Fig. 2: TRPML1 activation induces rapid CASM-dependent recruitment of DMXL1 to lysosomes.
From: DMXL1 promotes recruitment of V1-ATPase to lysosomes upon TRPML1 activation
a,b, Live-cell imaging of WT (a) or ATG16L1-KO (b) U2OS cells expressing DMXL1–mNG and mScarlet–LAMP1 to mark lysosomes upon treatment with MLSA5. Images taken at 2-min intervals for 10 min are shown. c, As in a, but after pretreatment with BafA1 to block CASM. d, Live-cell imaging of WT U2OS cells stably expressing DMXL1–mNG and mScarlet–LAMP1 to mark lysosomes, upon treatment with the mTOR inhibitor, Torin1. Images taken at 2-min intervals for 10 min are shown. e, Fold change in DMXL1–mNG mean fluorescence intensity over 20 min. Quantification was performed on cells from three biological replicates: WT, n = 21; ATG16L1 KO, n = 17; pretreatment BafA1, n = 12; Torin1, n = 16; pretreatment BFA, n = 11. Data are presented as mean values ± s.d. Two-way repeated measures ANOVA with the Geisser–Greenhouse correction followed by Dunnett’s multiple comparison test was used to assess significance. The indicated statistical significance is for the 20-min timepoint. ****P ≤ 0.0001; n.s., not significant. Scale bar, 10 µm; inset scale bar, 3 µm. f, Immunoblots from ATG16L1-KO U2OS cells reconstituted with either WT ATG16L1 or ATG16L1-K490A, with or without MLSA5 (2 h), were probed with the indicated antibodies. α-GAPDH was used as a loading control. The blot is representative of three independent experiments. g, The indicated cell lines, with or without MLSA5 (4 h), were fixed before confocal microscopy to detect colocalization of DMXL1–mNG and LAMP1. Scale bar, 10 µm; inset scale bar, 3 µm. h, Quantification of colocalization between α-LAMP1 for lysosomes and DMXL1–mNG using Mander’s colocalization coefficient for data in g. Quantification was performed on the number of cells indicated on the plot, from three independent experiments. Boxes show the 25th to 75th percentiles, whiskers extend from minimum to maximum values and the line represents the median. Statistical significance for each cell line was assessed by unpaired two-sided Student’s t-test. ****P ≤ 0.0001. Also see Extended Data Figures 3 and 4 and Supplementary Video 1.
