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doi: 10.1242/10.1242/jcs.00113

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Insect lipoprotein follows a transferrin-like recycling pathway that is mediated by the insect LDL receptor homologue

Department of Biochemical Physiology and Institute of Biomembranes, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands

View larger version (47K): [in a new window]   Fig. 1. iLR expression in CHO cell lines. Membrane proteins were isolated from wild-type CHO, ldlA, ldlA(iLR) and CHO(iLR) cells as described in Materials and Methods. Samples were either denatured for 5 minutes at 95°C in Laemmli buffer (Laemmli, 1970) (A), or dissolved in Laemmli buffer containing 0.1% SDS and directly subjected to SDS-PAGE under non-reducing conditions (B,C,D). Following transfer to PVDF membrane, iLR was detected with anti-iLR antibody (A,B,D) and LDLR with anti-human LDLR antibody (C,D). The molecular mass markers (kDa) are indicated on the left of each panel.

View larger version (36K): [in a new window]   Fig. 2. Receptor-mediated endocytic uptake of fluorescently-labeled lipoproteins by CHO cells. CHO cells were incubated with fluorescently-labeled lipoproteins in incubation medium for 15 minutes at 37°C, fixed with paraformaldehyde and mounted in mowiol (A-F). Single cells were imaged using fluorescence microscopy to visualize the accumulation of fluorescently-labeled ligands in endocytic vesicles. CHO(iLR) (A) and ldlA(iLR) (B) cells were incubated with Dil-LDL. HDLp labeled with DiI (C,D) or OG (E,F) was used to incubate CHO(iLR) (C,E) and wild-type CHO (D,F) cells. Uptake of fluorescently-labeled HDLp is reduced by an excess of unlabeled HDLp. CHO(iLR) cells were also incubated for 15 minutes at 37°C (G) or 30 minutes at 18°C (H,I) in incubation medium containing OG-HDLp in the absence (G,H) or presence (I) of a tenfold excess of unlabeled HDLp. Bars, 20 µm.

View larger version (36K): [in a new window]   Fig. 3. LDL endocytosis by CHO(iLR) cells is not significantly reduced by an equimolar concentration of RAP; however, endocytic uptake of HDLp is completely inhibited. CHO(iLR) cells were incubated for 30 minutes at 18°C with DiI-LDL (A) or OG-HDLp (B) in the presence of an equimolar concentration of unlabeled RAP. RAP is internalized by iLR-expressing CHO cells. CHO(iLR) (C) and wild-type CHO (D) cells were incubated with OG-RAP for 30 minutes at 18°C. Bar, 10 µm.

View larger version (56K): [in a new window]   Fig. 4. HDLp colocalizes with LDL in early endocytic vesicles. CHO(iLR) cells were allowed to simultaneously internalize OG-HDLp (A) and DiI-LDL (B) in incubation medium for 30 minutes at 18°C. Fixed cells were analyzed using confocal laser microscopy to visualize the colocalization of the ligands in endosomes by overlaying the two images (C). Overlapping fluorescently labeled endosomes stain yellow after merging the layers. The HDLp-positive juxtanuclear compartment is depleted of LDL. CHO(iLR) cells were simultaneously preincubated with OG-HDLp and DiI-LDL. After the preincubation, the cells were transferred to an aluminium chamber and incubated in chase medium at 37°C. At 10 minutes, large amounts of HDLp concentrated in the juxtanuclear region (D), whereas LDL remained spatially distributed throughout the entire cell interior (F). Within a defined area (squares in D and F), the relative fluorescent intensity of the juxtanuclear-positioned structure was plotted on a relative scale (from 0 to 255, indicated by the vertical bar) for OG-HDLp (E) and DiI-LDL (G). Internalized HDLp accumulates in a non-lysosomal juxtanuclear compartment. CHO cells stably expressing iLR were preincubated with OG-HDLp, rinsed in HEPES buffer and mounted in an aluminium chamber. The cells were subsequently incubated at 37°C in chase medium that was supplemented with LT. Images were generated with multicolour imaging, using confocal laser microscopy to spatially visualize internalized HDLp and LT, simultaneously, in living cells. After a chase of 15 minutes, OG-HDLp-positive endocytic vesicles were highly concentrated in the juxtanuclear region (H), which was depleted of LT (I). Partial colocalization with LT was visualized by merging the two images (J). To enhance the visibility of the spatial distribution of HDLp and LT, a bright-field image of the observed cells was overlayed with fluorescent images. Additionally, detailed images of a single juxtanuclear structure were taken to visualize the minimal colocalization (K,L,M). Intracellular transport of ligands by iLR is microtubule-dependent. CHO(iLR) cells were preincubated with fluorescently-labeled ligand in the presence of 5 µM nocodazole. The cells were subsequently incubated for an additional 30 minutes at 37°C in chase medium supplemented with 5 µM nocodazole. Fixed cells were observed with confocal laser microscopy and showed a peripheral localization of vesicles that contained LDL (N), HDLp (O) or RAP (P). Bars, 20 µm.

View larger version (58K): [in a new window]   Fig. 5. HDLp colocalizes with internalized Tf in the ERC. Directly after preincubation for 20 minutes at 18°C with OG-HDLp and TMR-Tf, iLR-transfected CHO cells were rinsed in HEPES buffer and mounted in an aluminium chamber. The living cells were observed at 37°C in chase medium and imaged using confocal laser scanning microscopy. Digital multicolour images of OG-HDLp (left panel) and TMR-Tf (middle panel) were taken at defined time points as indicated in the left panel. Colocalization was visualized by merging the two images of the same time point (right panel). The insert in the upper right panel shows a bright-field image of the observed cells directly after preincubation. Bar, 20 µm.

View larger version (61K): [in a new window]   Fig. 6. Internalized HDLp is trapped intracellularly by monensin. CHO(iLR) cells were incubated with fluorescently-labeled ligands in the presence of 25 µM monensin for 30 minutes at 18°C. The cells were subsequently chased for 30 minutes at 37°C with an equal concentration of monensin and mounted in mowiol after fixation. LDL was scattered throughout the cell in vesicles (A); however, HDLp was predominantly located in the juxtanuclear area (B). The non-exchangeable protein matrix of HDLp is transported to the ERC. CHO(iLR) cells were allowed to take up OG-HDLp for 15 minutes at 37°C in the presence of 25 µM monensin. After a chase of 30 minutes with an equal concentration of monensin, the cells were washed and labeled with antibodies against apoLp-I and -II, which were visualized with a Cy5-labeled second antibody. The fluorescent label OG that represents intracellular HDLp (C,D) colocalized with apoLp-I (E) and -II (F) in the juxtanuclear area (G,H). Bars, 10 µm.

View larger version (78K): [in a new window]   Fig. 7. Internalized RAP accumulates in the juxtanuclear area. CHO(iLR) cells preincubated with OG-RAP and chased for 30 minutes at 37°C in the presence of 25 µM monensin were fixed and mounted in mowiol. The cells were observed with fluorescence microscopy to visualize RAP that was predominantly located in the juxtanuclear region (A). RAP follows a transferrin-like intracellular pathway. CHO(iLR) cells were simultaneously preincubated with OG-RAP and TMR-Tf and chased for 30 minutes in the presence of 25 µM monensin. Digital images of fixed cells containing RAP (B) and Tf (C) were generated with laser scanning microscopy and the colocalization in the juxtanuclear area was visualized by merging the two images (D). HDLp colocalizes with iLR in the ERC. To determine the localization of iLR after preincubation with OG-HDLp and chase for 30 minutes in the presence of 25 µM monensin, CHO(iLR) cells were fixed and labeled with antibodies against iLR which were visualized with a Cy5-labeled second antibody. OG-HDLp (E) and iLR (F) show significant overlap in the ERC (G). iLR is also abundantly located in the ERC in the absence of ligand or monensin. CHO(iLR) cells were fixed after treatment with incubation medium for 15 minutes at 37°C and iLR was visualized as described above (H). Bars, 10 µm.

View larger version (43K): [in a new window]   Fig. 8. HDLp exits CHO(iLR) cells with a t of 13 minutes. Living CHO(iLR) cells, preincubated with DiI-LDL and OG-HDLp, were observed in an aluminium chamber with confocal laser microscopy during a chase for 20 minutes. Digital photos were collected 10 minutes after initiating the chase at 2 minute time intervals (A-F) to visualize the intracellular distribution of LDL (red) and HDLp (green). To quantify the re-secretion of internalized fluorescently-labeled lipoproteins, CHO(iLR) cells were fixed directly after preincubation with DiI-LDL (filled circles) or OG-HDLp (open circles), and after a chase for 10, 30 or 60 minutes (G). Images were recorded using confocal laser scanning microscopy and analyzed with Scion Image software. The data points are geometric means of the measured relative intensity of total cells. Of each time point, 10 to 17 images were taken with 4 to 33 cells per image, corresponding to more than 200 cells per data point. The mean values were normalized to the measurement directly after the preincubation. The data were processed with Microsoft Excel and plotted with SigmaPlot. Bar, 20 µm; values are means±s.e.m. of 10 or more digital images.