Fig. 2. PtdIns4P drives ORP-tethered ER–lysosomal contacts for rapid membrane repair.
a, ORP family members are enriched on damaged lysosomes. Note that ORP10 is absent from S20 (supernatant after centrifugation at 20,000 g) (arrow head). Asterisk indicates a nonspecific band. b, Endogenous ORP9 is recruited to LAMP1-positive lysosomes upon brief LLOME treatment in U2OS cells. c, Punctate intensities of endogenous ORP9 and ORP11 before and after LLOME treatment. More than 30 random cells were quantified for each condition. Data are mean ± s.e.m.; n = 4 trials. See fluorescence images in Extended Data Fig. 5m. d, LLOME induces extensive wrapping of endogenous LAMP1 by eGFP–VAPA in U2OS cells. Right, schematic illustration of the process. e, Percentage of U2OS cells with more than five lysosomes extensively wrapped by eGFP–VAPA. More than 100 cells were counted for each condition. Data are mean ± s.e.m.; n = 3 per condition. See fluorescence images in Extended Data Fig. 6a. f, Representative microscopy and quantification of ER–lysosome wrapping in various ORP-knockout cells. More than 100 cells were counted for each condition. Data are mean ± s.e.m.; n = 3. See more details in Extended Data Fig. 6c–f. g, ORP-QKO cells stably expressing eGFP–VAPA and mCherry–ORP proteins were stimulated with LLOME for 10 min and the percentages of cells showing VAPA clusters on ORP proteins were quantified. Data are mean ± s.e.m.; n = 3 per condition. See more details in Extended Data Fig. 6j–m. ORP9/11 (FYAA/WT), co-expression of mCherry–ORP9(FYAA) and mCherry–ORP11(WT). h, Representative images and quantification of eGFP–galectin-3 puncta in wild-type and ORP-QKO cells. More than 100 cells were quantified per condition. Data are mean ± s.e.m.; n = 3 per condition. i, Quantification of eGFP–galectin-3 puncta in ORP-QKO cells stably expressing eGFP–galectin-3 and mCherry–ORP proteins. Approximately 50–100 cells were quantified for each condition. Data are mean ± s.e.m.; n = 5 per condition. Images are presented in Extended Data Fig. 6o. j, Schematic illustration of PtdIns4P-stimulated, ORP-mediated membrane tethering between the ER and damaged lysosomes. ORP9 carries a stronger FFAT motif than ORP10 and ORP11 and thus provides stronger ER tethering. Unpaired, two-tailed t-tests. NS, not significant. Scale bars, 10 μm.