Fig 2: LIMP-2 mediates the endocytosis of liposomes in cells. a Immunocytochemistry of LIMP-2 in MDCK monolayer culture. Endogenous LIMP-2 proteins (green, nuclear content blue) were found not only inside the cell but also enriched on the cell surface that formed borders between cells. b, c Time-lapse images of a MDCK cell transfected with a LIMP-2-HA construct (enlarged view in insets; time stamp shown in yellow). LIMP-2-HA located on the cell surface was visualized by surface labeling of mouse anti-HA antibody and anti-mouse Fab fragment conjugated to AlexaFluor488. A LIMP-2 containing endocytic vesicle (arrowhead) formed within 14 s was detected in c but not in b. d Western blotting with anti-LIMP-2 antibody. e–g Surface labeling of LIMP-2 in WT or KO MEF. h Quantification of surface labeling with LIMP-2 or IgG isotype antibody. i–k 30 min uptake of PC or PS liposomes in MEF. i WT MEF with few intracellular PC particles (arrow). j WT MEF with intracellular PS particles (green). k LIMP-2 KO MEF with few intracellular PS particles (arrow). l Quantification of liposome uptake in WT and LIMP-2 KO MEF. m, n Dyngo-2a (100 μM, 15 min) blocks both PS liposome (green) uptake and transferrin (red) endocytosis. o Nystatin (100 μM, 15 min) inhibits the endocytosis of PS liposome (green) but not transferrin (red). p Quantification of PS fluorescence intensity in WT MEF treated with endocytosis inhibitor Dyngo-2a or nystatin. q–t Endocytosed PS vesicles (green) were partially localized to lysosomes (lysotracker, red) after 60 min of uptake. t Enlarged view of the square in s. Results are expressed as means ± SE. *, t test P = 3.8E−4; **, t test P < 2.2E−14. Blue, DAPI. Scale bar: a–k, 12 µm; m–o, 25 µm; q–t, 12 µm

Fig 3: Structure of lipids bound LIMP-2 dimer. a Lipid-bound LIMP-2 adopts a dimeric structure. The twofold axis of the dimer is perpendicular to the plane of the figure at the center. Protein ribbons are colored green and cyan for different subunits. Endogenous ligands, recognized as phosphatidylcholine (PC) and cholesterol (CLR), are shown in stick model, with carbon atoms colored magenta, oxygen red, nitrogen blue, and phosphorus orange. Carbon atoms in glycan moieties are colored yellow. Image prepared using pymol60. The N and C termini and the helical bundles are labeled. b Zoomed-in view of one of the hydrophobic tunnels (calculated using program caver61) in the LIMP-2 dimer; the entrances/exit to the hydrophobic tunnel are indicated by arrows. Bound lipids with their relative orientation to the cell membrane, and the helical bundle of the second subunit are indicated. Exit and entrance 2 are the proximal and distal openings highlighted in Fig. 2d. c PC and CLR molecules in the hydrophobic tunnel are shown embedded in 2mFo-DFc composite omit map (blue mesh) contoured at 1.0σ. d–f Surface electrostatic potential presentations of the LIMP-2 dimer. Color bar indicates blue positive and red negative charges, with neutral shown in white. Bound ligands in view are shown in yellow spheres for CLR, and cyan for PC, and cleft bound PC is indicated. Surface cationic patches are enclosed in dashed boxes and residues forming these patches are labeled. d The putative distal face of the LIMP-2 dimer. e Side view of the dimer. Orientation of the luminal domain relative to the cell membrane is indicated. f The putative cell membrane proximal face of the LIMP-2 dimer. Notice that CLR molecules are visible through openings on both the distal (d) and the proximal (f) faces. a and f, and c and e have the same views, respectively. For clarity, glycans are omitted in d–f

Fig 4: Proposed mechanism of lipids trafficking by LIMP-2. Lipids (green spheres) binding of LIMP-2 monomer promote dimer formation. In the LIMP-2 dimer the luminal domains are closer to the cell membrane, with the proximal face of LIMP-2 luminal domain able to interact with the cell membrane while its distal face able to interact with extracellular lipid vesicles, respectively, via their cationic patches. Within the tethers of the protein on the luminal side, between the ends of the structurally defined polypeptides and the transmembrane helices, there are sufficient residues that are flexible and lack defined secondary structure to enable this dimerization on cell surface: 24 residues after the last residue of the N-terminal transmembrane helix and 6 before the beginning of the C-terminal transmembrane helix. If fully extended, these tethers can stretch at least 20 Å. Notice the channels in LIMP-2 ectodomain dimer connect bound lipid vesicles and cell membrane, with openings near cationic patches in both faces that may facilitate lipids exchange between vesicles and outer leaflet of the cell membrane. LIMP-2 bound lipid vesicles can be trafficked into cell cytosol via endocytosis

Fig 5: Detection of LIMP-2 dimer in live cells using bimolecular fluorescence complementation method. a Schematic illustration of constructs designed for bimolecular fluorescence complementation in examining dimerization of LIMP-2. b High-resolution confocal images of Hela cells expressing LIMP-2 (WT, top, or H150A/F151D, bottom) fused to the N-terminal and C-terminal fragments of Venus fluorescent protein along with Lamp1-RFP co-transfected as a lysosome colocalization marker. c Western blot showing expression levels of the LIMP-2 WT and H150A/F151D variants using anti-myc and anti-HA antibodies. d Quantification of the ratio of mean Venus fluorescence intensity relative to ER-localized mCherry for cells transfected with LIMP-2 (WT and H150A/F151D) fused to the N-terminal and C-terminal fragments of Venus fluorescent protein fluorescence. Data is expressed as mean ± SEM, ~60 cells each experiment, n = 3, ****P < 0.0001, t test. Scale bar 20 µm