosomes using the TMEM192 Lyso-Tag. b, Representative confocal images showing colocalization of lysosome-targeted MTM1 (FLAG) with Lamp2 and mTOR in MTM1-KOCas9 myotubes expressing wild-type (FL) or phosphatase-dead (C375S) MTM1. Data are shown as mean ± s.d., n = 10 (upper) and n = 35 (lower) myotube areas per condition from three biologically independent experiments. Two-sided Mann–Whitney test. Scale bar, 100 µm. c, Representative immunoblots showing the effects of lysosome-targeted MTM1-FL or MTM1-C375S on mTORC1 activity (p-S6K) in MTM1-KOCas9 myotubes from three biologically independent experiments. d, Representative images of MHC-stained myotubes expressing lysosome-targeted MTM1-FL or MTM1-C375S, with the quantification of fusion index (n = 20) and myotube area (n = 22). Data are shown as mean ± s.d. from three biologically independent experiments. One-way ANOVA with Dunnett’s multiple-comparisons test. Scale bar, 100 µm. e, The strategy to target MTM1 (FL or C375S) to ER–lysosome MCS using the MOSPD2 transmembrane domain. f, Representative confocal images showing ER localization of MOSPD2–MTM1 and contacts with lysosomes (Lamp2). ER–lysosome contacts per region of interest were quantified. Data are shown as mean ± s.d., n = 93 myotubes per condition from three biologically independent experiments. Two-sided Mann–Whitney test. Scale bar, 100 µm. g, Representative immunoblots showing effects of ER-targeted MTM1-FL or MTM1-C375S on mTORC1 signalling (p-S6K) in MTM1-KOCas9 myotubes from three biologically independent experiments. h, Representative images of MHC-stained myotubes expressing ER-targeted MTM1-FL or MTM1-C375S, with quantification of fusion index and myotube area. Data are shown as mean ± s.d., n = 21 myotubes per condition from three biologically independent experiments. One-way ANOVA with Dunnett’s multiple-comparisons test. Scale bar, 100 µm. i,j, Lyso-IP immunoblots (i) and quantifications (j) showing that disruption of ER–lysosome contacts by OSW1 in CTRL myotubes (5–6 days of differentiation) retains lysosomal LAMTOR–Rag complexes, enhances mTORC1 activity and increases lysosomal PI3P and PI(3,5)P2, whereas OSW1 has no obvious effects in MTM1-KOCas9 myotubes. Data are shown as mean ± s.d. from three biologically independent experiments. Two-sided Mann–Whitney test. k, Representative MHC images showing effects of OSW1 on the differentiation of CTRL and MTM1-KOCas9 myotubes. Data are shown as mean ± s.d., n = 34 (fusion index) and n = 40 (myotube area) from three biologically independent experiments. One-way ANOVA with Dunnett’s multiple-comparisons test. Scale bar, 100 µm. For c, g and i, protein molecular weight is indicated in kDa. Illustrations in a and e created in BioRender; Karim, H. https://biorender.com/n6ucxpz (2026).
