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First published online 24 June 2008
doi: 10.1242/jcs.025452

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Lipid droplets are arrested in the ER membrane by tight binding of lipidated apolipoprotein B-100

Department of Anatomy and Molecular Cell Biology, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan

View larger version (50K): [in this window] [in a new window]   Fig. 1. ApoB lipidated by MTP activity is necessary for ApoB-crescent formation. (A) Incubation with MTP inhibitors (100 nM BAY13-9952 or WM1159) for 12 hours significantly decreased the number of ApoB-crescents in Huh7 cells. Triple labeling for ApoB (red), LD (green), and nucleus (blue) is shown. ApoB was labeled by a mouse anti-ApoB (6H12) antibody. The same result was obtained in HepG2 cells (data not shown). The number of ApoB-crescent-positive cells was reduced by the MTP inhibitors (left half of the bar graph), and it did not increase even after further treatment with 10 µM ALLN for 12 hours (right half of the bar graph). More than ten pictures were randomly taken, and the ratio of ApoB-crescent-positive cells was quantified. The results from three independent experiments were averaged, and the statistical difference from the control was examined by the Student's t-test (*P<0.001). Bars, 10 µm. (B) Knockdown of MTP or ApoB by RNAi reduced the number of ApoB-crescents in Huh7 cells. The ratio of ApoB-crescent-positive cells was measured three days after RNAi, and after treatment with 10 µM ALLN for the final 12 hours. For western blotting, an equal amount (30 µg) of each cell lysate was electrophoresed. The results from three independent experiments were averaged, and the statistical difference from the control was examined using the Student's t-test (*P<0.001). (C) The ApoB-crescent was increased by ALLN treatment even after protein translation was inhibited using 5 µg/ml cycloheximide (CHX). The results from three independent experiments were averaged, and the statistical difference was examined by the Student's t-test (*P<0.01). (D) Treatment with either 0.4 mM DHA complexed to BSA or 1 µg/ml cyclosporin A for 12 hours considerably increased ApoB-crescent formation. Representative immunofluorescence micrographs using a mouse anti-ApoB (6H12) antibody are presented. The ratio of ApoB-crescent-positive cells is shown in the bar graph. The results from three independent experiments were averaged, and the statistical difference from the controls was examined by a Student's t-test (*P<0.01, **P<0.001).

View larger version (99K): [in this window] [in a new window]   Fig. 2. Immunofluorescence microscopy of ApoB. (A) Labeling of ApoB with four monoclonal antibodies and one polyclonal antibody. Huh7 cells treated with 10 µM ALLN for 12 hours were fixed and permeabilized with 0.01% digitonin for 30 minutes. ApoB (red) and LD (green) are doubly labeled. One monoclonal antibody (6H12) labeled only the ApoB-crescent, whereas the other four antibodies labeled both ApoB-crescents and lysosomes (Ohsaki et al., 2006a) but did not label the ER. Bars, 10 µm. (B) Labeling of ApoB in ALLN-treated Huh7 cells that were fixed and permeabilized with 0.1% Triton X-100 for 5 minutes before labeling. One monoclonal antibody (6H12) exclusively labeled the ApoB-crescent, whereas the other four antibodies also labeled the ER network in addition to the ApoB-crescent. Bars, 10 µm. (C) Huh7 cells treated with 10 µM ALLN for 12 hours were fixed, permeabilized with 0.01% digitonin for 30 minutes, and subjected to triple labeling for ER proteins (red), ApoB (green), and LD (blue). PDI, calnexin, and transferrin showed a marked colocalization with ApoB around LDs (arrowheads). ApoB was labeled by either a goat polyclonal or mouse monoclonal (6H12) antibody. The nuclear membrane was also labeled for the ER proteins. Bars, 5 µm. (D) Huh7 cells incubated with 10 µM ALLN for 12 hours were fixed and permeabilized with 0.1% Triton X-100 for 5 minutes and labeled by PDI (red) and ApoB (green; goat polyclonal), and LD (blue). PDI was found in a network pattern throughout the cytoplasm, and labeling around LDs was not conspicuous. Bars, 5 µm.

View larger version (59K): [in this window] [in a new window]   Fig. 3. EM revealed that the ApoB-crescent is a flat ER cistern fused to an LD. (A) Pre-embedding immunoEM of Huh7 cells treated with 0.4 mM DHA. The labeling for both ApoB and PDI was localized to the thin cisternal lumen (arrowheads) adhering to the LD. ApoB was labeled by a goat anti-ApoB antibody. High magnification pictures clearly show the presence of the cisternal membrane (arrows). Bars, 500 nm. (B) Conventional EM of Huh7 cells treated with 10 µM ALLN (a,b,e) or 0.4 mM DHA (c,d,f) for 2-12 hours. (a-d) A flat membrane cistern (arrowheads) was observed along the LD perimeter. Some of the cistern was continuous with the rough ER (arrows) or decorated with ribosomes (c). The electron density of the LD is high in DHA-treated samples because osmium tetroxide reacts with unsaturated bonds of DHA. (e,f) High magnification micrographs delineated that the cisternal membrane with a unit membrane appearance (blue arrowheads) ends on the LD surface that lacks the visible membrane (green arrowheads). (g) A scheme of the membrane leaflet continuity between the cisternal membrane and the LD surface. Bars, 500 nm. (C) The ratio of cells with an LD-associated membrane cistern was quantified in EM pictures and compared with the ratio of cells with ApoB-crescents by immunofluorescence microscopy. The two ratios showed a good correlation in Huh7 cells treated with 0.4 mM DHA for 2, 6, and 12 hours. (D) The width of the LD-associated membrane cistern. Ten cisterns were randomly selected from Huh7 cell samples treated with 10 µM ALLN or 0.4 mM DHA for 12 hours. The width of each cistern was measured at four different points. (E) Direct continuity of the cisternal membrane and the LD surface was maintained in the isolated LD preparation. The cisternal membrane (arrowheads) was disrupted in most samples. Bar, 500 nm. (F) Glucose-6-phosphatase activity was visualized by enzyme histochemistry using lead nitrate. The reaction product was found in the LD-fused membrane cistern (arrowheads) and in the ER (arrows). Bar, 500 nm. (G) The ApoB-crescents in cells treated with 0.4 mM DHA for 2, 6, and 12 hours were photographed by EM, and the ratio of crescents that were directly continuous with the ER was quantified. The diameter of LD that contributed to the ApoB-crescent was also measured for the same sample and averaged.

View larger version (75K): [in this window] [in a new window]   Fig. 4. The ApoB-crescent membrane is enriched with free cholesterol. (A) Huh7 cells treated with 10 µM ALLN for 12 hours were incubated with filipin (green) to label free cholesterol-enriched membrane. Filipin stained a crescent-shaped area around LDs (arrowheads). In most LDs, filipin staining was found on the same side as ApoB (red, goat polyclonal; the left panel), but on the opposite side from ADRP (red; the right panel). LDs were stained by BODIPY493/503 (blue). Bars, 5 µm. (B) The ALLN-treated Huh7 cells were treated with filipin and observed by EM. High-magnification images are shown in the right. Note that the membrane of the ApoB-crescent shows wavy deformation (arrowheads in the lower right figure), indicating the presence of filipin-cholesterol complexes. The membranes of mitochondria (Mt) and sER were smooth and not deformed (arrows in the upper right figure). Bar, 200 nm.

View larger version (43K): [in this window] [in a new window]   Fig. 5. PAT proteins suppress ApoB-crescent formation. (A) Overexpression of ADRP and TIP47 suppressed the formation of ApoB-crescents in Huh7 cells. To identify transfected cells, EGFP cDNA was introduced together with empty vector, ADRP cDNA, or TIP47 cDNA. Two days after transfection, cells were treated by 10 µM ALLN for 12 hours and labeled by mouse anti-ApoB (6H12) antibody (red, arrowheads). Asterisks in images indicate the nuclei of EGFP-negative cells. The results from three independent experiments were averaged, and the statistical difference from the control that was transfected with empty vector was examined by the Student's t-test (*P<0.05, **P<0.01). Bars, 10 µm. (B) Knockdown of ADRP and TIP47 by RNAi increased the number of ApoB-crescents. Huh7 cells were examined three days after RNAi without any further treatment. Western blotting of cell lysates (10 µg) confirmed that there was a significant decrease in the levels of the targeted proteins. The results from three independent experiments were averaged, and the statistical difference from the control transfected with control RNA was examined by the Student's t-test (*P<0.001).

View larger version (36K): [in this window] [in a new window]   Fig. 6. ApoB in the ApoB-crescent tightly adheres to the LD surface through non-ionic interactions. (A) Gallery of isolated LDs labeled for ApoB (green; goat polyclonal) and ADRP (red). LDs were isolated from Huh7 cells treated with 0.4 mM DHA for 12 hours. ApoB and ADRP were observed in complementary areas of LDs. Bar, 5 µm. (B) LDs isolated from DHA-treated cells were incubated with 0.1 M sodium carbonate for 30 minutes at 4°C, and fractionated by the second round of density-gradient centrifugation. Using western blotting, most ApoB and ADRP were recovered from the top floating LD fraction, whereas cyclophilin B was shifted to the bottom soluble fraction.

View larger version (47K): [in this window] [in a new window]   Fig. 7. A putative mechanism for ApoB-crescent formation. (Top) Under normal conditions, lipid esters synthesized in the ER membrane continuously leave the ER membrane by forming LDs. LDs might leave the ER membrane by hatching, budding, or other mechanisms. In hepatocytes, LDs might recycle to the ER to supply lipids for VLDL formation. ApoB is co-translationally lipidated and acquires additional lipids to become mature VLDL. (Bottom) Abnormalities in lipidated ApoB might cause it to bind tightly to the ER membrane and arrest the LD departure from the ER. This results in the accumulation of lipid esters in the ER membrane and induces formation of the ApoB-crescent structure.

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