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First published online 19 September 2006
doi: 10.1242/jcs.03191

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Adipophilin-enriched domains in the ER membrane are sites of lipid droplet biogenesis

¹ Leibniz Institute for Arteriosclerosis Research, University of Münster, Domagkstr. 3, 48149 Münster, Germany
² Department of Medicine, Division of Cardiology, Pulmonary Diseases and Angiology, Heinrich Heine University, Düsseldorf, Germany
³ National Heart and Lung Institute, Imperial College, London, UK

View larger version (154K): [in a new window]   Fig. 1. Conventional thin-section electron microscopy of macrophages lipid laden by treatment with AcLDL for 24 hours. (A) The cells contain accumulations of lipid droplets (LD) in the cytoplasm. (B,C) Examples of close association between lipid droplets (LD) and ER membranes. At the sites of association with the lipid droplet, the ER lumen becomes narrowed and ribosomes are absent. G, Golgi apparatus; M, mitochondrion; N, nucleus. Bars, 1 µm (A); 0.2 µm (B,C).

View larger version (17K): [in a new window]   Fig. 2. Western blot analysis of adipophilin in whole nonlipid-laden (–AcLDL) and lipid-laden (+AcLDL) macrophages (A) and in isolated fractions from cells (B).

View larger version (63K): [in a new window]   Fig. 3. Immunofluorescence confocal microscopy and cryothin-section immunogold label electron microscopy of adipophilin in lipid droplets of AcLDL-treated macrophages. (A) BODIPY staining for neutral lipids. (B) Immunofluorescence labeling of adipophilin. (C) Merged image of A and B. High magnification views (inset in C) show a rim of adipophilin labeling around the neutral lipid core of the droplet. Immunogold labeling (18 nm gold) for adipophilin in cryothin sections examined by electron microscopy reveals prominent labeling at the surface of the lipid droplet (LD), consistent with the immunofluorescence results. Bars, 5 µm (A-C); 0.2 µm (D).

View larger version (145K): [in a new window]   Fig. 4. Survey freeze-fracture view of a lipid-laden macrophage immunogold labeled (18 nm gold) for adipophilin. Prominent labeling is seen on the P-faces of plasma membrane (PL) and ER membranes (ER), and on lipid droplets (LD). The E-face of the ER membranes, mitochondrial membranes (M) and vesicle membranes are devoid of label. Bar, 0.2 µm.

View larger version (169K): [in a new window]   Fig. 5. Freeze-fractured ER membranes in a lipid-laden macrophage after immunogold labeling (18 nm gold) for adipophilin. This example illustrates the consistency of labeling on the P-face, and its absence from the E-face. LD, lipid droplet; M, mitochondria. Bar, 0.5 µm.

View larger version (123K): [in a new window]   Fig. 6. Freeze-fractured nuclear membrane in a lipid-laden macrophage after immunogold labeling (18 nm gold) for adipophilin. The P-face of the outer nuclear membrane (N) is prominently labeled. The E-face of the outer nuclear membrane, and both fracture faces of the inner nuclear membrane are typically devoid of label. The periphery of cross-fractured lipid droplets (LD) and the P-face of the plasma membrane (PL) are also labeled. Bar, 0.5 µm.

View larger version (138K): [in a new window]   Fig. 7. Freeze-fracture views of lipid droplet-ER membrane associations in lipid-laden macrophages. (A) Lipid droplet (LD) situated in a cup formed from ER membranes. The lipid droplet has been convexly fractured to reveal the E-face equivalent (eq) of its outermost monolayer. Portions of the P-face and the E-face of the ER membranes are revealed; in this instance, the membrane adjacent to the lipid droplet is seen in P-face view, and portions of the partner ER membrane in E-face view. Note that both ER membranes constitute the cup that partially envelops the lipid droplet. The lipid droplet extends beneath both the ER membranes at the bottom of the field. As the ER membranes extend from the area of association with the lipid droplet, the lumen of the ER becomes larger. (B) `Bird's-eye' view into an ER cup at a site of lipid droplet association. The fracture plane has followed the plane of the ER membranes within a cup, making a concave fracture, and scooping away the lipid droplet so that only the ER membranes remain. Portions of the E-face of the ER membrane closest to the lipid droplet, and of the P-face of the partner membrane distant, are revealed. This image again confirms that at the site of lipid-droplet association, both ER membranes participate in the cup, with the lipid droplet itself lying close to but external to them. As in A, the two membranes of the ER are much closer together at the site of apposition to the lipid droplet. Bars, 0.2 µm.

View larger version (40K): [in a new window]   Fig. 8. Schematic diagram of fracture planes through ER membranes and lipid droplets. (A) In the electron microscope lipid droplets (LD) are frequently seen cupped by the ER. (B) One ER membrane lies adjacent to the droplet whereas the other membrane is farther removed. At the molecular level both membranes are comprised of cytoplasmic and endoplasmic leaflets of phospholipids, and the lumen of the ER intervenes between the two membranes. The lipid droplet core is enveloped by a phospholipid monolayer. (C) Upon fracturing between membrane leaflets as indicated by dashed lines, P-faces of the inner ER membrane, E-faces of outer ER membrane and the E-face equivalent of the lipid droplet core are revealed together with convexly fractured droplets. (D) Concave (E-faceeq) fractures of droplets, by contrast, are accompanied by E-faces of the inner ER membrane, P-faces of the outer ER membrane and the P-face of the lipid droplet monolayer. Phospholipid molecules (and immunoreactive integral membrane proteins, not shown) remaining on the replicas after SDS washing are depicted in blue (P-face) and purple (E-face). Gray areas represent residual membrane monolayers removed by SDS washing. Asterisks represent the physical locations of gold particles in the replicas marking specific membrane proteins following immunogold labeling. Arrows indicate the direction of viewing of the replicas in the electron microscope.

View larger version (200K): [in a new window]   Fig. 9. Freeze-fracture views of lipid droplet-ER membrane associations after immunogold labeling (18 nm gold) for adipophilin in lipid-laden macrophages. (A,B) In convex fractures of lipid droplets and associated ER membranes, prominent labeling for adipophilin is visible on the P-face of the ER membrane adjacent to the lipid droplet, whereas the partner membrane, seen in E-face view in A is devoid of label. The view of the lipid droplet is the E-face equivalent (eq) of the monolayer; this aspect is devoid of label. (C,D) Concave fractures of the lipid droplet revealing the P-face of its outermost monolayer show prominent adipophilin labeling. The E-face of the ER membrane immediately adjacent to the lipid droplet is unlabeled, whereas the P-face of the partner ER membrane shows substantial labeling. In D, the ER cup associated with the lipid droplet has been cross-fractured. Bars, 0.2 µm.

View larger version (189K): [in a new window]   Fig. 10. Adipophilin immunogold labeling (12 nm gold) in side-by-side convex and concave fractures of ER cups. The example at the top left of the field (magnified at top right) has been concavely fractured, showing the E-face of the ER membrane adjacent to the lipid droplet and the P-face of its partner membrane. The example at the bottom right (magnified at bottom left) has been convexly fractured to reveal the P-face of the membrane closest to the lipid droplet and the E-face of its partner membrane. No labeling is observed on either E-face view. The P-face of the ER membrane closest to the lipid droplet (lower example) is more heavily labeled than that of the partner membrane (upper example). Bar, 0.5 µm.

View larger version (22K): [in a new window]   Fig. 11. Summary of principal features of putative sites of lipid droplet biogenesis in ER. Lipid droplet biogenesis takes place at specialized cup-shaped regions of the ER. The lipid droplet is cupped by closely apposed ER membrane outside the ER. Adipophilin (dark red) is highly concentrated in the cytoplasmic leaflet of the ER membrane of the cup immediately adjacent to the lipid droplet, and in the envelope of the lipid droplet. Lower concentrations of adipophilin (pink) are found in the cytoplasmic leaflets of ER membranes, but not in endoplasmic leaflets (gray).

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