Extended Data Fig. 8: In parallel with ORP9/10/11-mediated PS transport, OSBP drives lysosomal cholesterol transfer as an auxiliary mechanism for rapid lysosomal repair.
From: A phosphoinositide signalling pathway mediates rapid lysosomal repair
a, Rapid lysosomal repair in ORP-QKO cells was only rescued by re-expressing wild type ORP9/11 but not their mutants defective in PS-transport. Cells stably expressing EGFP-Galectin3 and indicated mCherry-ORPs were treated with 1 mM LLOME for 60 min to continuously damage lysosomes. Images were taken using live cells. See image quantification in Fig. 3h. b, Reconstitution of OSBP alone in ORP-QKO cells fully rescues ER tethering to damaged lysosomes. ORP-QKO cells stably expressing EGFP-VAPA and the indicated mCherry-OSBP proteins were treated with LLOME for 10 min before being fixed for confocal microscopy. Similar to ORP mutations in Extended Data Fig. 7k, l, OSBP-2RE loses lysosomal recruitment; OSBP-HHAA does not perform PtdIns4P/cholesterol exchanges between membranes, despite its strong lysosomal recruitment. c, OSBP chimeric proteins rescues ER tethering to damaged lysosomes in ORP-QKO cells. See Fig. 3i for the details of the chimeric proteins. OSBP-Kes1 is constructed similarly to OSBP-Osh6. Cells stably expressing EGFP-VAPA and mCherry-OSBP-X chimeric proteins were stimulated with LLOME for 10 min and then fixed for the staining of endogenous LAMP1. Note, all cells expressing mCherry chimeric proteins showed extensive EGFP-VAPA recruitment to lysosomes, with three-channel colocalization. d, Rapid lysosomal repair in ORP-QKO cells was rescued by re-expressing the indicated chimeric proteins but not the OSBP-Osh6-HHAA mutant defective in PtdIns4P/PS counter transport. Cells stably expressing EGFP-Galectin3 and indicated chimeric proteins were treated as in (a). See image quantification in Fig. 3j. e, LLOME triggers PI4K2A- and OSBP-dependent lysosomal transport of cholesterol. U2OS cells with indicated genetic modifications were stimulated with LLOME for 60 min and then fixed for the staining of endogenous IST1. KDAA is a kinase dead mutant of PI4K2A. 2RE is a mutant of OSBP that is no longer recruited to damaged lysosomes due to loss of PtdIns4P binding to its PH ___domain. f, The colocalization of the cholesterol probe GFP-D4H and IST1 in (e) was quantified. Data show mean ± sem of Pearson’s correlation coefficient; n = 25 cells over 3 trials for each condition. Note that the PS transporters ORP9/11 cannot rescue lysosomal cholesterol accumulation. g, Reconstitution of wild type OSBP, but not its cholesterol transport defective mutants, appears to rescue rapid lysosomal repair in ORP-QKO cells. The same OSBP mutants in (e) and (f) were tested here. Cells stably expressing EGFP-Galectin3 and mCherry-OSBP were continuously treated with 1 mM LLOME and the EGFP-Galectin3 puncta in live cells were analyzed in the right panel. About 50–100 random cells were quantified for each condition. Mean ± sem; n = 3. h, Similar to OSBP-Osh6 in panel c, d, OSBP-Kes1 also rescues rapid lysosomal repair, whereas the cholesterol transport defective HHAA mutant does not. Left, cells stably expressing EGFP-Galectin3 and mCherry-OSBP-Kes1 were continuously treated with 1 mM LLOME and the EGFP-Galectin3 puncta were analyzed with live cells. Right, quantification of the Galectin3 intensities above threshold. About 50–100 random cells were quantified for each condition. Mean ± sem; n = 3. DAPI stains the nuclei. Bar, 10 μm. NS, not significant. Source data for graphs in this Figure are provided.
