Fig. 2

Lipoprotein-derived cholesterol can translocate through the LIMP-2 ectodomain. a Confocal microscopy of HeLa cells transiently expressing C-terminally GFP-tagged wild-type (WT.GFP) or chimeric LIMP-2 (cmr.GFP). Cells were labeled with dextran to visualize late endosomal organelles. Cell boundaries are indicated by dotted lines. b Plasmalemmal expression of chimeric C-terminally GFP-tagged LIMP-2 (LIMP-2.cmr.GFP) in CHO cells was detected using Western blotting of biotinylated cell surface proteins after immunoprecipitation using streptavidin-beads. LIMP-2.GFP was used as a negative control. β-actin was used as a loading control for the input samples. c Confocal microscopy and quantification (d) of DiI-LDL binding to LIMP-2.cmr.GFP in CHO cells. Cells transiently expressing LIMP-2.cmr.GFP were incubated with DiI-LDL at pH 5 (upper panel) and pH 7 (lower panel). pH 5: n = 78, pH 7: n = 9 cells from two experiments. t-test (****P < 0.0001). e Confocal microscopy of CHO cells transiently expressing LIMP-2.cmr.mCherry and incubated with doubly labeled LDL, AF647-LDL(BC), after binding (upper panel) and washing with HBSS (lower panel). Note that AF647 was removed upon washing but BC was not. f Representation of the tunnel-blocking mutations of the human LIMP-2 luminal tunnel, shown as a slice through the body of the protein (sliced solid shown in orange). Residues A379 and V415, which point towards the cavity of the tunnel, are shown in blue. g, h Confocal microscopy (g) and quantification (h) of AF647-LDL(BC)-derived BC transport in CHO cells expressing LIMP-2.cmr.mCherry or LIMP-2.cmr.A379W/V415W.mCherrry proteins. Cells were pre-incubated with doubly labeled LDL (AF647-LDL(BC)) and washed with HBSS. The ratio of green (BC) over far-red (AF647-labeled LDL) fluorescence, measured in ZEN Lite (Zeiss), was used to estimate uptake (h); LIMP-2.cmr.mCherry n = 59, LIMP-2.cmr.A379W/V415W.mCherrry n = 50 cells from 3 experiments). i Quantification of BC uptake from SapA(BC) by CHO cells transiently expressing C-terminally mCherry-tagged-wild type (LIMP-2.cmr.mCherry) or tunnel-blocking mutant LIMP-2 chimera (LIMP-2.cmr.A379W/V415W.mCherrry). Also presented is the total fluorescence intensity of non-transfected cells. The total cellular fluorescence intensity of BC was measured using Volocity (LIMP-2.cmr.mCherry n = 67, LIMP-2.cmr.A379W/V415W.mCherrry n = 90, non-transfected cells n = 152 cells from 3 experiments). BC data in h, and i are the mean ± SEM of triplicate samples (****P < 0.0001, unpaired two-tailed Student’s t-test). a.u., arbitrary units. Scale bars, 10 μm. Source data are provided as a Source Data file